LTBP complex-specific inhibitors of TGFβ and uses thereof

This application is a continuation of U.S. patent application Ser. No. 17/530,786, filed Nov. 19, 2021, allowed, which is a continuation of U.S. patent application Ser. No. 16/997,438, filed Aug. 19, 2020, issued as U.S. Pat. No. 11,214,614, which is a continuation application filed under 35 U.S.C. § 111 (a) of International Application No. PCT/US2020/015915, filed on Jan. 30, 2020, which in turn claims the benefit of and priority to U.S. Provisional Application No. 62/798,927, filed Jan. 30, 2019, the contents of each of which are expressly incorporated herein by reference in their entireties.

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on May 17, 2022, is named 127036 03007SL.txt and is 376,925 bytes in size.

Transforming growth factor beta (TGFβ) superfamily of growth factors are involved in a number of signaling cascades that regulate diverse biological processes including, but not limited to: inhibition of cell growth, tissue homeostasis, extracellular matrix (ECM) remodeling, endothelial to mesenchymal transition, cell migration and invasion, and immune modulation/suppression, as well as mesenchymal to epithelial transition. In relation to ECM remodeling, TGFβ signaling may increase fibroblast populations and ECM deposition (e.g., collagen). In the immune system, TGFβ ligand modulates T regulatory cell function and maintenance of immune precursor cell growth and homeostasis. In normal epithelial cells, TGFβ is a potent growth inhibitor and promoter of cellular differentiation. However, as tumors develop and progress, they frequently lose their negative growth response to TGFβ. In this setting, TGFβ may become a promoter of tumor development due to its ability to stimulate angiogenesis, alter the stromal environment, and induce local and systemic immunosuppression. For these and other reasons, TGFβ has been a therapeutic target for a number of clinical indications. Despite much effort made to date by a number of groups, clinical development of a TGFβ therapeutic has been challenging.

Observations from preclinical studies, including in rats and dogs, have revealed certain toxicities associated with inhibition of TGFβ in vivo. Moreover, although several TGFβ inhibitors have been developed to date, most clinical programs targeting TGFβ have been discontinued due to side effects or risk of toxicity.

For example, Anderton et al. (Toxicology Pathology, 39: 916-24, 2011) reported that small molecule inhibitors of TGFβ type I (ALK5) receptor induced heart valve lesions characterized by hemorrhage, inflammation, degeneration and proliferation of valvular interstitial cells in a preclinical animal model. The toxicity was observed in all heart valves at all doses tested. Frazier et al. (Toxicology Pathology, 35: 284-295, 2007) reported that administration of the small molecule inhibitor of TGFβ type I (ALK5) receptor GW788388 induced physeal dysplasia in rats.

Stauber et al. (J. Clin. Practice 4:3, 2014) reported that a chronic (≥3 months) administration of the inhibitor of TGFβ receptor I kinase, LY2157299, which is being investigated for certain cancer treatments, caused multiple organ toxicities involving the cardiovascular, gastrointestinal, immune, bone/cartilage, reproductive, and renal systems, in rats and dogs.

Fresolimumab (GC1008), a “pan” TGFβ antibody capable of neutralizing all human isoforms of TGFβ, has been reported to induce an epithelial hyperplasia of the gingiva, bladder, and of the nasal turbinate epithelium after multiple administrations in studies with cynomolgus macaques (Lonning et al., Current Pharmaceutical Biotechnology 12: 2176-89, 2011). Similarly, a variety of skin rashes/lesions, gingival bleeding and fatigue have been reported in clinical trials after administration of multiple doses of the drug. The most notable adverse reaction to fresolimumab includes the induction of cutaneous keratoacanthomas and/or squamous cell carcinomas in human cancer patients (see, for example: Lacouture et al., 2015, Cancer Immunol Immunother, 64: 437-46; Stevenson et al., 2013, Oncolmmunology, 2:8, e26218; and Lonning et al., 2011). Additional evidence from a clinical trial suggests that in some cases this antibody may accelerate tumor progression (Stevenson et al., 2013, Oncolmmunology, 2:8, e26218).

Thus, new methods and compositions for modulating TGFβ signaling are necessary that can be used to effectively and safely treat diseases and disorders involving TGFβ, including, for example, cancer, fibrosis and inflammation.

With an increasing recognition of potentially dangerous adverse effects associated with broad inhibition of TGFβ, a number of groups have more recently turned to identifying inhibitors that target a subset—but not all—of the isoforms and still retain sufficient efficacy. For example, WO 2016/161410 discloses neutralizing antibodies that bind both TGFβ1 and TGFβ2 (i.e., TGFβ1/2 inhibitors). WO 2006/116002 provides neutralizing antibodies that bind both TGFβ1 and TGFβ3 (i.e., TGFβ1/3 inhibitors), albeit preferentially to the former. In addition to traditional monoclonal antibodies, some groups are developing engineered fusion proteins that function as so-called “ligand traps” (see, for example, WO 2018/158727, WO 2018029367 and WO 2018129331), at least some of which may be selective for TGFβ1/3. Another class of TGFβ1/3 inhibitors include inhibitors of alpha-V (αv) integrins such as antibodies against αvβ6, which is an integrin known to activate both TGFβ1 and TGFβ3 (i.e., TGFβ1/3). Yet others continue to pursue “better” pan-inhibitors that inhibit all three isoforms (i.e., TGFβ1/2/3 or pan-inhibitors) (see, for example, WO 2018/134681).

From an efficacy standpoint, however, the prevailing view of the field remains to be that it is advantageous to inhibit multiple isoforms of TGFβ to achieve therapeutic effects, and to accommodate this, toxicity management by “careful dosing regimen” is suggested as a solution (Brennan et al. (2018) mAbs, 10:1, 1-17).

Recently, Applicant described isoform-selective TGFβ1 inhibitors which were demonstrated to be both safe and efficacious in animal models (see, for example: WO 2017/156500 and WO 2018/129329, incorporated by reference), supporting the notion that selectively targeting the TGFβ1 isoform, as opposed to broadly antagonizing all TGFβ isoforms, may provide an advantageous approach to achieving efficacy with acceptable toxicity.

Whilst the observed safety profile achieved by selective inhibition of TGFβ1 at doses that were shown efficacious in vivo is a promising step towards developing a TGFβ1 inhibitor for clinical applications, identification of TGFβ1 inhibitors that are capable of selectively affecting a defined subset of TGFβ1 effects (e.g., TGFβ inhibitors that are selective to LTBP-presented complexes) remained elusive. More recently, Applicant demonstrated that such “LTBP context-specific” inhibitors can be generated (WO 2019/023661, incorporated herein by reference) using the methods previously described by Applicant (see, for example, WO 2014/074532 and WO 2014/182676). However, the LTBP-selective TGFβ1 inhibitors described in the aforementioned international publication showed modest affinities and inhibitory activities, coupled with suboptimal cross-species reactivity.

The present disclosure provides improved TGFβ inhibitors capable of selectively targeting matrix-associated proTGFβ complexes, such as LTBP1-proTGFβ1 and LTBP3-proTGFβ1.

These inhibitors bind and inhibit LTBP1- and/or LTBP3-presented proTGFβ at high affinities (at least nanomolar range) but do not bind and inhibit immune cell-associated TGFβ, e.g., GARP- and/or LRRC33-presented proTGFβ1, or the binding is below meaningful levels (e.g., at least 50 times affinities for the LTBP complexes over GARP or LRRC33 complex). Thus, these inhibitors can selectively inhibit activation of TGFβ in a context-dependent manner, such that they selectively bind, thereby inhibiting the TGFβ signaling axis associated with the ECM. In particular, the present disclosure includes selective inhibitors of matrix-associated (e.g., LTBP1 and/or LTBP3-associated) TGFβ activation. In some embodiments, such inhibitors specifically bind a particular isoform of TGFβ (e.g., proTGFβ1, proTGFβ2, and/or proTGFβ3) associated with LTBP1 and/or LTBP3, thus also providing TGFβ isoform specificity. In a particular embodiment, such inhibitors specifically bind to LTBP1/3-proTGFβ1. In any of the embodiments of the present invention, such inhibitors do not inhibit activation of TGFβ1 associated with immune cell function, mediated by GARP and/or LRRC33. The improved antibodies encompassed by the present disclosure have affinities towards human LTBP1-proTGFβ1 and/or human LTBP3-proTGFβ1 in at least a nanomolar range (i.e., 1×10 9 M to 10×10 −9 M). In some embodiments, such antibodies also have affinities towards murine LTBP1-proTGFβ1 and/or murine LTBP3-proTGFβ1 in at least a nanomolar range (i.e., 1×10 −9 M to 10×10 −9 M).

Rationale for the therapeutic use of a TGFβ1 inhibitor that does not target the GARP-proTGFβ1 complex on regulatory T cells is at least threefold:

First, regulatory T cells play a crucial role in maintaining immune tolerance to self-antigens and in preventing autoimmune disease. Since Tregs generally suppress, dampen or downregulate induction and proliferation of effector T cells, systemic inhibition of this function may lead to overactive or exaggerated immune responses in the host by disabling the “break” that is normally provided by Treg cells. Thus, the approach taken here (e.g., TGFβ1 inhibition without disabling Treg function) is aimed to avoid the risk of eliciting autoimmunity. Furthermore, patients who already have a propensity for developing over-sensitive immune responses or autoimmunity may be particularly at risk of triggering or exacerbating such conditions, without the availability of normal Treg function; and therefore, the inhibitors that selectively target the matrix TGFβ1 may advantageously minimize such risk.

Second, evidence suggests that an alteration in the Th17/Treg ratio leads to an imbalance in pro-fibrotic Th17 cytokines, which correlate with severity of fibrosis, such as liver fibrosis (see, for example, Shoukry et al. (2017) J Immunol 198 (1 Supplement): 197.12). The present inventors reasoned that perturbation of the GARP arm of TGFβ1 function may directly or indirectly exacerbate fibrotic conditions.

Third, regulatory T cells are indispensable for immune homeostasis and the prevention of autoimmunity. It was reasoned that, particularly for a TGFβ1 inhibition therapy intended for a long-term or chronic administration, it would be desirable to avoid potential side effects stemming from perturbation of normal Treg function in maintaining immune homeostasis (reviewed in, for example, Richert-Spuhler and Lund (2015) Prog Mol Biol Transl Sci. 136: 217-243). This strategy is at least in part aimed to preserve normal immune function, which is required, inter alia, for combatting infections.

To this end, the inventors of the present disclosure set out to generate isoform-specific, context-selective inhibitors of TGFβ1 that selectively target matrix-associated TGFβ1 activation but not immune cell-associated TGFβ1 activation.

Technical challenges that exist to date include limited ability to discern and selectively modulate these subpools of TGFβ1 present in various contexts (or “niches”) in vivo.

In an effort to address this challenge, the present inventors have identified isoform-specific monoclonal antibodies that bind the latent TGFβ1 prodomain, with no detectable binding to latent TGFβ2 or TGFβ3, and that inhibit integrin-mediated activation of latent TGFβ1 in vitro with the context-dependency as described herein. The discovery and characterization of such antibodies was made possible, at least in part, by the development of context-dependent cell-based assays of TGFβ1 activation. In the process of this novel assay development and validation, it was demonstrated that, like the αVβ6 integrin, αVβ8 can also activate LTBP1-proTGFβ1. It was further demonstrated that, similar to the LTBP1 complex, LTBP3-proTGFβ1 can be activated by αVβ6. Antibodies discovered by screening in these assays revealed a class of antibodies that binds and inhibits TGFβ1 only when presented by LTBP1 or LTBP3. Such LTBP-specific antibodies do not inhibit TGFβ1 in the context of the immune-associated TGFβ1 presenters GARP and LRRC33. Such antibodies are therapeutic candidates for the treatment of disorders including, e.g., fibrotic conditions, and could allow chronic dosing that would avoid TGFβ-related immune system activation. Methods of selecting a context-specific or context-independent TGFβ1 inhibitor for various fibrotic conditions are also provided herein.

Accordingly, in one aspect, the invention provides isoform-specific TGFβ antibodies, or antigen-binding fragments thereof, characterized in that they bind selectively to an LTBP1-TGFβ1 complex and/or a LTBP3-TGFβ1 complex with a K D ≤50 nM. In one embodiment, the invention provides isoform-specific TGFβ antibodies, or antigen-binding fragments thereof, characterized in that they bind selectively to an LTBP1-TGFβ1 complex and/or a LTBP3-TGFβ1 complex with a K D ≤25 nM. In one embodiment, the invention provides isoform-specific TGFβ antibodies, or antigen-binding fragments thereof, characterized in that they bind selectively to an LTBP1-TGFβ1 complex and/or a LTBP3-TGFβ1 complex with a K D ≤10 nM. In one embodiment, the invention provides an isolated antibody, or antigen-binding portion thereof, that selectively binds to a LTBP1-proTGFβ1 complex and a LTBP3-proTGFβ1 complex, wherein the antibody, or antigen-binding portion thereof, does not bind to one or more of the following targets: (a) LTBP1 alone; (b) proTGFβ1 alone; (c) a GARP-proTGFβ1 complex; and (d) a LRRC33-proTGFβ1 complex. In further embodiments, the invention provides isoform-specific TGFβ antibodies, or antigen-binding fragments thereof, characterized in that they bind selectively to an LTBP1-TGFβ1 complex and/or a LTBP3-TGFβ1 complex with a K D ≤5 nM.

In one aspect, the invention provides inhibitors of extracellular matrix-associated TGFβ activation, which selectively bind a LTBP1/3-presented proTGFβ latent complex. In one embodiment, the inhibitor does not inhibit immune cell-associated TGFβ1 activation, for example, immune cell-associated TGFβ1 activation that results from activation of a GARP-presented proTGFβ1 latent complex. In exemplary embodiments, the inhibitor is an antibody, or antigen-binding portion thereof.

In other aspects, the invention provides TGFβ antibodies, or antigen-binding fragments thereof, characterized in that they bind selectively to an LTBP1-TGFβ complex and/or a LTBP3-TGFβ complex. In some embodiments, the antibodies, or antigen-binding fragments thereof, selectively bind to LTBP1-TGFβ1. In some embodiments, such antibodies bind both human and murine counterparts.

In one aspect, the invention provides an isolated antibody, or antigen-binding portion thereof, that selectively binds an LTBP1-proTGFβ latent complex and/or an LTBP3-proTGFβ latent complex, thereby modulating release of mature TGFβ growth factor from the latent complex, wherein the antibody, or antigen-binding portion thereof, does not bind mature TGFβ1 alone or a GARP-proTGFβ1 latent complex. In one embodiment, the antibody, or antigen-binding portion thereof, does not bind an LRRC33-proTGFβ1 latent complex. Alternatively, in one embodiment, the antibody, or antigen-binding portion thereof, binds an LRRC33-proTGFβ1 latent complex.

In some embodiments, the antibody, or antigen-binding portion thereof, is specific to an LTBP1-proTGFβ1 latent complex. In other embodiments, the antibody, or antigen-binding portion thereof, is specific to an LTBP3-proTGFβ1 latent complex. In one embodiment, the antibody, or antigen-binding portion thereof, binds an LTBP1-proTGFβ1 complex and/or a LTBP3-proTGFβ1 complex with a dissociation constant (K D ) of at least about 10 −8 M. In one embodiment, the antibody, or antigen-binding portion thereof, binds a human LTBP1-proTGFβ1 complex and/or a human LTBP3-proTGFβ1 complex with a K D of <50 nM as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI). In one embodiment, the antibody, or antigen-binding portion thereof, binds a human LTBP1-proTGFβ1 complex and/or a human LTBP3-proTGFβ1 complex with a K D of <10 nM as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI). In one embodiment, the antibody, or antigen-binding portion thereof, binds a mouse LTBP1-proTGFβ1 complex and/or a mouse LTBP3-proTGFβ1 complex with a K D of <50 nM as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI). In one embodiment, the antibody, or antigen-binding portion thereof, binds a mouse LTBP1-proTGFβ1 complex and/or a mouse LTBP3-proTGFβ1 complex with a K D of <10 nM as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI).

The present disclosure further provides antibodies and antigen binding fragments thereof, which selectively bind an LTBP1-proTGFβ complex and/or an LTBP3-proTGFβ complex and have one or more yet further advantageous properties. Indeed, the inventors surprisingly found that such antibodies could be provided which bind a human LTBP1-proTGFβ complex and a human LTBP3-proTGFβ complex with high affinity, and advantageously slow dissociation rates, while also being cross-reactive with mouse LTBP1-proTGFβ complex and mouse LTBP3-proTGFβ complex and displaying no significant binding to human GARP-proTGFβ complex (or indeed to human LRRC33-proTGFβ complex).

Further still, antibodies disclosed herein (including antibodies having one or more, or even all of the aforementioned advantageous properties) exhibit potent inhibition of TGFβ1 signaling in cell-based assays, and significantly reduce markers of fibrosis and TGFβ signaling in multiple animal models of fibrosis.

Thus, in some embodiments, the antibody, or antigen-binding fragment thereof binds a human LTBP1-proTGFβ1 complex and/or a human LTBP3-TGFβ1 complex with a K D of <5 nM as measured by BLI, and has one or more of the following properties:

i) is cross-reactive with mouse LTBP1-proTGFβ1 complex; ii) is cross-reactive with mouse LTBP3-proTGFβ1 complex; iii) binds a mouse LTBP1-proTGFβ1 complex with a K D of <10 nM as measured by BLI; iv) binds a mouse LTBP3-proTGFβ1 complex with a K D of <10 nM as measured by BLI; v) binds a human LTBP1-proTGFβ1 complex and/or a human LTBP3-TGFβ1 complex with a K D that is at least 50 times lower than the K D when binding to a human GARP-proTGFβ1 complex under the same assay conditions; vi) does not show detectable binding to a human GARP-proTGFβ1 complex, as measured by BLI, under the same assay conditions as used to measure binding to human LTBP1-proTGFβ1 complex and/or a human LTBP3-TGFβ1 complex; vii) does not show detectable binding to an LRRC33-proTGFβ1 complex (e.g., a human LRRC33-proTGFβ1 complex) as measured by BLI, under the same assay conditions as used to measure binding to human LTBP1-proTGFβ1 complex and/or human LTBP3-TGFβ1 complex.

In some embodiments, the antibody or antigen-binding fragment has at least properties (i)-(v) above, and optionally (vii). In some embodiments, the antibody or antigen-binding fragment has at least properties (i)-(iv) and (vi) above, and optionally (vii). In some embodiments, the antibody or antigen-binding fragment has at least properties (i), (iii) and (v) above, and optionally (vii). In some embodiments, the antibody or antigen-binding fragment has at least properties (ii), (iv) and (v) above, and optionally (vii). In some embodiments, the antibody or antigen-binding fragment has at least properties (i), (iii) and (vi) above, and optionally (vii). In some embodiments, the antibody or antigen-binding fragment has at least properties (ii), (iv) and (vi) above, and optionally (vii). In some embodiments, the antibody or antigen-binding fragment has at least properties (i)-(iii) and (v) above, and optionally (vii). In some embodiments, the antibody or antigen-binding fragment has at least properties (i)-(iii) and (vi) above, and optionally (vii).

In some preferred embodiments, the antibody or antigen-binding fragment binds a human LTBP1-proTGFβ1 complex and a human LTBP3-TGFβ1 complex with a K D of <5 nM as measured by BLI, and has all of the above properties (i)-(vii).

The antibody or antigen-binding fragment may selectively bind a LTBP1/3-presented proTGFβ latent complex and inhibit extracellular matrix-associated TGFβ activation.

Further still, further advantageous isoform-selective inhibitors of TGFβ1 activation may include monoclonal antibodies (including immunoglobulins and antigen-binding fragments or portions thereof) that exhibit slow dissociation rates (i.e., off-rates, k OFF ). Thus, the invention is further based on the recognition that treatment of chronic and progressive disease such as fibrosis may require inhibitors with superior durability, which may be reflected on the dissociation rate of such antibody.

The affinity of an antibody to its antigen is typically measured as the equilibrium dissociation constant, or K D . The ratio of the experimentally measured off- and on-rates (k OFF /k ON ) can be used to calculate the K D value. The k OFF value represents the antibody dissociation rate, which indicates how quickly it dissociates from its antigen, whilst the k ON value represents the antibody association rate which provides how quickly it binds to its antigen. The latter is typically concentration-dependent, while the former is concentration-independent. The K D value relates to the concentration of antibody (the amount of antibody needed for a particular experiment) and so the lower the K D value (lower concentration) and thus the higher the affinity of the antibody. With respect to a reference antibody, a higher affinity antibody may have a lower k OFF rate, a higher k ON rate, or both.

Both the k OFF and k ON rates contribute to the overall affinity of a particular antibody to its antigen, and relative importance or impact of each component may depend on the mechanism of action of the antibody. For example, neutralizing antibodies, which bind mature growth factors (e.g., soluble, transient TGFβ1 ligand liberated from a latent complex), must compete with the endogenous high-affinity receptors for ligand binding in vivo. Because the ligand-receptor interaction is a local event and because the ligand is short-lived, such antibodies must be capable of rapidly targeting and sequestering the soluble growth factor before the ligand finds its cellular receptor—thereby activating the TGFβ1 signaling pathway—in the tissue. Therefore, for ligand-targeting neutralizing antibodies to be potent, the ability to bind the target growth factor fast, i.e., high association rates (k ON ), may be especially important.

By contrast, Applicant reasoned that antibodies that inhibit the TGFβ1 signaling by preventing the activation (e.g., release) of mature growth factor from the latent complex (“activation inhibitors”) may preferentially benefit from having slow dissociation rates once the antibody is engaged with the target antigen (e.g., proTGFβ1 complexes). Unlike neutralizing antibodies, such antibodies do not directly compete with cellular receptors; rather, they work upstream of the signaling by targeting inactive precursor forms (e.g., latent proTGFβ1 complexes) that remain dormant within a tissue environment thereby preemptively preventing the activation of TGFβ1. Such antibodies may exert their inhibitory activity by preventing mature growth factor from being liberated from the latent complex. For example, such antibodies may function like a “clamp” to lock the active growth factor in the prodomain cage structure to keep it in an inactive (e.g., “latent”) state. Indeed, structural analyses, including epitope mapping, provided insight into the molecular mechanism underlining the ability of these antibodies to block TGFβ1 activation. In this regard, the Latency Lasso region of the prodomain may be a particularly useful target.

Upon target engagement, antibodies that are able to remain bound to the target (e.g., dissociate very slowly from the latent complex) are expected to be advantageous in achieving superior in vivo potency, due to enhanced durability of effects and/or avidity. Based on this recognition, Applicant of the present disclosure sought to identify isoform-selective activation inhibitors of TGFβ1 with particularly low k OFF values as compared to previously described antibodies. Thus, according to the invention, preferred antibodies have high affinities primarily attributable to a slow dissociation rate (k OFF ), as opposed to fast association rate (k ON ). Accordingly, in some embodiments, the antibody, or antigen-binding fragment thereof binds a human LTBP1-proTGFβ1 complex and/or a human LTBP3-TGFβ1 complex with a K D of <5 nM as measured by BLI, and has one or more of the following properties (which may be in addition to one of properties (i)-(vii), or combinations thereof set out above):

(viii) low dissociation rates (k OFF ) of <5×10 −4 (1/s), when binding human LTBP1-proTGFβ1 complex and/or human LTBP3-TGFβ1 complex (e.g., as measured by a suitable in vitro binding/kinetics assay, such as by BLI, e.g., Octet-based systems); and/or (ix) long half-binding time (t½) of ≥45 minutes when bound to human LTBP1-proTGFβ1 and/or human LTBP3-proTGFβ1 complex (e.g., as measured by SPR).

In some preferred embodiments, the antibody or antigen binding fragment comprises the following six CDRs:

a) CDR-H1 comprising the amino acid sequence FTFRSYVMH (SEQ ID NO: 166); b) CDR-H2 comprising the amino acid sequence VISHEGS(X 1 )KYYADSVKG, wherein: X 1 is L or G (SEQ ID NO: 366); and c) CDR-H3 comprising the amino acid sequence A(X 1 )PRIAARRGGFG(X 2 ), wherein: X 1 is V, R or L; and X 2 is Y, S or T (SEQ ID NO: 367); d) CDR-L1 comprising the amino acid sequence TRS(X 1 )G(X 2 )ID(X 3 )NYVQ, wherein, X 1 is S or H; X 2 is N, L, S or A; and X 3 is N, D or Y (SEQ ID NO: 368); e) CDR-L2 comprising the amino acid sequence ED(X 1 )(X 2 )RPS, wherein: X 1 is N, F or A; and X 2 is Q, I or V (SEQ ID NO: 369); and f) CDR-L3 comprising the amino acid sequence Q(X 1 )YD(X 2 )(X 3 )(X 4 )Q(X 5 )VV, wherein: X 1 is S or G; X 2 is S, F, Y, D, H or W; X 3 is N, D or S; X 4 is N, A, L, E or T; and X 5 is G, R, A or L (SEQ ID NO: 370).

In some preferred embodiments, the antibody or antigen-binding fragment thereof competes or cross-competes with an antibody having a heavy chain variable region sequence as set forth in SEQ ID NO: 318 and light chain variable region sequence as set forth in SEQ ID NO: 319 (e.g., Ab42). The antibody may comprise a heavy chain variable region having an amino acid sequence that is at least 90% identical to SEQ ID NO: 318 and a light chain variable region having an amino acid sequence that is at least 90% identical to SEQ ID NO: 319.

The antibody or antigen-binding fragment thereof provided herein may, in some preferred embodiments, comprise the following six CDRs (e.g., those of Ab42):

CDR-H1 comprising the amino acid sequence FTFRSYVMH (SEQ ID NO: 166); CDR-H2 comprising the amino acid sequence VISHEGSLKYYADSVKG (SEQ ID NO: 167); CDR-H3 comprising the amino acid sequence ARPRIAARRGGFGY (SEQ ID NO: 168); CDR-L1 comprising the amino acid sequence TRSSGNIDNNYVQ (SEQ ID NO: 169); CDR-L2 comprising the amino acid sequence EDNQRPS (SEQ ID NO: 170); and CDR-L3 comprising the amino acid sequence QSYDYDTQGVV (SEQ ID NO: 171).

The antibody or antigen-binding fragment may further comprise a heavy chain variable region having an amino acid sequence that is at least 95% identical (optionally at least 98% identical) to SEQ ID NO: 318 and a light chain variable region having an amino acid sequence that is at least 95% identical (optionally at least 98% identical) to SEQ ID NO: 319.

In some alternative embodiments, the antibody, or antigen-binding fragment thereof, comprises the following six CDRs:

a) CDR-H1 comprising the amino acid sequence GSIRSSSYYWG (SEQ ID NO: 292); b) CDR-H2 comprising the amino acid sequence SISYSATTYY (SEQ ID NO: 293); c) CDR-H3 comprising the amino acid sequence A(X 1 )DPSYDS(X 2 )AGM(X 3 )V, wherein: X 1 is S or G; X 2 is A or I; and X 3 is D or Q (SEQ ID NO: 371); d) CDR-L1 comprising the amino acid sequence RAS(X 1 )(X 2 )IS(X 3 )YLN, wherein: X 1 is K or Q; X 2 is V or S; and X 3 is S or Y (SEQ ID NO: 389); e) CDR-L2 comprising the amino acid sequence (X 1 )AS(X 2 )(X 3 )QS, wherein: X 1 is Y, A or S; X 2 is S or N; and X 3 is L or R (SEQ ID NO: 390); f) CDR-L3 comprising the amino acid sequence QQ(X 1 )(X 2 )D(X 3 )P(X 4 )T, wherein: X 1 is S or G; X 2 is F or N; X 3 is W or F; and X 4 is F or L (SEQ ID NO: 391).

In some embodiments, the antibody or antigen-binding fragment thereof competes or cross-competes with an antibody having a heavy chain variable region sequence as set forth in SEQ ID NO: 360 and light chain variable region sequence as set forth in SEQ ID NO: 361 (e.g., Ab63). The antibody may comprise a heavy chain variable region having an amino acid sequence that is at least 90% identical to SEQ ID NO: 360 and a light chain variable region having an amino acid sequence that is at least 90% identical to SEQ ID NO: 361.

The antibody or antigen-binding fragment thereof provided herein may comprise the following six CDRs (e.g., those of Ab63):

CDR-H1 comprising the amino acid sequence GSIRSSSYYWG (SEQ ID NO: 292); CDR-H2 comprising the amino acid sequence SISYSATTYY (SEQ ID NO: 293); CDR-H3 comprising the amino acid sequence AGDPSYDSIAGMQV (SEQ ID NO: 294); CDR-L1 comprising the amino acid sequence RASQSISSYLN (SEQ ID NO: 295); CDR-L2 comprising the amino acid sequence AASNLQS (SEQ ID NO: 296); and CDR-L3 comprising the amino acid sequence QQSFDWPLT (SEQ ID NO: 297).

The antibody or antigen-binding fragment may further comprise a heavy chain variable region having an amino acid sequence that is at least 95% identical (optionally at least 98% identical) to SEQ ID NO: 360 and a light chain variable region having an amino acid sequence that is at least 95% identical (optionally at least 98% identical) to SEQ ID NO: 361.

In one aspect, the invention provides an antibody, or antigen-binding fragment thereof, for use in a method for treating a fibrotic disorder in a subject, wherein the antibody, or antigen-binding fragment thereof, specifically binds a human LTBP1-proTGFβ complex and/or a human LTBP3-proTGFβ complex, and does not bind a human GARP-proTGFβ1 complex; wherein: a) the fibrotic disorder comprises chronic inflammation; b) the subject benefits from immune suppression; c) the subject has or is at risk of developing an autoimmune disease; d) the subject is a candidate for or has received an allograft transplant; e) the subject has an elevated Th17/Treg ratio; and/or, f) the subject is in need of a long-term or chronic administration of the TGFβ1 inhibitor. In some embodiments, the the subject has or is at risk of developing a metabolic disorder (and the subject is optionally a subject according to one or more of a)-f)). In some embodiments, the the antibody, or antigen-binding fragment thereof, is an isoform-specific LTBP1-proTGFβ1 inhibitor and/or LTBP3-proTGFβ1 inhibitor.

The antibodies or antigen-binding fragments thereof provided herein may be used in a method for treating a fibrotic disorder in a subject. The fibrotic disorder may comprise chronic inflammation. The subject may benefit from immune suppression. The subject may have or be at risk of developing an autoimmune disease. The subject may be a candidate for or may have received an allograft transplant.

Alternatively, or in addition, the subject may have an elevated Th17/Treg ratio. The subject may be in need of a long-term or chronic administration of the TGFβ1 inhibitor.

Alternatively, or in addition, the subject may have or be at risk of developing a metabolic disorder.

In another aspect, the invention provides a method for making a composition comprising an antibody, or antigen-binding fragment thereof, that specifically binds a human LTBP1-proTGFβ complex and/or a human LTBP3-proTGFβ complex, and does not bind a human GARP-proTGFβ1 complex; wherein the antibody, or antigen-binding fragment thereof, inhibits TGFβ1 but does not inhibit TGFβ2 or TGFβ3, the method comprising steps of i) providing at least one antigen comprising LTBP1-proTGFβ1 and/or LTBP3-proTGFβ1, ii) selecting a first pool of antibodies, or antigen-binding fragments thereof, that specifically bind the at least one antigen of step (i) so as to provide specific binders of LTBP1-proTGFβ1 and/or LTBP3-proTGFβ1; iii) selecting a second pool of antibodies, or antigen-binding fragments thereof, that inhibit activation of TGFβ1, so as to generate specific inhibitors of TGFβ1 activation; iv) formulating an antibody, or antigen-binding fragment thereof, that is present in the first pool of antibodies and the second pool of antibodies into a pharmaceutical composition, thereby making the composition comprising the antibody, or antigen-binding fragment thereof.

In one embodiment, the method further comprises a step of removing from the first pool of antibodies, or antigen-binding fragments thereof, any antibodies, or antigen-binding fragments thereof, that bind GARP-proTGFβ1, LRRC33-proTGFβ1, mature TGFβ1, GARP-proTGFβ2, LRRC33-proTGFβ2, mature TGFβ2, GARP-proTGFβ3, LRRC33-proTGFβ3, mature TGFβ3, or any combinations thereof. In one embodiment, the method further comprises a step of determining or confirming isoform-specificity of the antibodies, or antigen-binding fragments thereof, selected in steps (ii) and/or (iii). In one embodiment, the method further comprises a step of selecting for antibodies, or antigen-binding fragments thereof, that are cross-reactive to human and rodent antigens. In one embodiment, the method further comprises a step of generating a fully human or humanized antibody, or antigen-binding fragment thereof, of the antibody, or antigen-binding fragment thereof, that is present in the first pool of antibodies and the second pool of antibodies.

In one embodiment, the method further comprises a step of subjecting the antibody, or antigen-binding fragment thereof, that is present in the first pool of antibodies and the second pool of antibodies to affinity maturation and/or optimization, so as to provide an affinity matured and/or optimized antibody or fragment thereof. In one embodiment, the affinity maturation/optimization comprises a step of subjecting the antibody, or antigen-binding fragment thereof, that is present in the first pool of antibodies and/or the second pool of antibodies to light chain shuffling as described herein. In one embodiment, the affinity maturation/optimization comprises the step of subjecting the antibody, or antigen-binding fragment thereof, that is present in the first, second, and/or third pool of antibodies to CDR H1/H2 diversification as described herein. In one embodiment, the affinity maturation/optimization comprises the step of subjecting the antibody, or antigen-binding fragment thereof, to CDR-H3 mutagenesis as described herein. In one embodiment, the affinity maturation/optimization comprises the step of subjecting the antibodies, or antigen-binding fragment thereof, to light chain CDR mutagenesis as described herein. In one embodiment, the affinity maturation/optimization comprises the step of subjecting the antibodies, or antigen-binding fragment thereof, to light chain CDR L1/L2 diversification as described herein.

In one embodiment, the method further comprises a step of determining affinity of the antibodies, or antigen-binding fragments thereof, from the first and/or second pools of antibodies to human LTBP1-proTGFβ1 and/or human LTBP3-proTGFβ1. In some embodiments, the method further comprises a step of removing from the first and/or second pools of antibodies, or antigen-binding fragments thereof, any antibodies, or antigen-binding fragments thereof, that bind to human LTBP1-proTGFβ1 and/or human LTBP3-proTGFβ1 with a K D of >100 nM, >50 nM, >25 nM, or >10 nM, as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI).

In one embodiment, the method further comprises a step of determining affinity of the antibodies, or antigen-binding fragments thereof, from the first and/or second pools to mouse LTBP1-proTGFβ1 and/or mouse LTBP3-proTGFβ1. In some embodiments, the method further comprises a step of removing from the first and/or second pools of antibodies, or antigen-binding fragments thereof, any antibodies, or antigen-binding fragments thereof, that bind to mouse LTBP1-proTGFβ1 and/or mouse LTBP3-proTGFβ1 with a K D of >100 nM, >50 nM, or >10 nM, as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI).

In one embodiment, the method further comprises a step of removing from the first and/or second pools of antibodies, or antigen-binding fragments thereof, any antibodies, or antigen-binding fragments thereof, that do not bind mouse LTBP1-proTGFβ1 and/or mouse LTBP3-proTGFβ1.

In one embodiment, the method further comprises a step of determining the IC 50 of the antibodies, or antigen-binding fragments thereof, from of the first and/or second pools of antibodies, or antigen-binding fragments thereof, as measured by a suitable functional in vitro cell-based assay such as a caga assay, as described herein. In some embodiments, the method comprises the step of removing antibodies, or antigen-binding fragments thereof, from the first and/or second pools of antibodies, or antigen-binding fragments thereof, that have an IC 50 of greater than 100 nM, 50 nM, 25 nM, 10 nM, or 5 nM as measured by a cell-based assay (such as a caga assay) as described herein.

In some embodiments, the method comprises the step of removing antibodies, or antigen-binding fragments thereof, from the first and/or second pools, antibodies, or antigen-binding fragments thereof, that have an IC 50 of greater than 50 nM or 10 nM as measured by an endogenous LTBP caga assay as described herein.

In some embodiments, the method comprises the step of removing antibodies, or antigen-binding fragments thereof, from the first and/or second pools, antibodies, or antigen-binding fragments thereof, that have an IC 50 of greater than 50 nM, 25 nM, or 10 nM, as measured by a human LTBP overexpression caga assay as described herein.

In some embodiments, the method comprises the step of removing antibodies, or antigen-binding fragments thereof, from the first and/or second pools, antibodies, or antigen-binding fragments thereof, that have an IC 50 of greater than 50 nM, 25 nM, 10 nM, or 5 nM, as measured by a murine LTBP overexpression caga assay as described herein.

Processes and methods for identifying or selecting TGFβ1-selective inhibitors suitable for therapeutic use are encompassed by the invention, as are methods for making a composition comprising a TGFβ1-selective inhibitor. In preferred embodiments, a TGFβ1 inhibitor (e.g., a selected inhibitor) includes one or more antibodies or antigen-binding fragments with particularly advantageous kinetics criteria characterized by: i) high affinities to each of human LTBP1/3-proTGFβ1 complexes (e.g., K D ≤5 nM), and, ii) low dissociation rates (k OFF ), e.g., ≤5×10 −4 (1/s), as measured by a suitable in vitro binding/kinetics assay, such as by BLI, e.g., Octet-based systems. The low dissociation rate criterion may be reflected in long dissociation half-time t½), e.g., >45 minutes from human LTBP1-proTGFβ1 and/or human LTBP3-proTGFβ1 complexes. Preferably, the long dissociation half-time of an antibody or antigen-binding fragment thereof for the matrix-associated complex(es) is coupled with short dissociation half-time with respect to cell-associated complexes, e.g., human GARP-proTGFβ1 and/or human LRRC33-proTGFβ1 complexes. In particular, preferred antibodies or fragments dissociate from human GARP-proTGFβ1 complex with t½ of no more than 10 minutes, more preferably no more than 5 minutes. Likewise, methods for making a composition comprising a TGFβ1-selective inhibitor as described herein may further include a step of selecting such antibodies. The selected antibody or the plurality of antibodies are evaluated in preclinical studies comprising an efficacy study and a toxicology/safety study, employing suitable preclinical models. Effective amounts of the antibody or the antibodies determined in the efficacy study are below the level that results in undesirable toxicities determined in the toxicology/safety study. Preferably, the antibody or antibodies are selected which has/have at least 3-fold, 6-fold, and more preferably 10-fold therapeutic window. Effective amounts of the antibodies according to the present disclosure may be between about 0.1 mg/kg and about 30 mg/kg when administered weekly. In preferred embodiments, the maximally tolerated dose (MTD) of the antibodies according to the present disclosure is >100 mg/kg when dosed weekly for at least 4 weeks. In some embodiments, in a preclinical toxicology study, the antibodies show a NOAEL of >100 mg/kg/week, >200 mg/kg/week or >300 mg/kg/week, wherein optionally the toxicology study is a 4-week study, 8-week study, or a 12-week study. For example, the NOAEL is >100 mg/kg/week in a 12-week sub-chronic dosing regimen in healthy mice or rats.

The present disclosure also includes a surprising finding that inhibition of TGFβ3 with a TGFβ3-selective inhibitor produced pro fibrotic effects in mice. Similarly, concurrent inhibition of both TGFβ1 and TGFβ3 in the same model with a combination of a TGFβ1-selective inhibitor and a TGFβ3-selective inhibitor resulted in attenuated anti-fibrotic effects of the TGFβ1 inhibitor. These observations raise the possibility that non-selective TGFβ inhibitors (such as pan-inhibitors and TGFβ1/3 inhibitors) may in fact exacerbate fibrosis. Advantageously, the antibodies disclosed herein (e.g., Ab42 and variants thereof, as described herein) are isoform-selective in that they specifically target the latent TGFβ1 complex and do so with low dissociation rates. Thus, the invention includes the recognition that when selecting a particular TGFβ inhibitor for patients with a fibrotic condition (e.g., disease involving ECM dysregulation), isoform selectivity should be carefully considered so as to avoid risk of exacerbating ECM dysregulation. Accordingly, the present disclosure includes therapeutic methods comprising selecting a TGFβ inhibitor that does not inhibit TGFβ3 to treat a subject with a fibrotic condition, (including preferred fibrotic conditions, as described herein).

The isoform-selective LTBP1/3-proTGFβ1 complex-selective inhibitor as used herein may in some embodiments be selected from Ab31, Ab34, Ab37, Ab38, Ab39, Ab40, Ab41, Ab42, Ab43, Ab44, Ab45, Ab62, Ab63, and Ab64 (optionally Ab42 or Ab63) (i.e., an antibody or antigen-binding fragment having the heavy and light chain variable regions of the corresponding Ab, as provided herein), a variant/derivative or antigen-binding fragment thereof, or an engineered molecule comprising an antigen-binding fragment thereof. In some preferred embodiments, the LTBP1/3-proTGFβ1 complex-selective inhibitor inhibitor is Ab42, a variant/derivative or antigen-binding fragment thereof, or an engineered molecule comprising an antigen-binding fragment thereof. In preferred embodiments, the LTBP1/3-proTGFβ1 complex-selective inhibitor is Ab42 or an antigen-binding fragment thereof.

FIG. 1 graphically depicts that targeting of the latent form of TGFβ1 provides isoform and context specificity.

FIGS. 2 A- 2 B demonstrate the identification of isoform-specific and LTBP complex-specific binders of latent TGFβ1. FIG. 2 A demonstrates that SR-AB1 binds latent TGFβ1, independent of the presenting molecule. SR-AB1 is a human monoclonal antibody that was discovered by yeast display, which selectively binds latent TGFβ1, without detectable binding to latent TGFβ2, TGFβ3, or mature TGFβ1. SR-AB1 cross-reacts with mouse, rat, and cynomolgus monkey proteins and binds to all four latent TGFβ1 complexes. FIG. 2 B demonstrates that SR-AB2, an anti-LTBP1-proTGFβ1 antibody, does not bind GARP-proTGFβ1 or mature TGFβ1. SR-AB2 cross-reacts with rodent LTBP1-proTGFβ1.

FIGS. 3 A- 3 B demonstrate functional assays (potency assays) to detect the inhibition of activated recombinant latent TGFβ1. FIG. 3 A depicts the activation of latent TGFβ1 deposited in the extracellular matrix (ECM). In this assay, presenting molecules are co-transfected with proTGFβ1 in integrin-expressing cells. Transiently transfected cells are seeded in assay plates in the presence of inhibitors. Latent LTBP-proTGFβ1 complex is embedded in the ECM. TGFβ reporter cells are then added to the system; free growth factor (released by integrin) signals and is detected by luciferase assay. FIG. 3 B depicts the activation of latent TGFβ1 presented on the cell surface. Presenting molecules are co-transfected with proTGFβ1 in integrin-expressing cells. Latent TGFβ1 is expressed on the cell surface by GARP or LRRC33. TGFβ reporter cells and inhibitors are then added to the system; free growth factor (released by integrin) signals and is detected by luciferase assay.

FIGS. 4 A- 4 B depict the optimization of the recombinant functional assays. FIG. 4 A depicts the relative contribution of presenting molecule and/or proTGFβ1 activation upon co-transfection of presenting molecule and proTGFβ1. FIG. 4 B depicts the optimization of co-transfection: the ratio of plasmid DNAs for presenting molecule and proTGFβ1. Equivalent amounts of each plasmid were optimal for co-transfection.

FIG. 5 demonstrates that fibronectin promotes integrin activation of LTBP-presented latent TGFβ1. Assay plates were pre-coated with fibronectin purified from human plasma. Fibronectin increases integrin-mediated activation of latent TGFβ1 presented by LTBP1 and/or LTBP3.

FIG. 6 is a graph demonstrating that SR-AB1 is a context-independent inhibitor of TGFβ1 activation. SR-AB1 was shown to inhibit integrin-dependent activation of TGFβ1 independent of the presenting molecule.

FIGS. 7 A, 7 B, and 7 C present data confirming LTBP-selective inhibition of TGFβ1 large latent complex (LLC). FIG. 7 A demonstrates that SR-AB2 specifically binds LTBP-proTGFβ1 complex; it does not bind proTGFβ1 or LTBP1 alone. SR-AB2 also does not bind GARP-proTGFβ1. FIG. 7 B depicts that SR-AB2 inhibits integrin activation of LTBP1-proTGFβ1 (human and mouse complexes). FIG. 7 C depicts that SR-AB2 inhibits integrin activation of LTBP3-proTGFβ1.

FIG. 8 presents the heavy chain and light chain variable region sequences of SR-AB2 (SEQ ID NOs: 7-8, respectively, in order of appearance). Complementary determining regions (CDRs) are underlined.

FIG. 9 is a graph demonstrating the binding specificity of SR-AB2 to LTBP1-proTGFβ1 and LTBP3-proTGFβ1 complexes.

FIGS. 10 A- 10 B provide data showing context-selective inhibition of matrix-associated TGFβ1 activation by SR-AB2. FIG. 10 A demonstrates that SR-AB2 inhibits LTBP-proTGFβ, wherein the transfected proTGFβ1 is presented by endogenous LTBP1/3. FIG. 10 B demonstrates that SR-AB2 does not inhibit GARP-presented TGFβ1 activation. These assays were performed in LN229 cells, which express high LTBP3 mRNA, low LTBP1 mRNA, undetectable GARP, and undetectable LRRC33. TGFβ activity, normalized to vehicle, is shown on the y-axis.

FIG. 11 presents binding profiles and affinity data for LTBP complex-specific antibodies SR-AB10, SR-AB2, and SR-13.

FIGS. 12 A and 12 B are graphs showing improved potency of optimized LTBP complex-specific antibodies. FIG. 12 A provides a graph showing improved inhibitory potency of SR-AB14 (an optimized SR-AB10) as measured by cell-based TGFβ reporter assays. FIG. 12 B provides a graph showing improved inhibitory potency of SR-AB15 (an optimized SR-AB13) as measured by cell-based TGFβ assays.

FIGS. 13 A and 13 B are graphs showing improved potency of optimized LTBP complex-specific antibodies after CDR-H3 mutagenesis (i.e., SR-AB20, SR-AB21, SR-AB22, and SR-AB23), as measured by cell-based TGFβ reporter assays.

FIGS. 14 A and 14 B are graphs showing improved potency of optimized LTBP complex-specific antibodies after CDR-H3 mutagenesis (i.e., SR-AB24, SR-AB25, SR-AB26, SR-AB27, SR-AB28, and SR-AB29), as measured by cell-based TGFβ reporter assays.

FIG. 15 is a graph that shows affinity matured antibodies show specific binding to the LTBP-proTGFβ1 complex.

FIG. 16 is a graph showing improved potency of optimized LTBP complex-specific antibodies after cycles 1, 2 and 3 of antibody optimization as measured by cell-based TGFβ reporter assays.

FIG. 17 depicts the results of an enzyme-linked immunosorbent assay (ELISA) showing antibody binding to baculovirus (BV) particles, which tests antibody polyspecificity.

FIG. 18 depicts the results of affinity-capture Self-interaction Nanoparticle Spectroscopy (AC-SINS) assay, which tests antibody self-interaction. Increased plasmon wavelength indicates self-interaction.

FIG. 19 is a graph showing treatment with SR-AB42 and SR-AB31 inhibited the increase in hydroxyproline (HYP) (μg/mg tissue) in liver tissue in animals on a choline-deficient high fat diet (CDHFD).

FIG. 20 A is a graph showing relative ratios of phosphorylated versus total (phosphorylated and unphospohrylated) Smad2/3 (pSMAD2/3:tSMAD2/3) in an Alport mouse model. A single dose of SR-AB42 or SR-AB63 was sufficient to significantly inhibit pSmad2/3 signaling in whole kidney lysates. FIG. 20 B is a graph showing the amount of phosphorylated SMAD2/3 (pSMAD2/3) as determined by ELISA, and FIG. 20 C is a graph showing the amount of total SMAD2/3 (tSMAD2/3) protein as determined by ELISA. As shown by FIG. 20 B and FIG. 20 C , reduction of pSMAD is contributing to the change in ratio shown in FIG. 20 A .

FIG. 21 is a graph showing that the lead cycle 3 antibodies show no inhibition in the LTBP-TGFβ33 assay.

FIG. 22 provides 5 representative PSR-stained images from controls, CDHFD mice treated with Reference Ab, a TGFβ3 inhibitor, or both (left). A graph showing picosirius red area (%) in liver sections of CDHFD mice treated with Reference Ab, a TGFβ3 inhibitor, or both, as compared to control, is also provided (right).

FIG. 23 shows LTBP-complex antibodies such as SR-AB63 are highly specific and have picomolar monovalent affinities.

FIG. 24 shows LTBP-complex antibodies such as SR-AB42 are highly specific and have picomolar monovalent affinities.

The present invention provides compositions that are useful for reducing activation of TGFβ. Inhibitors that target latent proTGFβ complexes, upstream of growth factor-receptor interaction, are generally referred to as activation inhibitors of TGFβ.

To date, four presenting molecules for TGFβ have been identified: latent TGF beta-binding protein 1 (“LTBP1”), latent TGF beta-binding protein 3 (“LTBP3”), glycoprotein A repetitions predominant (“GARP”) and leucine-rich repeat-containing protein 33 (“LRRC33”). Each of these presenting molecules can form disulfide bonds with a homodimeric pro-protein complex of the TGFβ precursor, i.e., proTGFβ. The proTGFβ complex remains dormant (latent) in the respective extracellular niche (e.g., ECM and immune cell surface) until activation events trigger the release of soluble growth factor from the complex.

As compared to the TGFβ growth factors and the receptors, which are expressed broadly, the presenting molecules show more restricted or selective (e.g., tissue-specific) expression patterns, giving rise to functional compartmentalization of TGFβ activities by virtue of association. The four presenting molecule-proTGFβ complexes, namely, LTBP1-proTGFβ, LTBP3-proTGFβ, GARP-proTGFβ and LRRC33-proTGFβ3, therefore, provide discrete “contexts” of TGFβ signaling within the tissue in which the presenting molecules are expressed. These contexts may be divided into two broad categories: i) TGFβ signaling associated with the ECM (e.g., matrix-associated TGFβ function); and ii) TGFβ signaling associated with cells (particularly certain immune cell function). The LTBP1-proTGFβ3 and LTBP3-proTGFβ complexes fall under the first category, while GARP-proTGFβ and LRRC33-proTGFβ complexes fall under the second category. Thus, disclosed herein are inhibitors of TGFβ that are capable of selectively inhibiting the activation of TGFβ that is associated with the ECM. In some embodiments, the inhibitors are also selective for a particular TGFβ isoform (e.g., proTGFβ1, proTGFβ2, and/or proTGFβ33).

In exemplary embodiments, the compositions described herein are useful for selectively reducing activation of TGFβ1 in the context of an LTBP protein, e.g., a LTBP1 and/or a LTBP3 protein. Such compositions advantageously inhibit activation of extracellular matrix-associated TGFβ1, without inhibiting TGFβ1 in the context of the immune-associated TGFβ1 presenting molecules GARP and LRRC33. The compositions described herein are useful for treating disorders associated with TGFβ1 activation, e.g., fibrotic disorders. Accordingly, in embodiments, the invention provides compositions for reducing activation of TGFβ1, methods of use thereof, methods of manufacture, and treatment methods. Methods of selecting a TGFβ1 inhibitor for subjects exhibiting symptoms of a fibrotic disorder are also provided.

In order that the disclosure may be more readily understood, certain terms are first defined. These definitions should be read in light of the remainder of the disclosure and as understood by a person of ordinary skill in the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art. Additional definitions are set forth throughout the detailed description.

Affinity: Affinity is the strength of binding of a molecule (such as an antibody) to its ligand (such as an antigen). It is typically measured and reported by the equilibrium dissociation constant (K D ). K D is the ratio of the antibody dissociation rate (“off rate” or K off ), how quickly it dissociates from its antigen, to the antibody association rate (“on rate” or K on ) of the antibody, how quickly it binds to its antigen. For example, an antibody with an affinity of ≤1 μM has a K D value that is 1 μM or lower (i.e., 1 μM or higher affinity) determined by a suitable in vitro binding assay. Suitable in vitro assays, such as Biolayer Interferometry (e.g., Octet) or surface plasmon resonance (e.g., Biacore System) can be used to assess affinities, as measured by K D values based on well-known methods.

Affinity maturation: Affinity maturation is a type of antibody optimization and is a process of improving the affinity of an antibody or a fragment to its antigen and typically involves making one or more changes to the amino acid sequence of the antibody or the fragment to achieve greater affinity. Typically, a parental antibody and an affinity-matured counterpart retain the same epitope. Affinity maturation may include diversification and/or mutagenesis of one or more CDR sequences.

Antibody: The term “antibody” encompasses any naturally-occurring, recombinant, modified or engineered immunoglobulin or immunoglobulin-like structure or antigen-binding fragment or portion thereof, or derivative thereof, as further described elsewhere herein. Thus, the term refers to an immunoglobulin molecule that specifically binds to a target antigen, and includes, for instance, chimeric, humanized, fully human, and bispecific antibodies. Unless otherwise specified to the contrary, the term “antibody” as used herein shall encompass antigen-binding fragments and variants thereof. An intact antibody will generally comprise at least two full-length heavy chains and two full-length light chains, but in some instances can include fewer chains such as antibodies naturally occurring in camelids which can comprise only heavy chains. Antibodies can be derived solely from a single source, or can be “chimeric,” that is, different portions of the antibody can be derived from two different antibodies. Antibodies, or antigen-binding portions thereof, can be produced in hybridomas, by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact antibodies. The term antibodies, as used herein, includes monoclonal antibodies, bispecific antibodies, minibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as “antibody mimetics”), chimeric antibodies, humanized antibodies, human antibodies, antibody fusions (sometimes referred to herein as “antibody conjugates”), respectively. In some embodiments, the term also encompasses peptibodies.

Antigen: The term “antigen” broadly includes any molecules comprising an antigenic determinant within a binding region(s) to which an antibody or a fragment specifically binds. An antigen can be a single-unit molecule (such as a protein monomer or a fragment) or a complex comprised of multiple components. An antigen provides an epitope, e.g., a molecule or a portion of a molecule, or a complex of molecules or portions of molecules, capable of being bound by a selective binding agent, such as an antigen-binding protein (including, e.g., an antibody). Thus, a selective binding agent may specifically bind to an antigen that is formed by two or more components in a complex. In some embodiments, the antigen is capable of being used in an animal to produce antibodies capable of binding to that antigen. An antigen can possess one or more epitopes that are capable of interacting with different antigen-binding proteins, e.g., antibodies. In the context of the present disclosure, a suitable antigen is a complex (e.g., multimeric complex comprised of multiple components in association) containing a proTGF dimer (“small latent complex” or SLC) preferably in association with a presenting molecule (together “large latent complex” or LLC). Each monomer of the proTGF dimer comprises a prodomain and a growth factor domain, separated by a furin cleavage sequence. Two such monomers form the proTGF dimer complex. This in turn is covalently associated with a presenting molecule via disulfide bonds, which involve a cysteine residue present near the N-terminus of each of the proTGF monomer. This multi-complex formed by a proTGF dimer bound to a presenting molecule is generally referred to as a large latent complex. An antigen complex suitable for screening antibodies or antigen-binding fragments, for example, includes a presenting molecule component of a large latent complex. Such presenting molecule component may be a full-length presenting molecule or a fragment(s) thereof. Minimum required portions of the presenting molecule typically contain at least 50 amino acids, but more preferably at least 100 amino acids of the presenting molecule polypeptide, which comprises two cysteine residues capable of forming covalent bonds with the proTGFβ1 dimer.

Antigen-binding portion/fragment: The terms “antigen-binding portion” or “antigen-binding fragment” of an antibody, as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., LTBP1-proTGFβ1 and LTBP3-proTGFβ1). Antigen-binding portions include, but are not limited to, any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex. In some embodiments, an antigen-binding portion of an antibody may be derived, e.g., from full antibody molecules using any suitable standard techniques such as proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding antibody variable and optionally constant domains. Non-limiting examples of antigen-binding portions include: (i) Fab fragments, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) F(ab′)2 fragments, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) Fd fragments consisting of the VH and CH1 domains; (iv) Fv fragments consisting of the VL and VH domains of a single arm of an antibody; (v) single-chain Fv (scFv) molecules (see, e.g., Bird et al. (1988) SCIENCE 242:423-426; and Huston et al. (1988) PROC. NAT′L. ACAD. SCI. USA 85:5879-5883); (vi) dAb fragments (see, e.g., Ward et al. (1989) NATURE 341: 544-546); and (vii) minimal recognition units consisting of the amino acid residues that mimic the hypervariable region of an antibody (e.g., an isolated complementarity determining region (CDR)). Other forms of single chain antibodies, such as diabodies are also encompassed. The term antigen-binding portion of an antibody includes a “single chain Fab fragment” otherwise known as an “scFab,” comprising an antibody heavy chain variable domain (VH), an antibody constant domain 1 (CH1), an antibody light chain variable domain (VL), an antibody light chain constant domain (CL) and a linker, wherein said antibody domains and said linker have one of the following orders in N-terminal to C-terminal direction: a) VH-CH1-linker-VL-CL, b) VL-CL-linker-VH-CH1, c) VH-CL-linker-VL-CH1 or d) VL-CH1-linker-VH-CL; and wherein said linker is a polypeptide of at least 30 amino acids, preferably between 32 and 50 amino acids.

Advanced fibrosis: As used herein, a subject suffers from advanced fibrosis if s/he has an advanced stage of a fibrotic disorder, particularly organ fibrosis, which renders the patient a candidate for receiving, or in need of, an allograft transplant.

As needed: In the context of dosing regimens, the term “as needed” refers to a dosing regimen that is not based on a predetermined dosing schedule but instead based on one or more parameters or markers measured or monitored periodically during treatment, which provides information or guidance as to whether additional doses should be beneficial to the subject/patient. For instance, a pharmaceutical composition comprising a TGFβ inhibitor such as TGFβ1/2/3 inhibitors (“pan” inhibitors), TGFβ1/2 inhibitors and TGFβ1/3 inhibitors, may be administered, intermittently, on an “as needed” basis in a therapeutically effective amount sufficient to achieve and/or maintain clinical benefit (e.g., reduction of one or more clinical markers of fibrosis). In some embodiments, administration of a LTBP1/3-complex selective TGFβ inhibitor such as any one of the antibodies disclosed herein (e.g., Ab31, Ab34, Ab37, Ab38, Ab39, Ab40, Ab41, Ab42, Ab43, Ab44, Ab45, Ab62, Ab63, or Ab64 (optionally Ab42)) may be used in combination with a method of determining or monitoring therapeutic efficacy. In some embodiments, the LTBP1/3-complex selective TGFβ inhibitor is administered in patients only when clinical benefit from additional doses of the TGFβ inhibitor is expected. It is contemplated that, in order to manage toxicities, intermittent or “as-needed” dosing regimen may be required more frequently with isoform-non-selective inhibitors of TGFβ, as compared to TGFβ1-selective inhibitors, such as those disclosed herein.

Bias: In the context of the present disclosure, the term “bias” refers to skewed or uneven affinity towards or against a subset of antigens to which an antibody is capable of specifically binding. For example, an antibody is said to have bias when the affinity for one antigen complex and the affinity for another antigen complex are not equivalent (e.g., more than five-fold difference in affinity). Antibodies characterized as “unbiased” have approximately equivalent affinities towards such antigen complexes (e.g., less than five-fold difference in affinity). Antibodies of the present disclosure “selectively” bind EMC-associated complexes (LTBP1-proTGFβ1 and LTBP3-proTGFβ). Such selective binding may in some embodiments comprise binding such that relative affinities between at least one of the matrix-associated complexes and at least one (preferably both) of the cell-associated complexes (GARP-proTGFβ1 and/or LRRC33-proTGFβ1 complexes) is greater than fifty-fold.

Biolayer Interferometry (BLI): BLI is a label-free technology for optically measuring biomolecular interactions, e.g., between a ligand immobilized on the biosensor tip surface and an analyte in solution. BLI provides the ability to monitor binding specificity, rates of association and dissociation, or concentration, with precision and accuracy. BLI platform instruments are commercially available, for example, from ForteBio and are commonly referred to as the Octet® System. BLI can be employed in carrying out in vitro binding assays as described herein.

Autoimmune disease: An autoimmune disease is a condition arising from an abnormal or overactive immune response to a normal body part Immunostimulating agents administered to such patients with autoimmune conditions may exacerbate the condition.

Cell-associated proTGFβ1: The term refers to TGFβ1 or its signaling complex (e.g., pro/latent TGFβ1) that is membrane-bound (e.g., tethered to cell surface). Typically, such cell is an immune cell. TGFβ1 that is presented by GARP or LRRC33 is a cell-associated TGFβ1. GARP and LRRC33 are transmembrane presenting molecules that are expressed on cell surface of certain cells. GARP-proTGFβ1 and LRRC33-proTGFβ1 may be collectively referred to as “cell-associated” (or “cell-surface”) proTGFβ1 complexes, that mediate cell-associated (e.g., immune cell-associated) TGFβ1 activation/signaling.

Chronic inflammation: In the context of the present disclosure, fibrotic disorders that involve chronic inflammation are characterized by continuous or persistent injury to a tissue such that it does not resolve in normal healing after an initial injury. Chronic inflammation refers to a prolonged inflammatory response that involves a progressive change in the type of cells present at the site of inflammation (e.g., fibrotic tissues). It is characterized by the simultaneous destruction and repair of the tissue from the inflammatory process. It can follow an acute form of inflammation or be a prolonged low-grade form.

Clinical benefit: As used herein, the term “clinical benefits” is intended to include both efficacy and safety of a therapy. Thus, therapeutic treatment that achieves a desirable clinical benefit is both efficacious and safe (e.g., with tolerable or acceptable toxicities or adverse events).

Combinatory or combinatorial epitope: A combinatorial epitope is an epitope that is recognized and bound by a combinatorial antibody at a site (i.e., antigenic determinant) formed by non-contiguous portions of a component or components of an antigen, which, in a three-dimensional structure, come together in close proximity to form the epitope. Thus, antibodies of the invention may bind an epitope formed by two or more components (e.g., portions or segments) of a pro/latent TGFβ1 complex. A combinatory epitope may comprise amino acid residue(s) from a first component of the complex, and amino acid residue(s) from a second component of the complex, and so on. Each component may be of a single protein or of two or more proteins of an antigenic complex. A combinatory epitope is formed with structural contributions from two or more components (e.g., portions or segments, such as amino acid residues) of an antigen or antigen complex.

Complementary determining region: As used herein, the term “CDR” refers to the complementarity determining region within antibody variable sequences. There are three CDRs in each of the variable regions of the heavy chain and the light chain, which are designated CDR1, CDR2 and CDR3, for each of the variable regions. The exact boundaries of these CDRs have been defined differently according to different systems. The system described by Kabat (Kabat et al. (1987; 1991) Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md.) not only provides an unambiguous residue numbering system applicable to any variable region of an antibody, but also provides precise residue boundaries defining the three CDRs on each of the heavy and light chains. These CDRs may be referred to as Kabat CDRs.

Conformational epitope: A conformational epitope is an epitope that is recognized and bound by a conformational antibody in a three-dimensional conformation, but not in an unfolded peptide of the same amino acid sequence. A conformational epitope may be referred to as a conformation-specific epitope, conformation-dependent epitope, or conformation-sensitive epitope. A corresponding antibody or fragment thereof that specifically binds such an epitope may be referred to as conformation-specific antibody, conformation-selective antibody, or conformation-dependent antibody. Binding of an antigen to a conformational epitope depends on the three-dimensional structure (conformation) of the antigen or antigen complex.

Context-specific: Context-specific (or context-selective) antibodies of the invention (as opposed to “context-independent” antibodies) are capable of binding selectively to a subset, but not all, of proTGFβ1 complexes associated with a particular biological context. For example, matrix-selective targeting enables specific inhibition of TGFβ1 function associated with the ECM. ECM-selective inhibition can be achieved by the use of antibodies or fragments thereof that selectively target the ECM components, LTBP1-proTGFβ1 and/or LTBP3-proTGFβ1. Antibodies and fragments disclosed herein therefore represent a class of context-specific antibodies. LTBP1-specific and LTBP3-specific inhibitors of TGFβ1 activation are also context-specific antibodies.

Cross-block/cross-blocking: a first antibody or antigen-binding portion thereof and a second antibody or antigen-binding portion thereof cross-block with each other with respect to the same antigen, for example, as assayed by as measured by Biolayer Interferometry (such as Octet) or surface plasmon resonance (such as Biacore System), using standard test conditions, e.g., according to the manufacturer's instructions (e.g., binding assayed at room temperature, —20-25° C.). The first antibody or fragment thereof and the second antibody or fragment thereof may have the same epitope; may have non-identical but overlapping epitopes; or, may have separate (different) epitopes which are in close proximity in a three-dimensional space, such that antibody binding is cross-blocked via steric hindrance. “Cross-block” means that binding of the first antibody to an antigen prevents binding of the second antibody to the same antigen, and similarly, binding of the second antibody to an antigen prevents binding of the first antibody to the same antigen.

Dissociation rate: The term dissociation rate as used herein has the meaning understood by the skilled artisan in the pertinent art (e.g., antibody technology) as refers to a kinetics parameter measured by how fast/slow a ligand (e.g., antibody or fragment) dissociates from its binding target (e.g., antigen). Dissociation rate is also referred to as the “off” rate (“k OFF ”). Relative on/off rates between an antibody and its antigen (i.e., k ON and k OFF ) determine the overall strength of the interaction, or affinity, typically expressed as a dissociation constant, or K D . Therefore, equivalent affinities (e.g., K D values) may be achieved by having fast association (high k ON ), slow dissociation (low k OFF ), or contribution from both factors. Monovalent interactions may be measured by the use of monovalent antigen-binding molecules/fragments, such as fAb (Fab), whilst divalent interactions may be measured by the use of divalent antigen-binding molecules such as whole immunoglobulins (e.g., IgGs). Dissociation kinetics may be expressed in terms of dissociation half-time (sometimes referred to as half binding time), or t½, defined as a duration of time it takes for one half the number of antibody molecules (e.g., mAb, Fab, etc.) to dissociate from bound antigen. Thus, antibodies with slow dissociation rates have long dissociation half-time, and antibodies with fast dissociation rates have short dissociation half-time.

Dosage: As used herein, typical therapeutic dosage of an antibody of the present invention ranges between about 1-30 mg/kg per dose. A typical dosing regimen may include once a week, every 2 weeks, every 3 weeks, every 4 weeks, once a month, every 6 weeks, etc.

ECM-associated (or “matrix-associated”) TGFβ1: The term refers to TGFβ1 or its signaling complex (e.g., pro/latent TGFβ31) that is a component of (e.g., deposited into) the extracellular matrix. TGFβ1 that is presented by LTBP1 or LTBP3 is an ECM-associated TGFβ1.

Effective amount: An “effective amount” (or therapeutically effective amount) is a dosage or dosing regimen that achieves statistically significant clinical benefits in a patient population.

Fibrotic disorder: The term “fibrosis” or “fibrotic condition/disorder” refers to the process or manifestation characterized by the pathological accumulation of extracellular matrix (ECM) components, such as collagens, within a tissue or organ. Fibrosis can include primary fibrosis, as well as secondary fibrosis that are associated with a disease or disorder.

GARP-proTGFβ1: As used herein, the term “GARP-proTGFβ1” refers to a protein complex comprising a pro-protein form or latent form of a transforming growth factor-β1 (TGFβ1) protein associated with a glycoprotein-A repetitions predominant protein (GARP) or fragment or variant thereof. The proTGFβ1 homodimer is capable of forming covalent association with a single molecule of GARP via disulfide bonds. The term “GARP-TGFβ1” may be used interchangeably. GARP-proTGFβ1 expression is limited to certain cell types, such as regulatory T cells (Treg).

Human antibody: The term “human antibody,” as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the present disclosure may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs and in particular CDR3 (e.g., CDR-H3 or CDR-L3 mutagenesis).

Humanized antibody: The term “humanized antibody” refers to antibodies, which comprise heavy and light chain variable region sequences from a non-human species (e.g., a mouse) but in which at least a portion of the VH and/or VL sequence has been altered to be more “human-like,” i.e., more similar to human germline variable sequences. One type of humanized antibody is a CDR-grafted antibody.

Immune suppression/immunosuppression: The term immunosuppression refers to suppression or reduction of the strength of the body's immune system. Patients who “benefit from immunosuppression” include those who have advanced stages of organ fibrosis and are candidates for, being considered for, or have undergone transplantation.

Isoform-specific: The term “isoform specificity” refers to an agent's ability to discriminate one isoform over other structurally related isoforms (i.e., selectivity). An isoform-specific TGFβ inhibitor exerts its inhibitory activity towards one isoform of TGFβ but not the other isoforms of TGFβ at a given concentration. For example, an isoform-specific TGFβ1 antibody selectively binds TGFβ1. A TGFβ1-specific inhibitor (antibody) preferentially targets (binds thereby inhibits) the TGFβ1 isoform over TGFβ2 or TGFβ3 with substantially greater affinity. For example, the selectivity in this context may refer to at least a 500-1000-fold difference in respective affinities as measured by an in vitro binding assay such as Octet and Biacor. In some embodiments, the selectivity is such that the inhibitor when used at a dosage effective to inhibit TGFβ1 in vivo does not inhibit TGFβ2 and TGFβ3. Context-specific inhibitors of the present disclosure are also isoform-specific.

Isolated: An “isolated” antibody as used herein, refers to an antibody that is substantially free of other antibodies having different antigenic specificities. In some embodiments, an isolated antibody is substantially free of other unintended cellular material and/or chemicals.

Long-term or chronic administration: As used herein, a therapeutic regimen that involves over six months of treatment is considered long-term. In some patient populations, long-term therapeutic regimens involve administration of a drug (such as context-selective TGFβ1 inhibitors) for an indefinite duration of time.

LRRC33-proTGFβ1: As used herein, the term “LRRC33-TGFβ1 complex” refers to a complex between a pro-protein form or latent form of transforming growth factor-β1 (TGFβ1) protein and a Leucine-Rich Repeat-Containing Protein 33 (LRRC33; also known as Negative Regulator Of Reactive Oxygen Species or NRROS) or fragment or variant thereof. In some embodiments, a LRRC33-TGFβ1 complex comprises LRRC33 covalently linked with pro/latent TGFβ1 via one or more disulfide bonds. In other embodiments, a LRRC33-TGFβ1 complex comprises LRRC33 non-covalently linked with pro/latent TGFβ1. In some embodiments, a LRRC33-TGFβ1 complex is a naturally-occurring complex, for example a LRRC33-TGFβ1 complex in a cell.

LTBP1-TGFβ1: As used herein, the term “LTBP1-TGFβ1 complex” (or “LTBP1-proTGFβ1 complex”) refers to a protein complex comprising a pro-protein form or latent form of transforming growth factor-β1 (TGFβ31) protein (may be referred to as “proTGFβ31” herein) and a latent TGF-beta binding protein 1 (LTBP1) or fragment or variant thereof. In some embodiments, a LTBP1-TGFβ1 complex comprises LTBP1 covalently linked with pro/latent TGFβ1 via one or more disulfide bonds. In other embodiments, a LTBP1-TGFβ1 complex comprises LTBP1 non-covalently linked with pro/latent TGFβ1. In some embodiments, a LTBP1-TGFβ1 complex is a naturally-occurring complex, for example a LTBP1-TGFβ1 complex in a cell. An exemplary LTBP1-TGFβ1 complex is shown in FIG. 3 .

LTBP3-TGFβ1: As used herein, the term “LTBP3-TGFβ1 complex” (or “LTBP3-proTGFβ1 complex”)refers to a protein complex comprising a pro-protein form or latent form of transforming growth factor-β1 (TGFβ31) protein (may be referred to as “proTGFβ31” herein) and a latent TGF-beta binding protein 3 (LTBP3) or fragment or variant thereof. In some embodiments, a LTBP3-TGFβ1 complex comprises LTBP3 covalently linked with pro/latent TGFβ1 via one or more disulfide bonds. In other embodiments, a LTBP3-TGFβ1 complex comprises LTBP1 non-covalently linked with pro/latent TGFβ1. In some embodiments, a LTBP3-TGFβ1 complex is a naturally-occurring complex, for example a LTBP3-TGFβ1 complex in a cell. An exemplary LTBP3-TGFβ1 complex is shown in FIG. 3 .

Macrophages: Macrophages are a type of white blood cells of the immune system and includes heterogeneous, phenotypically diverse subpopulations of myeloid cells. Some macrophages differentiate from bone marrow-derived, circulating monocytes, while others are tissue-specific macrophages that reside within particular anatomical or tissue locations (“resident” macrophages). Tissue-specific macrophages include but are not limited to: Adipose tissue macrophages; Kupffer cells (Liver); Sinus histiocytes (Lymph nodes); Alveolar macrophages (or dust cells, Pulmonary alveoli of lungs); Tissue macrophages (histiocytes) leading to giant cells (Connective tissue); Langerhans cells (Skin and mucosa); Microglia (Central nervous system); Hofbauer cells (Placenta); Intraglomerular mesangial cells (Kidney); Osteoclasts (Bone); Epithelioid cells (Granulomas); Red pulp macrophages (or Sinusoidal lining cells, Red pulp of spleen); Peritoneal macrophages (Peritoneal cavity); and, LysoMac (Peyer's patch). Macrophages, e.g., bone-marrow derived monocytes, can be activated by certain stimuli (such as cytokines) resulting in polarized phenotypes, e.g., M1 and M2. M2-biased activated macrophages are further classified into several phenotypically distinct subtypes, such as M2a, M2b, M2c (e.g., pro-fibrotic) and M2d (pro-tumor or TAM-like).

Matrix-associated proTGF LTBP1 and LTBP3 are presenting molecules that are components of the extracellular matrix (ECM). LTBP1-proTGFβ1 and LTBP3-proTGFβ1 may be collectively referred to as “ECM-associated” (or “matrix-associated”) proTGFβ1 complexes, that mediate ECM-associated TGFβ1 activation/signaling.

Maximally tolerated dose (MTD): The term MTD generally refers to, in the context of safety/toxicology considerations, the highest amount of a test article (such as a TGFβ1 inhibitor) evaluated with no observed adverse effect level (NOAEL). For example, the NOAEL for Ab2 in rats was the highest dose evaluated (100 mg/kg), suggesting that the MTD for Ab2 is >100 mg/kg, based on a four-week toxicology study.

Myeloid-derived suppressor cell: Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of cells generated during various pathologic conditions and thought to represent a pathologic state of activation of monocytes and relatively immature neutrophils. MDSCs include at least two categories of cells termed i) “granulocytic” (G-MDSC) or polymorphonuclear (PMN-MDSC), which are phenotypically and morphologically similar to neutrophils; and ii) monocytic (M-MDSC) which are phenotypically and morphologically similar to monocytes. MDSCs are characterized by a distinct set of genomic and biochemical features, and can be distinguished by specific surface molecules. For example, human G-MDSCs/PMN-MDSCs typically express the cell-surface markers CD11b, CD33, CD15 and CD66. In addition, human G-MDSCs/PMN-MDSCs may also express HLA-DR and/or Arginase. By comparison, human M-MDSCs typically express the cell surface markers CD11b, CD33 and CD14. The MDSCs may also express CD39 and CD73 to mediate adenosine signaling involved in organ fibrosis (such as liver fibrosis, and lung fibrosis), cancer and myelofibrosis). In addition, human M-MDSCs may also express HLA-DR. In addition to such cell-surface markers, MDSCs are characterized by the ability to suppress immune cells, such as T cells, NK cells and B cells Immune suppressive functions of MDSCs may include inhibition of antigen-non-specific function and inhibition of antigen-specific function. MDSCs can express cell surface LRRC33 and/or LRRC33-proTGFβ1.

Myofibroblast: Myofibroblasts are cells with certain phenotypes of fibroblasts and smooth muscle cells and generally express vimentin, alpha-smooth muscle actin (α-SMA; human gene ACTA2) and paladin. In many disease conditions involving extracellular matrix dysregulations (such as increased matrix stiffness), normal fibroblast cells become de-differentiated into myofibroblasts in a TGFβ-dependent manner.

Off rate (k OFF ): The off rate is a kinetic parameter of how fast or how slowly an antibody (such as mAb) or antigen-binding fragment (such as fAb) dissociates from its antigen and may be also referred to as the dissociation rate. Dissociation rates can be experimentally measured in suitable in vitro binding assays, such as BLI (Octet®)- and/or SPR (Biacore)-based systems. In the context of antibody-antigen binding kinetics, the term “half-binding-time” (T½) or “dissociation half-time” refers to the duration of time required for half the number of antibody molecules (e.g., mAb, Fab) to dissociate from the bound antigen (e.g., LTBP1-proTGFβ1, LTBP3-proTGFβ31). Thus, an antibody that dissociates slowly (i.e., low off rates) from its antigen has a long T½. Conversely, an antibody that dissociates rapidly (i.e., high off rates) from its antigen has a short T½.

Pan-TGFβ inhibitor/pan-inhibition of TGFβ: The term “pan-TGFβ inhibitor” refers to any agent that is capable of inhibiting or antagonizing all three isoforms of TGFβ. Such an inhibitor may be a small molecule inhibitor of TGFβ isoforms. The term includes pan-TGFβ antibody which refers to any antibody capable of binding to each of TGFβ isoforms, i.e., TGFβ1, TGFβ2, and TGFβ3. In some embodiments, a pan-TGFβ antibody binds and neutralizes activities of all three isoforms, i.e., TGFβ1, TGFβ2, and TGFβ3 activities.

Potency: The term “potency” as used herein refers to activity of a drug, such as a functional antibody (or fragment) having inhibitory activity, with respect to concentration or amount of the drug to produce a defined effect. For example, an antibody capable of producing certain effects at a given dosage is more potent than another antibody that requires twice the amount (dosage) to produce equivalent effects. Potency may be measured in cell-based assays, such as TGFβ activation/inhibition assays. In some cases, the degree of TGFβ activation, such as activation triggered by integrin binding, can be measured in the presence or absence of test article (e.g., inhibitory antibodies) in a cell-based system. Typically, antibodies with higher affinities tend to show higher potency than antibodies with lower affinities.

Presenting molecule: Presenting molecules are proteins that form covalent bonds with latent pro-proteins (e.g., proTGFβ31) and “present” the inactive complex in an extracellular niche (such as ECM or immune cell surface) thereby maintaining its latency until an activation event occurs. Known presenting molecules for proTGFβ1 include: LTBP1, LTBP3, GARP and LRRC33, which can form presenting molecule-proTGFβ1 complexes, namely, LTBP1-proTGFβ1, LTBP3-proTGFβ1, GARP-proTGFβ1 and LRRC33-proTGFβ1, respectively. LTBP1 and LTBP3 are components of the extracellular matrix (ECM); therefore, LTBP1-proTGFβ1 and LTBP3-proTGFβ1 may be collectively referred to as “ECM-associated” (or “matrix-associated”) proTGFβ1 complexes, that mediate ECM-associated TGFβ1 signaling/activities. GARP and LRRC33, on the other hand, are transmembrane proteins expressed on cell surface of certain cells; therefore, GARP-proTGFβ1 and LRRC33-proTGFβ1 may be collectively referred to as “cell-associated” (or “cell-surface”) proTGFβ1 complexes, that mediate cell-associated (e.g., immune cell-associated) TGFβ1 signaling/activities.

ProTGFβ1: The term “proTGFβ1” as used herein is intended to encompass precursor forms of inactive TGFβ1 complex that comprises a prodomain sequence of TGFβ1 within the complex. Thus, the term can include the pro-, as well as the latent-forms of TGFβ1. The expression “pro/latent TGFβ1” may be used interchangeably. The “pro” form of TGFβ1 exists prior to proteolytic cleavage at the furin site. Once cleaved, the resulting form is said to be the “latent” form of TGFβ1. The “latent” complex remains associated until further activation trigger, such as integrin-driven activation event. The proTGFβ1 complex is comprised of dimeric TGFβ1 pro-protein polypeptides, linked with disulfide bonds. The latent dimer complex is covalently linked to a single presenting molecule via the cysteine residue at position 4 (Cys4) of each of the proTGFβ1 polypeptides. The adjective “latent” may be used generally to describe the “inactive” state of TGFβ1, prior to integrin-mediated or other activation events. The proTGFβ1 polypeptide contains a prodomain (LAP) and a growth factor domain (SEQ ID NO: 12).

Regulatory T cell (Treg): “Regulatory T cells,” or Tregs, are a type of immune cells characterized by the expression of the biomarkers, CD4, forkhead box P3 (FOXP3), and CD25, as well as STAT5. Tregs are sometimes referred to as suppressor T cells and represent a subpopulation of T cells that modulate the immune system, maintain tolerance to self-antigens, and prevent autoimmune disease. Tregs are immunosuppressive and generally suppress or downregulate induction and proliferation of effector T (Teff) cells. Tregs can develop in the thymus (so-called CD4+Foxp3+“natural” Tregs) or differentiate in the periphery upon priming of naïve CD4+ T cells by antigen-presenting cells (APCs), for example, following exposure to TGFβ or retinoic acid. Treg cells produce and secrete cytokines including IL-10 and TGFβ1. Generally, differentiation of Treg and Th17 cells is negatively correlated.

Specific binding: As used herein, the term “specific binding” or “specifically binds” means that the interaction of the antibody, or antigen-binding portion thereof, with an antigen is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope). For example, the antibody, or antigen-binding portion thereof, binds to a specific protein rather than to proteins generally. In some embodiments, an antibody, or antigen-binding portion thereof, specifically binds to a target, e.g., TGFβ1, if the antibody has a K D for the target of at least about 10 −6 M. More preferably, the measured K D values of such antibody range between 10-100 nM. More preferably, the measured K D values of such antibody range between 0.1-10 nM.

Subject: The term “subject” in the context of therapeutic applications refers to an individual who receives clinical care or intervention, such as treatment, diagnosis, etc. Suitable subjects include vertebrates, including but not limited to mammals (e.g., human and non-human mammals). Where the subject is a human subject, the term “patient” may be used interchangeably. In a clinical context, the term “a patient population” or “patient subpopulation” is used to refer to a group of individuals that falls within a set of criteria, such as clinical criteria (e.g., disease presentations, disease stages, susceptibility to certain conditions, responsiveness to therapy, etc.), medical history, health status, gender, age group, genetic criteria (e.g., carrier of certain mutation, polymorphism, gene duplications, DNA sequence repeats, etc.) and lifestyle factors (e.g., smoking, alcohol consumption, exercise, etc.).

TGFβ inhibitor: The term “TGFβ inhibitor” refers to any agent capable of antagonizing biological activities or function of TGFβ growth factor (e.g., TGFβ31, TGFβ2 and/or TGFβ3). The term is not intended to limit its mechanism of action and includes, for example, neutralizing inhibitors, receptor antagonists, soluble ligand traps, and activation inhibitors of TGFβ.

T helper 17 cell: T helper 17 cells (Th17) are a subset of pro-inflammatory T helper cells characterized by the markers STAT3 and RORγt and the production of cytokines including interleukin 17 (IL-17A/F) and IL-22. Th17 cells are differentiated when naïve T cells are exposed to TGFβ and IL-6. Th17 cells are generally associated with tissue inflammation, autoimmunity and clearance of certain pathogens. The differentiation of Th17 cells and Treg cells is generally inversely related. Imbalance in Th17-to-Treg ratios (e.g., “Th17/Treg”) has been implicated in a number of pathologies, such as fibrotic conditions and autoimmune conditions.

Th17/Treg ratio: Th17-to-Treg ratios refer to measured ratios (relative proportions) of the number of Th17 cells versus the number of Treg cells in a tissue or sample of interest. Typically, known cell markers are used to identify, sort or isolate the cell types. Such markers include cell-surface molecules expressed on the particular cell type; a cytokine or a panel of cytokines produced (e.g., secreted) by the particular cell type, and/or mRNA expression of certain gene markers that serve as a signature/profile of the particular cell type. For example, the Th17/Treg ratio of one (1) means that there is an equal or equivalent number of each of the cell types within the tissue or sample being evaluated. The Th17/Treg ratio of two (2) means that there is approximately twice the number of Th17 cells as compared to Treg cells in the tissue or sample. An elevated Th17/Treg ratio may arise from an increased number of Th17 cells, a decreased number of Treg cells, or combination thereof.

Therapeutic window: The term “therapeutic window” refers to a range of doses/concentrations that produces therapeutic response without causing significant/observable/unacceptable adverse effect (e.g., within adverse effects that are acceptable or tolerable) in subjects. Therapeutic window may be calculated as a ratio between minimum effective concentrations (MEC) to the minimum toxic concentrations (MTC). To illustrate, a TGFβ1 inhibitor that achieves in vivo efficacy at 10 mg/kg and shows tolerability or acceptable toxicities at 100 mg/kg provides at least a 10-fold (e.g., 10×) therapeutic window. By contrast, a pan-inhibitor of TGFβ that is efficacious at 10 mg/kg but causes adverse effects at 5 mg/kg is said to have “dose-limiting toxicities.” For example, the applicants have found that a context-independent TGFβ1 inhibitor antibody is efficacious at dosage ranging between about <3 and 30 mg/kg/week and is free of observable toxicities associated with pan-inhibition of TGFβ at least 100 mg/kg/week for 4 weeks in preclinical models such as rats. Based on this, the context-independent TGFβ1 inhibitor antibody shows at minimum a 3.3-fold and up to 33-fold therapeutic window.

Toxicity: As used herein, the term “toxicity” or “toxicities” refers to unwanted in vivo effects in patients associated with a therapy administered to the patients, such as undesirable side effects and adverse events. “Tolerability” refers to a level of toxicities associated with a therapy or therapeutic regimen, which can be reasonably tolerated by patients, without discontinuing the therapy due to the toxicities (i.e., acceptable level of toxicities). Typically, toxicity/toxicology studies are carried out in one or more preclinical models prior to clinical development to assess safety profiles of a drug candidate (e.g., monoclonal antibody therapy). Toxicity/toxicology studies may help determine the “no observed adverse effect level (NOAEL)” and the “maximally tolerated dose (MTD)” of a test article, based on which a therapeutic window may be deduced. Preferably, a species that is shown to be sensitive to the particular intervention should be chosen as a preclinical animal model in which safety/toxicity study is to be carried out. In case of TGFβ inhibition, suitable species include rats, dogs, and cynos. Mice are reported to be less sensitive to pharmacological inhibition of TGFβ and may not reveal toxicities that are potentially dangerous in other species, including human, although certain studies report toxicities observed with pan-inhibition of TGFβ in mice. To illustrate, the NOAEL for a context-independent TGFβ1 inhibitor antibody in rats was the highest dose evaluated (100 mg/kg), suggesting that the MTD is >100 mg/kg per week, based on a four-week toxicology study.

Treat/treatment: The term “treat” or “treatment” includes therapeutic treatments, prophylactic treatments, and applications in which one reduces the risk that a subject will develop a disorder or other risk factor. Thus the term is intended to broadly mean: causing therapeutic benefits in a patient by, for example, enhancing or boosting the body's immunity; reducing or reversing immune suppression; reducing, removing or eradicating harmful cells or substances from the body; reducing disease burden (e.g., tumor burden); preventing recurrence or relapse; prolonging a refractory period, and/or otherwise improving survival. Treatment does not require the complete curing of a disorder and encompasses embodiments in which one reduces symptoms or underlying risk factors. In the context of combination therapy, the term may also refer to: i) the ability of a second therapeutic to reduce the effective dosage of a first therapeutic so as to reduce side effects and increase tolerability; ii) the ability of a second therapy to render the patient more responsive to a first therapy; and/or iii) the ability to effectuate additive or synergistic clinical benefits.

Variable region: The term “variable region” or “variable domain” refers to a portion of the light and/or heavy chains of an antibody, typically including approximately the amino-terminal 120 to 130 amino acids in the heavy chain and about 100 to 110 amino terminal amino acids in the light chain. In certain embodiments, variable regions of different antibodies differ extensively in amino acid sequence even among antibodies of the same species. The variable region of an antibody typically determines specificity of a particular antibody for its target.

TGFβ1

In mammals, the transforming growth factor-beta (TGFβ3) superfamily is comprised of at least 33 gene products. These include the bone morphogenetic proteins (BMPs), activins, growth and differentiation factors (GDFs), and the three isoforms of the TGFβ family: TGFβ1, TGFβ2, and TGFβ3. The TGFβ s are thought to play key roles in diverse processes, such as inhibition of cell proliferation, extracellular matrix (ECM) remodeling, and immune homeostasis. The importance of TGFβ1 for T cell homeostasis is demonstrated by the observation that TGFβ1−/− mice survive only 3-4 weeks, succumbing to multiorgan failure due to massive immune activation (Kulkarni, A. B., et al., Proc Natl Acad Sci USA, 1993. 90(2): p. 770-4; Shull, M. M., et al., Nature, 1992. 359(6397): p. 693-9). The roles of TGFβ2 and TGFβ3 are less clear. Whilst the three TGFβ isoforms have distinct temporal and spatial expression patterns, they signal through the same receptors, TGFβRI and TGFβ3, although in some cases, for example for TGFβ2 signaling, type III receptors such as betaglycan are also required (Feng, X. H. and R. Derynck, Annu Rev Cell Dev Biol, 2005. 21: p. 659-93; Massague, J., Annu Rev Biochem, 1998. 67: p. 753-91). Ligand-induced oligomerization of TGFβRI/II triggers the phosphorylation of SMAD transcription factors, resulting in the transcription of target genes, such as Collal, Col3al, ACTA2, and SERPINE1 (Massague, J., J. Seoane, and D. Wotton, Genes Dev, 2005. 19(23): p. 2783-810). SMAD-independent TGFβ signaling pathways have also been described, for example in cancer or in the aortic lesions of Marfan mice (Derynck, R. and Y. E. Zhang, Nature, 2003. 425(6958): p. 577-84; Holm, T. M., et al., Science, 2011. 332(6027): p. 358-61).

The biological importance of the TGFβ pathway in humans has been validated by genetic diseases. Camurati-Engelman disease results in bone dysplasia due to an autosomal dominant mutation in the TGFB1 gene, leading to constitutive activation of TGFβ1 signaling (Janssens, K., et al., J Med Genet, 2006. 43(1): p. 1-11). Patients with Loeys/Dietz syndrome carry autosomal dominant mutations in components of the TGFβ signaling pathway, which cause aortic aneurism, hypertelorism, and bifid uvula (Van Laer, L., H. Dietz, and B. Loeys, Adv Exp Med Biol, 2014. 802: p. 95-105). As TGFβ pathway dysregulation has been implicated in multiple diseases, several drugs that target the TGFβ pathway have been developed and tested in patients, but with limited success. Most TGFβ inhibitors described to date lack isoform specificity as briefly summarized below.

Fresolimumab, a humanized monoclonal antibody that binds and inhibits all three isoforms of TGFβ has been tested clinically in patients with focal segmental glomerulosclerosis, malignant melanoma, renal cell carcinoma, and systemic sclerosis (Rice, L. M., et al., J Clin Invest, 2015. 125(7): p. 2795-807; Trachtman, H., et al., Kidney Int, 2011. 79(11): p. 1236-43; Morris, J. C., et al., PLoS One, 2014. 9(3): p. e90353). Additional companies have developed monoclonal antibodies against the TGFβ growth factors with varying degrees of selectivity for TGFβ isoforms. Such agents likely elicit toxicities in vivo through residual activity against other TGFβ family members besides TGFβ1. This lack of isoform specificity may be due to the high degree of sequence identity between isoforms.

Other approaches to target the TGFβ pathway include ACE-1332, a soluble TGFβRII-Fc ligand trap from Acceleron (Yung, L. M., et al., A Am J Respir Crit Care Med, 2016. 194(9): p. 1140-1151), or small molecule inhibitors of the ALK5 kinase, such as Eli Lilly's galunisertib. ACE-1332 binds TGFβ1 and TGFβ3 with equally high affinity (Yung, L. M., et al., Am J Respir Crit Care Med, 2016. 194(9): p. 1140-1151), and ALK5 inhibitors block the activity of all growth factors that signal through TGFR1. Substantial toxicities have been found in preclinical studies using ALK5 inhibitors (Anderton, M. J., et al., Toxicol Pathol, 2011. 39(6): p. 916-24; Stauber, A., et al., Clinical Toxicology, 2014. 4(3): p. 1-10), and sophisticated clinical dosing schemes are required to maintain efficacy while reducing adverse events (Herbertz, S., et al., Drug Des Devel Ther, 2015. 9: p. 4479-99). In fact, the question of TGFβ signaling specificity and its possible effect on toxicity observed with the known TGFβ inhibitors has not been raised in most, if not all, of the candidate drugs that attempted to block TGFβ. For example, how much of the toxicities are due to inhibition of TGFβ1 versus TGFβ2 and/or TGFβ3 has not been addressed. Similarly, modes of TGFβ activation have not been taken into account in designing or developing ways to antagonize TGFβ signaling.

Recent structural insights into the activation mechanism of TGFβ1 (Shi, M., et al., Nature, 2011. 474(7351): p. 343-9) have enabled more specific approaches to TGFβ inhibition (see, e.g., PCT/US2017/21972, the entire contents of which are incorporated herein by reference). Unlike other cytokines, TGFβ superfamily members are not secreted as active growth factors, but as dimeric pro-proteins which consist of an N-terminal prodomain and a C-terminal growth factor domain. Cleavage of proTGFβ1 by furin proteases separates the homodimeric growth factor domain from its prodomain, also referred to as latency associated peptide (LAP). However, the growth factor and LAP remain noncovalently associated, forming a latent complex which is unable to bind its receptors and induce signaling. During translation, latent TGFβ1, also called the small latent complex (SLC), becomes linked to “presenting molecules” via disulfide bridges, forming the large latent complex (LLC). These molecules allow proTGFβ1 to be presented in specific cellular or tissue contexts. Two cysteines near the N-terminus of the latent TGFβ1 link to appropriately positioned cysteines on the presenting molecule. The identity of the presenting molecule depends on the environment and cell type producing latent TGFβ1. For example, fibroblasts secrete latent TGFβ1 tethered to latent TGFβ-binding proteins (LTBPs), which then associate with proteins in the extracellular matrix (ECM) (i.e., fibronectin, fibrillin-1) to link latent TGFβ3 to the ECM (Robertson et al. Matrix Biol 47: 44-53 (2015) ( FIG. 2 A ). On the surface of activated regulatory T cells latent TGFβ1 is covalently linked to the transmembrane protein GARP (glycoprotein-A repetitions predominant protein (GARP), and a protein closely related to GARP, LRRC33 (leucine-rich repeat-containing protein 33), serves as a presenting molecule for TGFβ1 on the surface of monocytes, macrophages and microglia (Wang, R., et al., Mol Biol Cell, 2012. 23(6): p. 1129-39 and T. A. Springer, Int. BMP Conference 2016).

A number of studies have shed light on the mechanisms of TGFβ1 activation. Three integrins, αVβ6, αVβ8, and αVβ1 have been demonstrated to be key activators of latent TGFβ1 (Reed, N. I., et al., Sci Transl Med, 2015. 7(288): p. 288ra79; Travis, M. A. and D. Sheppard, Annu Rev Immunol, 2014. 32: p. 51-82; Munger, J. S., et al., Cell, 1999. 96(3): p. 319-28). αV integrins bind the RGD sequence present in TGFβ1 and TGFβ1 LAPs with high affinity (Dong, X., et al., Nat Struct Mol Biol, 2014. 21(12): p. 1091-6). Transgenic mice with a mutation in the TGFβ1 RGD site that prevents integrin binding, but not secretion, phenocopy the TGFβ1−/− mouse (Yang, Z., et al., J Cell Biol, 2007. 176(6): p. 787-93). Mice that lack both 136 and 138 integrins recapitulate all essential phenotypes of TGFβ1 and TGFβ3 knockout mice, including multiorgan inflammation and cleft palate, confirming the essential role of these two integrins for TGFβ1 activation in development and homeostasis (Aluwihare, P., et al., J Cell Sci, 2009. 122(Pt 2): p. 227-32). Key for integrin-dependent activation of latent TGFβ1 is the covalent tether to presenting molecules; disruption of the disulfide bonds between GARP and TGFβ1 LAP by mutagenesis does not impair complex formation, but completely abolishes TGFβ1 activation by αVβ6 (Wang, R., et al., Mol Biol Cell, 2012. 23(6): p. 1129-39). The recent structure of latent TGFβ1 illuminates how integrins enable release of active TGFβ1 from the latent complex: the covalent link of latent TGFβ1 to its presenting molecule anchors latent TGFβ1, either to the ECM through LTBPs, or to the cytoskeleton through GARP or LRRC33. Integrin binding to the RGD sequence results in a force-dependent change in the structure of LAP, allowing active TGFβ1 to be released and bind nearby receptors (Shi, M., et al., Nature, 2011. 474(7351): p. 343-9). The importance of integrin-dependent TGFβ1 activation in disease has also been well validated. A small molecular inhibitor of αVβ1 protects against bleomycin-induced lung fibrosis and carbon tetrachloride-induced liver fibrosis (Reed, N. I., et al., Sci Transl Med, 2015. 7(288): p. 288ra79), and αVβ6 blockade with an antibody or loss of integrin 136 expression suppresses bleomycin-induced lung fibrosis and radiation-induced fibrosis (Munger, J. S., et al., Cell, 1999. 96(3): p. 319-28); Horan, G. S., et al., Am J Respir Crit Care Med, 2008. 177(1): p. 56-65). In addition to integrins, other mechanisms of TGFβ1 activation have been implicated, including thrombospondin-1 and activation by proteases such as matrix metalloproteinases (MMPs), cathepsin D or kallikrein. However, the majority of these studies were performed in vitro using purified proteins; there is less evidence for the role of these molecules from in vivo studies. Knockout of thrombospondin-1 recapitulates some aspects of the TGFβ1−/− phenotype in some tissues, but is not protective in bleomycin-induced lung fibrosis, known to be TGFβ3-dependent (Ezzie, M. E., et al., Am J Respir Cell Mol Biol, 2011. 44(4): p. 556-61). Additionally, knockout of candidate proteases did not result in a TGFβ1 phenotype (Worthington, J. J., J. E. Klementowicz, and M. A. Travis, Trends Biochem Sci, 2011. 36(1): p. 47-54). This could be explained by redundancies or by these mechanisms being critical in specific diseases rather than development and homeostasis.

TGFβ3 has been implicated in a number of biological processes, including fibrosis, immune-modulation and cancer progression. TGFβ1 was the first identified member of the TGFβ3 superfamily of proteins. Like other members of the TGFβ3 superfamily, TGFβ1 and the isoforms TGFβ2 and TGFβ3, are initially expressed as inactive precursor pro-protein forms (termed proTGF(3). TGFβ3 proteins (e.g., TGFβ1, TGFβ2 and TGF(33) are proteolytically cleaved by proprotein convertases (e.g., furin) to yield the latent form (termed latent TGF(3). In some embodiments, a pro-protein form or latent form of a TGFβ3 protein (e.g., TGFβ1, TGFβ2 and TGFβ3) may be referred to as “pro/latent TGFβ3 protein”. TGFβ1 may be presented to other molecules in complex with multiple molecules including, for example, GARP (to form a GARP-TGF(31 complex), LRRC33 (to form a LRRC33-TGF(31 complex), LTBP1 (to form a LTBP1-TGF(31 complex), and/or LTBP3 (to form a LTBP3-TGF(31 complex). The TGFβ1 present in these complexes may be in either latent form (latent TGF(31) or in precursor form (proTGF(31).

Isoform Selectivity and Mechanisms of Action of TGF Inhibitors

From a safety standpoint, there has been an increasing recognition that broad inhibition of TGFβ across isoforms may be a cause of observed toxicities, which underscores the fact that no TGFβ inhibitors have been successfully developed to this day. To circumvent potentially dangerous adverse effects, a number of groups have recently turned to identifying inhibitors that target a subset—but not all—of the isoforms and still retain efficacy. From an efficacy standpoint, however, the prevailing view of the field remains to be that it is advantageous to inhibit multiple isoforms of TGFβ3 to achieve therapeutic effects, and to accommodate this, toxicity management by “careful dosing regimen” is suggested as a solution (Brennan et al. (2018) mAbs, 10:1, 1-17). Consistent with this premise, numerous groups are developing TGFβ3 inhibitors that target more than one isoforms. These include low molecular weight antagonists of TGFβ3 receptors, e.g., ALK5 antagonists, such as Galunisertib (LY2157299 monohydrate); monoclonal antibodies (such as neutralizing antibodies) that inhibit all three isoforms (“pan-inhibitor” antibodies) (see, for example, WO 2018/134681); monoclonal antibodies that preferentially inhibit two of the three isoforms (e.g., antibodies against TGFβ1/2 (for example WO 2016/161410) and TGFβ1/3 (for example WO 2006/116002); and engineered molecules (e.g., fusion proteins) such as ligand traps (for example, WO 2018/029367; WO 2018/129331 and WO 2018/158727). Similarly, inhibitors of integrins such as αVβ6 also block integrin-dependent activation of both TGFβ1 and TGFβ3 and therefore may be considered as isoform-non-selective inhibitors of TGFβ signaling. In addition, examples of antibodies that selectively bind and neutralize both TGFβ1 and TGFβ2 (i.e., TGFβ1/2 inhibitors) include XOMA 089 (or NIS793) and variants (see, for example, WO 2016/161410).

Previously, Applicant demonstrated that inhibition of TGFβ1 alone was sufficient to sensitize immunosuppressive tumors to a checkpoint inhibitor therapy even in tumors where both TGFβ1/3 are co-expressed (PCT/US2019/041373). Similarly, TGFβ1-selective inhibitors are shown to mitigate fibrosis in preclinical models, including mouse liver fibrosis model where both the TGFβ1/3 isoforms are co-expressed in the fibrotic tissue, albeit in discrete cell types, as observed by immunohistochemistry (data now shown). Surprisingly, inhibition of TGFβ3 promoted pro fibrotic phenotypes. The exacerbation of fibrosis is observed when the TGFβ3 inhibitor is used alone. In addition, when used in combination with a TGFβ1-selective inhibitor, the TGFβ3 inhibitor attenuated the anti-fibrotic effect of the TGFβ1-selective inhibitor, as evidenced by increased collagen accumulation in the fibrotic liver. These results raise the possibility that inhibitory potency against TGFβ3 may be an undesirable feature of TGFβ inhibitors to be used as therapy in situations where fibrosis is a concern.

Beyond the fibrosis context, there is a broader implication to this unexpected finding since the pro-fibrotic phenotype (e.g., increased collagen deposit into the ECM) is associated not only with fibrosis, but also with aspects of cancer progression, such as tumor invasion and metastasis. See, for example, Chakravarthy et al. (Nature Communications, (2018) 9:4692. “TGF-β-associated extracellular matrix genes link cancer-associated fibroblasts to immune evasion and immunotherapy failure”). Diseased tissues with dysregulated ECM, including fibrotic tissues and stroma of various tumor types, can express both TGFβ1 and TGFβ3. As of today, multiple groups are making effort to develop TGFβ inhibitors that target both of these isoforms, such as ligand traps, neutralizing antibodies and integrin inhibitors. However, the finding presented herein cautions that such approach may in fact exacerbate (e.g., worsen) the disease.

Accordingly, the present disclosure provides the teaching that for the treatment of a disorder involving ECM dysregulation, such as fibrosis and cancer, a TGFβ inhibitor that does not specifically target TGFβ3 should be selected. Preferably, such inhibitor is an isoform-selective inhibitor of TGFβ1, such as inhibitors that selectively target LTBP1/3-associated TGFβ1 (e.g., as disclosed herein). Related methods include a method for selecting a TGFβ inhibitor for use in the treatment of a fibrotic disorder in a subject, wherein the method includes the steps of: testing potency of one or more candidate inhibitors for the ability to inhibit TGFβ1, TGFβ2 and TGFβ3, and selecting an inhibitor that inhibits TGFβ1 but does not inhibit TGFβ3, for therapeutic use. Related treatment methods can further comprise a step of administering to the subject the inhibitor that inhibits TGFβ1 but does not inhibit TGFβ3 in an amount sufficient to treat the fibrotic disorder or treat a subject having or at risk of developing a fibrotic disorder. Preferably, the selected inhibitor is an antibody or fragment thereof that selectively inhibits LTBP1- and/or LTBP3-associated TGFβ1 signaling (e.g., as disclosed herein). In some embodiments, subjects at risk of developing a fibrotic disorder may suffer from a metabolic disorder, such as diabetes, obesity and NASH. The proposed exclusion of the subpopulation of patients is aimed to reduce risk of triggering, facilitating or exacerbating a pro-fibrotic effect.

In addition to the possible concerns of inhibiting TGFβ3 addressed above, Takahashi et al. (Nat Metab. 2019, 1(2): 291-303) recently reported a beneficial role of TGFβ2 in regulating metabolism. The authors identified TGFβ2 as an exercise-induced adipokine, which stimulated glucose and fatty acid uptake in vitro, as well as tissue glucose uptake in vivo; which improved metabolism in obese mice; and, which reduced high fat diet-induced inflammation. Moreover, the authors observed that lactate, a metabolite released from muscle during exercise, stimulated TGFβ2 expression in human adipocytes and that a lactate-lowering agent reduced circulating TGFβ2 levels and reduced exercise-stimulated improvements in glucose tolerance. These observations suggest that therapeutic use of a TGFβ inhibitor with inhibitory activity towards the TGFβ2 isoform may be harmful at least in the metabolic aspect.

Without being bound by particular theory, it is contemplated that it is advantageous to select a TGFβ1-selective inhibitor as a TGFβ inhibitor for use in the treatment of a metabolic disease, such as liver fibrosis associated with NASH. In preferred embodiments, the TGFβ1-selective inhibitor selected for use in the treatment of the metabolic disease selectively inhibits LTBP1/3-associated TGFβ1, such as the antibodies and fragments disclosed herein. Accordingly, the invention includes a a TGFβ inhibitor for use in the treatment of a metabolic disease in a subject, wherein the treatment comprises selection of a TGFβ inhibitor that inhibits TGFβ1 but does not inhibit TGFβ2, optionally wherein the inhibitor is TGFβ1-selective, and administration of the inhibitor to a subject suffering from a metaboic disease. The metabolic disease may be a liver disease, such as liver fibrosis, NASH, NAFLD, optionally accompanied by obesity and/or type 2 diabetes. In preferred embodiments, the TGFβ1-selective inhibitor is an antibody or antigen-binding fragment thereof that selectively targets matrix-associated TGFβ1 (e.g., LTBP1-proTGFβ1 and LTBP3-proTGFβ31), such as those disclosed herein.

In preferred embodiments, a TGFβ inhibitor for use in the treatment of a fibrotic disorder is an isoform-selective activation inhibitor of TGFβ1 (such as the novel antibodies with low k OFF or long t½ disclosed herein) capable of targeting matrix-associated TGFβ1-containing latent complexes in vivo.

The antibodies of the present disclosure work by preventing the step of TGFβ1 activation. In some embodiments, such inhibitors can inhibit integrin-dependent (e.g., mechanical or force-driven) activation of TGFβ1. In some embodiments, such inhibitors can inhibit protease-dependent or protease-induced activation of TGFβ1. The latter includes inhibitors that inhibit the TGFβ1 activation step in an integrin-independent manner. In some embodiments, such inhibitors can inhibit TGFβ1 activation irrespective of the mode of activation, e.g., inhibit both integrin-dependent activation and protease-dependent activation of TGFβ1. Non-limiting examples of proteases which may activate TGFβ1 include serine proteases, such as Kallikreins, Chemotrypsin, Trypsin, Elastases, Plasmin, thrombin, as well as zinc metalloproteases (MMP family) such as MMP-2, MMP-9, MMP-12, MMP-13 and ADAM proteases (e.g., ADAM10 and ADAM17) Kallikreins include plasma-Kallikreins and tissue Kallikreins, such as KLK1, KLK2, KLK3, KLK4, KLKS, KLK6, KLK7, KLK8, KLK9, KLK10, KLK11, KLK12, KLK13, KLK14 and KLK15.

Latent TGFβ-Binding Proteins (LTBPs)

In mammals there are four known LTBPs, LTBP1-4, each with multiple splice variants (Robertson, I. B., et al., Matrix Biol, 2015. 47: p. 44-53). LTBP2 is the only LTBP that does not associate with latent TGFβ (Saharinen, J. and J. Keski-Oja, Mol Biol Cell, 2000. 11(8): p. 2691-704). While the association between LTBP1 or LTBP3 and latent TGFβ1 has been well validated, the role of LTBP4 in TGFβ presentation is less clear. The complex with LTBP4 and latent TGFβ1 appears to form much less efficiently, potentially due to the absence of several negatively charged residues in the TGFβ-binding domain of LTBP4 (Saharinen, J. and J. Keski-Oja, Mol Biol Cell, 2000. 11(8): p. 2691-704; Chen, Y., et al., J Mol Biol, 2005. 345(1): p. 175-86). Both LTBP4S−/− mice and Urban-Rifkin-Davis syndrome patients, who have null mutations in LTBP4, suffer from disrupted elastic fiber assembly (Urban, Z., et al., Am J Hum Genet, 2009. 85(5): p. 593-605; Dabovic, B., et al., J Cell Physiol, 2015. 230(1): p. 226-36). Additionally, while LTBP4S−/− mice have a lung septation and an elastogenesis defect, transgenic mice with an LTBP4 that cannot form a complex with latent TGFβ1 have no obvious phenotype (Dabovic, B., et al., J Cell Physiol, 2015. 230(1): p. 226-36). Whether LTBP4 is directly involved in regulation of latent TGFβ1 by functioning as a presenting molecule is unclear; LTBP4 may instead be required for proper formation of elastic fibrils in the ECM and its loss indirectly affect latent TGFβ1 activation through defects in the ECM.

In one aspect, the present invention is directed to inhibitors, e.g., immunoglobulins, e.g., antibodies, or antigen-binding portions thereof, that selectively bind to a complex containing a TGFβ pro-protein and a LTBP protein (e.g., LTBP1 or LTBP3). In a preferred embodiment, the TGFβ protein is TGFβ1. In some embodiments, the binding molecules disclosed herein bind selectively to a complex containing pro/latent TGFβ1 and LTBP1 or LTBP3. Such binding molecules can allow TGFβ1 activity to be selectively modulated in a context-dependent manner, i.e., by modulating TGFβ1 in the context of a LTPB protein, without modulating the activity of TGFβ1 complexed with other presenting molecules (e.g., GARP and/or LRRC33).

Antibodies that Selectively Inhibit LTBP-Mediated TGFβ Activation

The present invention provides novel, TGFβ inhibitors that selectively target matrix- or ECM-associated TGFβ activities. More specifically, such inhibitors include isoform-specific, context-selective inhibitors of TGFβ1 activation that specifically bind latent forms of TGFβ1 (e.g., proTGFβ1 complex) within the ECM environment and prevent release of mature growth factor from the complex at the niche. Such matrix-targeting inhibitors are context-specific in that they selectively bind proTGFβ1 associated with ECM presenting molecules, namely, LTBP1 and/or LTBP3. Thus, disclosed herein are monoclonal antibodies and fragments thereof capable of binding an epitope present in an LTBP1-proTGFβ1 complex and/or LTBP3-proTGFβ1 complex, whereas the epitope is not present in a GARP-proTGFβ1 complex and/or LRRC33-proTGFβ1 complex.

In some embodiments, the context-selective inhibitors of the present disclosure are capable of specifically binding both a human LTBP1-proTGFβ1 complex and a human LTBP3-proTGFβ1 complex, with affinities of <5 nM each (measured K D values) in a suitable in vitro binding assay, such as Octet. In preferred embodiments, such antibodies bind both a human LTBP1-proTGFβ1 complex and a human LTBP3-proTGFβ1 with affinities of <5 nM each (measured K D values) in a suitable in vitro binding assay, such as Octet. On the other hand, these context-specific antibodies do not show any detectable binding to a human GARP-proTGFβ1 complex or a human LRRC33-proTGFβ1 complex under the same assay conditions. Preferably, such antibody or the fragment binds each of the human LTBP1-proTGFβ1 complex and the human LTBP3-proTGFβ1 complex with KD of less than 1 nM.

In some embodiments, the context-selective inhibitors of the present disclosure are capable of specifically binding either a human LTBP1-proTGFβ1 complex or a human LTBP3-proTGFβ1 complex. Neither shows any detectable binding to a human GARP-proTGFβ1 complex or a human LRRC33-proTGFβ1 complex under the same assay conditions.

The art is familiar with suitable in vitro binding assays, including, for example, BLI-based assays such as Octet and SPR-based assays such as Biacore, which can be used to measure antibody-antigen interactions (e.g., binding kinetics). As used herein, “no binding” in these contexts may refer to no detectable binding by a particular assay, e.g., the binding, if any, is below the sensitivity of the assay. In some embodiments, “no binding” may refer to “no meaningful binding”, set by a cutoff, which defines the minimum level required to be considered meaningful as measured by a particular assay system. For example, in a BLI (Octet) assay, 0.1 nm of optical shift measured at predetermined analyte (e.g., target antigen) concentrations (e.g., 100 nM, 200 nM, etc.) may indicate meaningful binding, below which may be considered as no binding (see for example, Example 9). Similarly, in a typical SPR (Biacore) assay, the cutoff level may be 0.2 RU (resonance unit). In some embodiments, the signal at 0 nM antibody (e.g., background noise) is subtracted from all the sensorgrams obtained at higher concentrations. Under these conditions, using SPR (Biacore), 0.2 RUs may be a suitable cutoff.

In some embodiments, the context-selective inhibitors of the present disclosure are capable of specifically binding a human LTBP1-proTGFβ1 complex or a human LTBP3-proTGFβ1 complex without showing detectable binding to a human GARP-proTGFβ1 complex, as measured by BLI, under the same assay conditions as used to measure binding to human LTBP1-proTGFβ1 complex and/or human LTBP3-TGFβ1.

In some embodiments, the context-selective inhibitors of the present disclosure bind a human LTBP1-proTGFβ1 complex or a human LTBP3-proTGFβ1 complex with a K D that is at least 50 times lower (e.g., at least 75 times lower, at least 100 times lower) than the K D when binding to a human GARP-proTGFβ1 complex under the same assay conditions. In some embodiments, the K D is as determined by BLI or SPR. In some embodiments, the K D is as determined by SPR.

In some embodiments, the context-selective inhibitors of the present disclosure are capable of specifically binding a human LTBP1-proTGFβ1 complex or a human LTBP3-proTGFβ1 complex without showing detectable binding to an LRRC33-proTGFβ1 latent complex, as measured by BLI, under the same assay conditions as used to measure binding to human LTBP1-proTGFβ1 complex and/or human LTBP3-TGFβ1.

In some embodiments, the context-selective inhibitors of the present disclosure bind a human LTBP1-proTGFβ1 complex or a human LTBP3-TGFβ1 complex with a K D that is at least 50 times lower (e.g., at least 75 times lower, at least 100 times lower) than the K D when binding to a human LRRC33-proTGFβ1 complex under the same assay conditions. In some embodiments, the K D is as determined by BLI or SPR. In some embodiments, the K D is as determined by SPR.

The invention includes the recognition that preferred antibodies (e g, immunoglobulins and antigen-binding fragments such as Fabs, as well as engineered constructs incorporating such fragments), once bound to its target/antigen (e.g., human LTBP1-proTGFβ1, human LTBP3-TGFβ31), dissociates slowly from the antigen. Thus, the novel antibodies of the instant invention are selected not only for their high overall affinities (such as KD of no more than 5 nM) but especially for their low dissociation rates. According to the present disclosure, such antibodies have dissociation rates of <5×10 (1/s), as measured by BLI (e.g., when binding to human LTBP1-proTGFβ1 and/or human LTBP3-TGFβ31). Such dissociation rates of of <5×10 −4 (1/s) of the antibodies or antigen-binding fragments may be monovalent dissociation rates or divalent dissociation rates. In some embodiments, the antibody or the fragment dissociates slowly from human LTBP1-proTGFβ1 and/or human LTBP3-TGFβ1, preferably for both human LTBP1-proTGFβ1 and human LTBP3-TGFβ1, with a monovalent dissociation half-time (t½) of at least 45 minutes (e.g., >45, 60, 75, 90 minutes) as measured by SPR. On the other hand, should binding to a human GARP-proTGFβ1 and/or LRRC33-TGFβ1 complex be detectable, such antibody dissociates rapidly from a human GARP-proTGFβ1 and/or human LRRC33-TGFβ1 complex(es), particularly the human GARP-proTGFβ1. In some embodiments, the antibody dissociates from human GARP-proTGFβ1 with t′ of less than 5 minutes as measured by SPR. In particularly preferred embodiments, the antibody or the fragments show species cross-reactivity such that they bind murine counterparts with equivalent affinities.

The TGFβ1 present in these complexes may be in either latent form (latent TGFβ31) or in precursor form (proTGFβ31). In one embodiment, the inhibitors do not significantly bind to LTBP1 alone (e.g., when not complexed with TGFβ31). In another embodiment, the inhibitors do not significantly bind to LTBP3 alone (e.g., when not complexed with TGFβ31). In another embodiment, the inhibitors do not significantly bind to TGFβ1 alone (e.g., pro or latent TGFβ1 not complexed with LTBP1 or LTBP3, or mature TGFβ1). In another embodiment, the inhibitors that selectively bind a LTBP1-TGFβ1 complex and/or a LTBP3-TGFβ1 complex do not significantly bind to a complex containing TGFβ1 and another presenting molecule, e.g., a GARP-TGFβ1 complex (e.g., GARP complexed to pro- or latent TGFβ1) and/or a LRRC33-TGFβ1 complex (e.g., LRRC33 complexed to pro- or latent TGFβ31). In one embodiment, the inhibitors that selectively bind LTBP1/3-TGFβ1 do not significantly bind one or more (e.g., two or more, three or more, or all four) of the following: LTBP1 alone, TGFβ1 alone, a GARP-TGFβ1 complex, and a LRRC33-TGFβ1 complex. In addition, in some embodiments, the inhibitors do not significantly bind LTBP3 alone.

As used herein, the term “inhibitor” refers to any agent capable of blocking or antagonizing TGFβ1 signaling. Such agents may include small molecule antagonists of TGFβ1 and biologic antagonists of TGFβ1 (e.g., protein fragments and antibodies). In some embodiments, the inhibitor may be an antibody (including fragments thereof, such as Domain Antibodies (dAbs) as described in, for example, U.S. Pat. Nos. 6,291,158; 6,582,915; 6,593,081; 6,172,197; and 6,696,245), a small molecule inhibitor, an Adnectin, an Affibody, a DARPin, an Anticalin, an Avimer, a Versabody or a gene therapy. Use of inhibitors encompassed by the present invention also includes antibody mimetics, such as monobodies and single-domain antibodies. Monobodies are synthetic binding proteins that typically employ a fibronectin type III domain (FN3) as a molecular scaffold. Monobodies include Adnectins™ which are based on the 10th fibronectin type III domain.

In some aspects, the inhibitors, e.g., antibodies, or antigen-binding portions thereof, selectively bind to an epitope present on a LTBP1/3-TGFβ1 complex, that is not present on a GARP-TGFβ1 complex and/or a LRRC33-TGFβ1 complex. In some embodiments, the epitope is available due to a conformational change in LTBP1/3 and/or TGFβ1 that occurs when LTBP1/3 and TGFβ1 form a complex. In this embodiment, the epitope is not present in LTBP1/3 or TGFβ1 when the proteins are not associated in a complex. In one embodiment, the epitope is present on TGFβ1, when TGFβ1 is in a complex with LTBP1 or LTBP3. In another embodiment, the epitope is present on LTBP1, when LTBP1 is in a complex with TGFβ1. In another embodiment, the epitope is present on LTBP3, when LTBP3 is in a complex with TGFβ1. In another embodiment, the epitope comprises residues from both LTBP1 and TGFβ1. In another embodiment, the epitope comprises residues from both LTBP3 and TGFβ1.

Surprisingly, some of the LTBP1/3 complex-selective antibodies disclosed herein (e.g., Ab14, Ab20, Ab21-23, Ab17, and Ab24-29) are capable of binding to the small latent complex (proTGFβ1 C4S) in the absence of a presenting molecule (e.g., LTBP1/3) and yet exert context-selectivity. Without wishing to be bound by theory, this finding suggests that the antibodies (and variants thereof, or cross-competing antibodies) may bind an epitope that is available in the LTBP1/3-proTGFβ1 complex and in proTGFβ1 alone, but which is not available when an LRRC-type of presenting molecule (GARP or LRRC33) is present. The epitope might be entirely on latent TGFβ1, but gets occluded (directly or indirectly) when GARP or LRRC33 is complexed.

Alternatively, LTBP-selective inhibitors according to the present disclosure may bind a combinatorial epitope that comprises one or more amino acid residues of LTBP1 or LTBP3 and one or more amino acid residues of proTGFβ1, which confer the context-selectivity towards an LTBP-bound complex over GARP/LRRC33-bound complex. In these embodiments, selectivity towards the isoform (TGFβ31) as well as the context (ECM) is attributable to the combined contributions from both elements of the antigen complex.

In some embodiments, the inhibitors, e.g., antibodies, or antigen-binding portions thereof, are selective for the TGFβ1 isoform. In such embodiments, the inhibitors, e.g., antibodies, or antigen-binding portions thereof, do not bind to TGFβ2 and/or TGFβ3. For example, in one embodiment, the inhibitors, e.g., antibodies, or antigen-binding portions thereof, selectively bind a LTBP1/3-TGFβ1 complex, but do not bind TGFβ2, or a complex containing TGFβ2. In another embodiment, the inhibitors, e.g., antibodies, or antigen-binding portions thereof, selectively bind a LTBP1/3-TGFβ1 complex, but do not bind TGFβ3, or a complex containing TGFβ3.

In some embodiments, the inhibitors, e.g., antibodies, or antigen-binding portions thereof, do not prevent TGFβ1 from binding to integrin. For example, in some embodiments, the inhibitors, e.g., antibodies, or antigen-binding portions thereof, do not mask the integrin-binding site of TGFβ1.

In one aspect, the invention provides functional inhibitors, e.g., antibodies, that modulate TGFβ1 activity. In exemplary embodiments, the antibodies described herein are inhibitory antibodies, which inhibit the function or activity of TGFβ1. In some embodiments, the antibodies, or antigen-binding portions thereof, inhibit the activation (release) of TGFβ1 from a LTBP1-TGFβ1 complex and/or a LTBP3-TGFβ1 complex. The present disclosure provides, in exemplary embodiments, “context-specific” or “context-selective” inhibitors of TGFβ1 activation. Such inhibitors can bind a LTBP1/3-TGFβ1 complex and inhibit activation of TGFβ1 that is presented by LTBP1 or LTBP3, without inhibiting the activation of TGFβ1 presented by GARP and/or LRRC33. Accordingly, in some embodiments, the antibodies, or antigen-binding portions thereof, described herein inhibit the release of mature TGFβ1 from a LTBP1-TGFβ1 complex and/or a LTBP3-TGFβ1 complex, but do not inhibit the release of mature TGFβ1 from a GARP-TGFβ1 complex and/or a LRRC33-TGFβ1 complex. Due to the differential localization of LTBP, GARP, and LRRC33, the context-specific inhibitors of TGFβ1 provided by the present invention can block a particular subset of TGFβ1 activity in vivo. In one embodiment, the context-specific antibodies provided herein that inhibit LTBP1/3-TGFβ1 but do not inhibit GARP-TGFβ1 or LRRC33-TGFβ1 can be used to inhibit TGFβ1 localized to the extracellular matrix. In another embodiment, the context-specific antibodies can inhibit TGFβ1 without modulating TGFβ1-associated immune activity or immune response. In another embodiment, the context-specific antibodies can be used to inhibit TGFβ1 activity associated with the extracellular matrix without modulating TGFβ1 activity associated with hematopoietic cells. Accordingly, the context-specific antibodies can be used to inhibit LTBP1/3-associated TGFβ1 activity in applications in which TGFβ1 activation in the context of GARP and/or LRRC33 is undesirable, as described herein.

In some embodiments, the TGFβ1 comprises a naturally occurring mammalian amino acid sequence. In some embodiment, the TGFβ1 comprises a naturally occurring human amino acid sequence. In some embodiments, the TGFβ1 comprises a human, a monkey, a rat or a mouse amino acid sequence.

In some embodiments, an antibody, or antigen-binding portion thereof, described herein selectively binds to a complex comprising a TGFβ1 protein comprising the amino acid sequence set forth in SEQ ID NO: 9, and LTBP1 or LTBP3. In some embodiments, an antibody, or antigen-binding portion thereof, described herein selectively binds to a LTBP1/3-TGFβ1 complex which comprises a non-naturally-occurring TGFβ1 amino acid sequence (otherwise referred to herein as a non-naturally-occurring TGFβ1). For example, a non-naturally-occurring TGFβ1 may comprise one or more recombinantly generated mutations relative to a naturally-occurring TGFβ1 amino acid sequence.

In some embodiments, an antibody, or antigen-binding portion thereof, described herein does not bind TGFβ2 and/or TGFβ3, or to protein complexes containing TGFβ2 and/or TGFβ3. Exemplary TGFβ2 and TGFβ3 amino acid sequences are set forth in SEQ ID NOs: 10 and 11, respectively. In some embodiments, a TGFβ1, TGFβ2, or TGFβ3 amino acid sequence comprises an amino acid sequence as set forth in SEQ ID NOs: 12-23, as shown in Table 1. In some embodiments, a TGFβ1 amino acid sequence comprises an amino acid sequence as set forth in SEQ ID NOs: 24-31, as shown in Table 2.

In some embodiments, an antibody, or antigen-binding portion thereof, as described herein, is capable of selectively binding to an LTBP-TGFβ1 complex. In some embodiments, antigenic protein complexes (e.g., a LTBP-TGFβ1 complex) may comprise an LTBP protein selected from the following: LTBP1, LTBP2, LTBP3, and LTBP4.

In some embodiments, the antibody, or antigen-binding portion thereof, selectively binds an LTBP1-TGFβ1 complex. In some embodiments, the LTBP1 protein is a naturally-occurring protein. In some embodiments, the LTBP1 protein is a non-naturally occurring protein. In some embodiments, the LTBP1 protein is a recombinant protein. Such recombinant LTBP1 protein may comprise LTBP1, alternatively spliced variants thereof, and/or fragments thereof. Recombinant LTBP1 proteins may also be modified to comprise one or more detectable labels. In some embodiments, the LTBP1 protein comprises a leader sequence (e.g., a native or non-native leader sequence). In some embodiments, the LTBP1 protein does not comprise a leader sequence (i.e., the leader sequence has been processed or cleaved). Such detectable labels may include, but are not limited to biotin labels, polyhistidine tags, myc tags, HA tags and/or fluorescent tags. In some embodiments, the LTBP1 protein is a mammalian LTBP1 protein. In some embodiments, the LTBP1 protein is a human, a monkey, a mouse, or a rat LTBP1 protein. In some embodiments, the LTBP1 protein comprises an amino acid sequence as set forth in SEQ ID NO: 32 in Table 3. In some embodiments, the LTBP1 protein comprises an amino acid sequence as set forth in SEQ ID NOs: 33 or SEQ ID NO: 34 in Table 3.

In some embodiments, an antibody, or antigen-binding portion thereof, as described herein, is capable of binding to a LTBP3-TGFβ1 complex. In some embodiments, the LTBP3 protein is a naturally-occurring protein. In some embodiments, the LTBP3 protein is a non-naturally occurring protein. In some embodiments, the LTBP3 protein is a recombinant protein. Such recombinant LTBP3 protein may comprise LTBP3, alternatively spliced variants thereof and/or fragments thereof. In some embodiments, the LTBP3 protein comprises a leader sequence (e.g., a native or non-native leader sequence). In some embodiments, the LTBP3 protein does not comprise a leader sequence (i.e., the leader sequence has been processed or cleaved). Recombinant LTBP3 proteins may also be modified to comprise one or more detectable labels. Such detectable labels may include, but are not limited to biotin labels, polyhistidine tags, myc tags, HA tags and/or fluorescent tags. In some embodiments, the LTBP3 protein is a mammalian LTBP3 protein. In some embodiments, the LTBP3 protein is a human, a monkey, a mouse, or a rat LTBP3 protein. In some embodiments, the LTBP3 protein comprises an amino acid sequence as set forth in SEQ ID NO: 35. In some embodiments, the LTBP3 protein comprises an amino acid sequence as set forth in SEQ ID NOs: 36 or 37.

In an exemplary embodiment, inhibitors, e.g., antibodies, and antigen-binding portions thereof, that selectively bind LTBP1-TGFβ1 and/or LTBP3-TGFβ1 do not bind to a complex containing TGFβ1 and GARP or LRRC33. In one embodiment, the antibodies, or antigen-binding portions thereof, do not bind a GARP protein having a sequence set forth in SEQ ID NO:38 or SEQ ID NO:39, and do not bind to a complex containing said GARP protein. In another embodiment, the inhibitors, e.g., antibodies, or antigen-binding portions thereof, do not bind a GARP protein having a sequence set forth in SEQ ID NO:40 or SEQ ID NO:41, and do not bind to a complex containing said GARP protein. In one embodiment, the inhibitors, e.g., antibodies, or antigen-binding portions thereof, do not bind a LRRC33 protein having a sequence set forth in SEQ ID NO:42 or SEQ ID NO:43, and do not bind a complex containing said LRRC33 protein. In one embodiment, the inhibitors, e.g., antibodies, or antigen-binding portions thereof, do not bind a GARP/LRRC33 chimera, e.g., the GARP/LRRC33 chimera set forth in SEQ ID NO:44.

In another aspect, the invention provides methods of inhibiting TGFβ1 activation in the context of LTBP1 and/or LTBP3. In one embodiment, the method comprises exposing a LTBP1-proTGFβ1 complex or a LTBP3-proTGFβ1 complex an inhibitor, an antibody or antigen-binding portion thereof, and/or a pharmaceutical composition described herein. For example, in one embodiment, the inhibitor is an inhibitor of extracellular matrix-associated TGFβ1 activation, which selectively binds a LTBP1/3-presented proTGFβ1 latent complex. In one embodiment, the inhibitor does not inhibit immune cell-associated TGFβ1 activation, for example, immune cell-associated TGFβ1 activation that results from activation of a GARP-presented proTGFβ1 latent complex. In another embodiment, the antibody, or antigen-binding portion thereof, selectively binds an LTBP1-proTGFβ1 latent complex and/or an LTBP3-proTGFβ1 latent complex, thereby modulating release of mature TGFβ1 growth factor from the latent complex, wherein the antibody, or antigen-binding portion thereof, does not bind mature TGFβ1 alone or a GARP-proTGFβ1 latent complex. In one embodiment, the antibody, or antigen-binding portion thereof, inhibits the release of mature TGFβ1 from the LTBP1-proTGFβ1 complex and/or the LTBP3-proTGFβ1 complex. In one embodiment, the antibody, or antigen-binding portion thereof, does not inhibit the release of mature TGFβ1 from a GARP-proTGFβ1 complex or a LRRC33-proTGFβ1 complex.

In one embodiment, the method is performed in vitro. In another embodiment, the method is performed in vivo. In one embodiment, the LTBP1-proTGFβ1 complex or the LTBP3-proTGFβ1 complex is in an extracellular matrix. The extracellular matrix can comprise, for example, fibrillin and/or fibronectin. In some embodiments, the extracellular matrix comprises a protein comprising an RGD motif.

In some embodiments of the foregoing aspects, the antibody, or antigen-binding portion thereof, does not stimulate immune effector cells. In one embodiment, the antibody, or antigen-binding portion thereof, inhibits the release of mature TGFβ1 from a LTBP1-proTGFβ1 complex and/or a LTBP3-proTGFβ1 complex, and does not inhibit the release of mature TGFβ1 from a GARP-proTGFβ1 complex and/or an LRRC33-proTGFβ1 complex.

In some embodiments, inhibitors, e.g., antibodies, of the present disclosure that selectively bind to a LTBP1-TGFβ1 complex and/or a LTBP3-TGFβ1 complex can bind the complex with relatively high affinity, e.g., with a dissociation constant (K D ) less than 10 −6 M, 10 −7 M, 10 −8 M, 10 −9 M, 10 −10 M, 10 −11 M or lower. In one embodiment, an antibody, or antigen-binding portion thereof, binds a LTBP1-TGFβ1 complex and/or a LTBP3-TGFβ1 complex with a dissociation constant (K D ) of about 10 −8 M, about 10 −9 M, about 10 −10 M, about 10 −11 M, about 10 −12 M, or about 10 −13 M. For example, antibodies that selectively bind to a LTBP1-TGFβ1 complex and/or a LTBP3-TGFβ1 complex can bind the complex with an affinity between 5 pM and 500 nM, e.g., between 10 pM and 100 nM, e.g., between 50 pM and 50 nM. In one embodiment, the antibody, or antigen-binding fragment thereof, can bind a LTBP1-TGFβ1 complex and/or a LTBP3-TGFβ1 complex with an affinity of less than about 300 nm, for example about 20 nM or lower, about 10 nM or lower, about 500 pM or lower, or about 5 pM or lower. For example, the antibody, or antigen-binding fragment thereof, can bind a LTBP1-TGFβ1 complex and/or a LTBP3-TGFβ1 complex with an affinity of about 1 nm to about 350 nm, from about 10 nm to about 200 nm, from about 15 nm to about 250 nm, from about 20 nm to about 200 nm, about 1 nm, about 20 nm, about 25 nm, about 50 nm, about 100 nm, about 150 nm, about 200 nm, about 250 nm, about 300 nm, or about 500 pm.

The disclosure also includes antibodies or antigen-binding fragments that compete with any of the antibodies described herein for binding to a LTBP1-TGFβ1 complex and/or a LTBP3-TGFβ1 complex. In some embodiments, such antibodies have an affinity for the complex of 50 nM or lower (e.g., 20 nM or lower, 10 nM or lower, 500 pM or lower, 50 pM or lower, or 5 pM or lower). The affinity and binding kinetics of antibodies (or antigen-binding fragments thereof) that selectively bind to a LTBP1-TGFβ1 complex and/or a LTBP3-TGFβ1 complex can be tested using any suitable method, including but not limited to biosensor technology (e.g., OCTET or BIACORE).

In one embodiment, the antibodies, or antigen-binding fragments thereof, of the present disclosure do not compete with antibody SR-Abl for binding to a human LTBP1-proTGFβ1 complex.

Aspects of the disclosure relate to antibodies that compete or cross-compete with any of the antibodies provided herein. The term “compete”, as used herein with regard to an antibody, means that a first antibody binds to an epitope (e.g., an epitope of a LTBP1-TGFβ1 complex and/or an epitope of a LTBP3-TGFβ1 complex) in a manner sufficiently similar to the binding of a second antibody, such that the result of binding of the first antibody with its epitope is detectably decreased in the presence of the second antibody compared to the binding of the first antibody in the absence of the second antibody. The alternative, where the binding of the second antibody to its epitope is also detectably decreased in the presence of the first antibody, can, but need not be the case. That is, a first antibody can inhibit the binding of a second antibody to its epitope without that second antibody inhibiting the binding of the first antibody to its respective epitope. However, where each antibody detectably inhibits the binding of the other antibody with its epitope or ligand, whether to the same, greater, or lesser extent, the antibodies are said to “cross-compete” with each other for binding of their respective epitope(s). Both competing and cross-competing antibodies are within the scope of this disclosure. Regardless of the mechanism by which such competition or cross-competition occurs (e.g., steric hindrance, conformational change, or binding to a common epitope, or portion thereof), the skilled artisan would appreciate that such competing and/or cross-competing antibodies are encompassed and can be useful for the methods and/or compositions provided herein.

Aspects of the disclosure relate to antibodies that compete or cross-compete with any of the specific antibodies, or antigen-binding portions thereof, as provided herein, e.g., an antibody having one or more CDR sequences (1, 2, 3, 4, 5, or 6 CDR sequences) set forth in Table 5. In one embodiment, the invention provides antibodies, and antigen-binding fragments thereof, that compete or cross-compete with an antibody having heavy chain CDR sequences comprising SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3 as set forth in Table 5, and/or light chain CDR sequences comprising SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6 as set forth in Table 5. In one embodiment, the invention provides antibodies that compete or cross-compete with an antibody, or antigen-binding portion thereof, having a heavy chain variable region sequence comprising SEQ ID NO:7, and/or a light chain variable region sequence comprising SEQ ID NO:8. In some embodiments, an antibody, or antigen-binding portion thereof, binds at or near the same epitope as any of the antibodies provided herein. In some embodiments, an antibody, or antigen-binding portion thereof, binds near an epitope if it binds within 15 or fewer amino acid residues of the epitope. In some embodiments, any of the antibody, or antigen-binding portion thereof, as provided herein, binds within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acid residues of an epitope that is bound by any of the antibodies provided herein.

In another embodiment, provided herein is an antibody, or antigen-binding portion thereof, competes or cross-competes for binding to any of the antigens provided herein (e.g., a LTBP1-TGFβ1 complex and/or a LTBP3-TGFβ1 complex) with an equilibrium dissociation constant, K D , between the antibody and the protein of less than 10 −6 M. In other embodiments, an antibody competes or cross-competes for binding to any of the antigens provided herein with a K D in a range from 10 11 M to 10 −6 M. In other embodiments, an antibody competes or cross-competes for binding to a human LTBP1-TGFβ1 complex and/or a human LTBP3-TGFβ1 complex with a K D of <50 nM as determined by a suitable in vitro binding assay, e.g., BLI, such as Octet®. In other embodiments, an antibody competes or cross-competes for binding to a human LTBP1-TGFβ1 complex and/or a human LTBP3-TGFβ1 complex with a K D of <10 nM as determined by a suitable in vitro binding assay, e.g., BLI, such as Octet.

In some embodiments, the antibody or antigen-binding portion competes or cross-competes with an antibody having a heavy chain variable region sequence and light chain variable region sequence of Ab42, as set forth in Table 6 (e.g., SEQ ID NOs: 318 and 319, respectively). The antibody may compete or cross-compete for binding to a human LTBP1-TGFβ1 complex and/or a human LTBP3-TGFβ1 complex with a K D of <10 nM as determined by a suitable in vitro binding assay, e.g., BLI, such as Octet. The antibody may compete or cross-compete for binding to a human LTBP1-TGFβ1 complex and/or a human LTBP3-TGFβ1 complex with a K D of <5 nM as determined by a suitable in vitro binding assay, e.g., BLI, such as Octet. The antibody may bind to a human LTBP1-TGFβ1 complex and a human LTBP3-TGFβ1 complex with a K D of <5 nM as determined by a suitable in vitro binding assay, e.g., BLI, such as Octet. The antibody may not show any detectable binding to a human GARP-proTGFβ1 complex in a suitable in vitro binding assay, such as BLI (e.g., Octet). The antibody may not show detectable binding to a human GARP-proTGFβ1 complex, as measured by BLI, under the same assay conditions as used to measure binding to human LTBP1-proTGFβ1 complex and/or human LTBP3-TGFβ1 complex. Alternatively, or in addition, the antibody or antigen-binding portion may bind (e.g., selectively bind) a human LTBP1-proTGFβ1 complex and/or a human LTBP3-TGFβ1 complex with a K D that is at least 50 times lower than the K D when binding to a human GARP-proTGFβ1 complex (and optionally at least 50 times lower than the K D when binding to a human LRRC33-proTGFβ1 complex) under the same assay conditions.

In some embodiments, the antibody competes or cross-competes with an antibody having a heavy chain variable region sequence and light chain variable region sequence of Ab63, as set forth in Table 6 (e.g., SEQ ID NOs: 360 and 361, respectively). The antibody may compete or cross-compete for binding to a human LTBP1-TGFβ1 complex and/or a human LTBP3-TGFβ1 complex with a K D of <10 nM as determined by a suitable in vitro binding assay, e.g., BLI, such as Octet. The antibody may compete or cross-compete for binding to a human LTBP1-TGFβ1 complex and/or a human LTBP3-TGFβ1 complex with a K D of <5 nM as determined by a suitable in vitro binding assay, e.g., BLI, such as Octet. The antibody may bind to a human LTBP1-TGFβ1 complex and a human LTBP3-TGFβ1 complex with a K D of <5 nM as determined by a suitable in vitro binding assay, e.g., BLI, such as Octet. The antibody may not show any detectable binding to a human GARP-proTGFβ1 complex in a suitable in vitro binding assay, such as BLI (e.g., Octet). The antibody may not show detectable binding to a human GARP-proTGFβ1 complex, as measured by BLI, under the same assay conditions as used to measure binding to human LTBP1-proTGFβ1 complex and/or human LTBP3-TGFβ1 complex. Alternatively, or in addition, the antibody or antigen-binding portion may bind (e.g., selectively bind) a human LTBP1-proTGFβ1 complex and/or a human LTBP3-TGFβ1 complex with a K D that is at least 50 times lower than the K D when binding to a human GARP-proTGFβ1 complex (and optionally at least 50 times lower than the K D when binding to a human LRRC33-proTGFβ1 complex) under the same assay conditions.

In further embodiments, the antibody which selectively binds a human LTBP1-TGFβ1 complex and/or a human LTBP3-TGFβ1 complex may not show meaningful binding (e.g., may not show a response of more than 0.1 units (nm)) on exposure to a human GARP-proTGFβ1 complex in a BLI assay (e.g., Octet) when the human GARP-proTGFβ1 complex is at a concentration of 200 nM.

In some embodiments, provided herein is an anti-TGFβ1 antibody, or antigen-binding portion thereof, that competes for binding with an antibody, or antigen-binding portion thereof, described herein. In some embodiments, provided herein is an anti-TGFβ1 antibody, or antigen-binding portion thereof, that binds to the same epitope as an antibody, or antigen-binding portion thereof, described herein.

The antibodies provided herein can be characterized using any suitable methods. For example, one method is to identify the epitope to which the antigen binds, or “epitope mapping.” There are many suitable methods for mapping and characterizing the location of epitopes on proteins, including solving the crystal structure of an antibody-antigen complex, competition assays, gene fragment expression assays, and synthetic peptide-based assays, as described, for example, in Chapter 11 of Harlow and Lane, Using Antibodies, a Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1999. In an additional example, epitope mapping can be used to determine the sequence to which an antibody binds. The epitope can be a linear epitope, i.e., contained in a single stretch of amino acids, or a conformational epitope formed by a three-dimensional interaction of amino acids that may not necessarily be contained in a single stretch (primary structure linear sequence). In some embodiments, the epitope is a TGFβ1 epitope that is only available for binding by the antibody, or antigen-binding portion thereof, described herein, when the TGFβ1 is in a LTBP1-TGFβ1 complex and/or a LTBP3-TGFβ1 complex. In some embodiments, the epitope is present on a LTBP1/3-TGFβ1 complex, and is not present on a GARP-TGFβ1 complex and/or a LRRC33-TGFβ1 complex. In some embodiments, the epitope is available due to a conformational change in LTBP1/3 and/or TGFβ1 that occurs when LTBP1/3 and TGFβ1 form a complex. In this embodiment, the epitope is not present in LTBP1/3 or TGFβ1 when the proteins are not associated in a complex. In one embodiment, the epitope is present on TGFβ1, when TGFβ1 is in a complex with LTBP1 or LTBP3. In another embodiment, the epitope is present on LTBP1, when LTBP1 is in a complex with TGFβ1. In another embodiment, the epitope is present on LTBP3, when LTBP3 is in a complex with TGFβ1. In another embodiment, the epitope comprises residues from both LTBP1 and TGFβ1. In another embodiment, the epitope comprises residues from both LTBP3 and TGFβ1. Peptides of varying lengths (e.g., at least 4-6 amino acids long) can be isolated or synthesized (e.g., recombinantly) and used for binding assays with an antibody. In another example, the epitope to which the antibody binds can be determined in a systematic screen by using overlapping peptides derived from the target antigen sequence and determining binding by the antibody. According to the gene fragment expression assays, the open reading frame encoding the target antigen is fragmented either randomly or by specific genetic constructions and the reactivity of the expressed fragments of the antigen with the antibody to be tested is determined. The gene fragments may, for example, be produced by PCR and then transcribed and translated into protein in vitro, in the presence of radioactive amino acids. The binding of the antibody to the radioactively labeled antigen fragments is then determined by immunoprecipitation and gel electrophoresis. Certain epitopes can also be identified by using large libraries of random peptide sequences displayed on the surface of phage particles (phage libraries). Alternatively, a defined library of overlapping peptide fragments can be tested for binding to the test antibody in simple binding assays. In an additional example, mutagenesis of an antigen-binding domain, domain swapping experiments and alanine scanning mutagenesis can be performed to identify residues required, sufficient, and/or necessary for epitope binding. For example, domain swapping experiments can be performed using a mutant of a target antigen in which various fragments of the LTBP1-TGFβ1 complex or LTBP3-TGFβ1 complex have been replaced (swapped) with sequences from a closely related, but antigenically distinct protein, such as another member of the TGFβ protein family (e.g., GDF11). By assessing binding of the antibody to the mutant of the LTBP1-TGFβ1 complex and/or LTBP3-TGFβ1 complex, the importance of the particular antigen fragment to antibody binding can be assessed.

Alternatively, competition assays can be performed using other antibodies known to bind to the same antigen to determine whether an antibody binds to the same epitope as the other antibodies. Competition assays are well known to those of skill in the art.

Further, the interaction of the any of the antibodies provided herein with one or more residues in a LTBP1-TGFβ1 complex and/or a LTBP3-TGFβ1 complex can be determined by routine technology. For example, a crystal structure can be determined, and the distances between the residues in a LTBP1-TGFβ1 complex and/or a LTBP3-TGFβ1 complex, and one or more residues in the antibody, can be determined accordingly. Based on such distance, whether a specific residue in a LTBP1/3-TGFβ1 complex interacts with one or more residues in the antibody can be determined. Further, suitable methods, such as competition assays and target mutagenesis assays, can be applied to determine the preferential binding of a candidate antibody.

In some embodiments, the antibodies, or antigen-binding portions thereof, of the present invention that selectively bind to a LTBP1-TGFβ1 complex and/or a LTBP3-TGFβ1 complex include one or more of complementary determining regions (CDRs) shown in Table 5. In some embodiments, the invention provides a nucleic acid molecule that encodes an antibody, or antigen-binding portion thereof, that selectively binds to a LTBP1-TGFβ1 complex and/or a LTBP3-TGFβ1 complex, as described herein. In one embodiment, the nucleic acid molecules encode one or more of the CDR sequences shown in Table 5.

In some embodiments, antibodies of the present invention that selectively bind to a LTBP1-TGFβ3 complex and/or a LTBP3-TGFβ complex include any antibody, or antigen-binding portion thereof, comprising a CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, or CDRL3, or combinations thereof, as provided in Table 5. In some embodiments, antibodies that selectively bind to a LTBP1-TGFβ3 complex and/or a LTBP3-TGFβ complex include CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 as provided in Table 5.

The present invention also provides a nucleic acid sequence that encodes a molecule comprising CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, or CDRL3, or combinations thereof, as provided in Table 5.

Antibody heavy and light chain CDR3 domains may play a particularly important role in the binding specificity/affinity of an antibody for an antigen. Accordingly, in some embodiments, the antibodies, or antigen-binding portions thereof, that selectively bind to a LTBP1-TGFβ complex and/or a LTBP3-TGFβ complex, or the nucleic acid molecules that encode these antibodies, or antigen-binding portions thereof, can include at least the heavy and/or light chain CDR3 of the antibody shown in Table 5.

Aspects of the invention relate to a monoclonal antibody, or antigen-binding portion thereof, that binds selectively to a LTBP1-TGFβ complex and/or a LTBP3-TGFβ complex, and that comprises six complementarity determining regions (CDRs): CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3. The antibody, or antigen-binding portion thereof may have the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 of one of the antibodies (e.g., Ab42) shown in Table 5.

In some embodiments, CDRH1 comprises a sequence as set forth in SEQ ID NO: 1. In some embodiments, CDRH2 comprises a sequence as set forth in SEQ ID NO: 2. In some embodiments, CDRH3 comprises a sequence as set forth in SEQ ID NO: 3. In some embodiments, CDRL1 comprises a sequence as set forth in SEQ ID NO: 4. In some embodiments, CDRL2 comprises a sequence as set forth in SEQ ID NO: 5. In some embodiments, CDRL3 comprises a sequence as set forth in SEQ ID NO: 6.

In one aspect, the invention provides an isolated antibody, or an antigen-binding fragment thereof, that specifically binds a human LTBP1-proTGFβ complex and/or a human LTBP3-proTGFβ complex and does not bind a human GARP-proTGFβ1 complex; wherein the antibody or the antigen-binding fragment thereof does not bind mature TGFβ1, mature TGFβ2 or mature TGFβ3; wherein the antibody or the antigen-binding fragment thereof is a fully human or humanized antibody or antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof comprises at least three CDRs selected from the following, optionally comprising up to one or more amino acid changes for each of the CDRs: CDR-H1: SEQ ID NO:1; CDR-H2: SEQ ID NO:2; CDR-H3: SEQ ID NO:3; CDR-L1: SEQ ID NO:4; CDR-L2: SEQ ID NO:5; and, CDR-L3: SEQ ID NO:6. In some embodiments, the one or more amino acid changes comprises up to 1, 2, 3, 4, 5, or 6 amino acid changes for each of the CDRs.

In some embodiments (e.g., as for antibody SR-AB2, shown in Table 5), the antibody, or antigen-binding portion thereof, that selectively binds to a LTBP1-TGFβ complex and/or a LTBP3-TGFβ3 complex comprises: a CDRH1 comprising an amino acid sequence as set forth in SEQ ID NO: 1, a CDRH2 comprising an amino acid sequence as set forth in SEQ ID NO: 2, a CDRH3 comprising an amino acid sequence as set forth in SEQ ID NO: 3, a CDRL1 comprising an amino acid sequence as set forth in SEQ ID NO: 4, a CDRL2 comprising an amino acid sequence as set forth in SEQ ID NO: 5, and a CDRL3 comprising an amino acid sequence as set forth in SEQ ID NO: 6.

In some embodiments, the antibody, or antigen-binding portion thereof, comprises a heavy chain variable region comprising a complementarity determining region 3 (CDR3) having the amino acid sequence of SEQ ID NO: 3 and a light chain variable region comprising a CDR3 having the amino acid sequence of SEQ ID NO: 6. In some embodiments, the antibody, or antigen-binding portion thereof, comprises a heavy chain variable region comprising a complementarity determining region 2 (CDR2) having the amino acid sequence of SEQ ID NO: 2 and a light chain variable region comprising a CDR2 having the amino acid sequence of SEQ ID NO: 5. In some embodiments, the antibody, or antigen-binding portion thereof, comprises a heavy chain variable region comprising a complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 1 and a light chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 4.

The amino acid sequences of the heavy chain variable region (HCVR) and the light chain variable region (LCVR) of the antibody set forth in Table 5 (e.g., SR-AB2) are provided in Table 6.

In some embodiments, CDRH1 comprises a sequence as set forth in SEQ ID NO: 94. In some embodiments, CDRH2 comprises a sequence as set forth in SEQ ID NO: 95. In some embodiments, CDRH3 comprises a sequence as set forth in SEQ ID NO: 96. In some embodiments, CDRL1 comprises a sequence as set forth in SEQ ID NO: 97. In some embodiments, CDRL2 comprises a sequence as set forth in SEQ ID NO: 98. In some embodiments, CDRL3 comprises a sequence as set forth in SEQ ID NO: 99.

In one aspect, the invention provides an isolated antibody, or an antigen-binding fragment thereof, that specifically binds a human LTBP1-proTGFβ complex and/or a human LTBP3-proTGFβ complex and does not bind a human GARP-proTGFβ1 complex; wherein the antibody or the antigen-binding fragment thereof does not bind mature TGFβ1, mature TGFβ2 or mature TGFβ3; wherein the antibody or the antigen-binding fragment thereof is a fully human or humanized antibody or antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof comprises at least three CDRs selected from the following, optionally comprising one or more amino acid changes for each of the CDRs: CDR-H1: SEQ ID NO:94; CDR-H2: SEQ ID NO:95; CDR-H3: SEQ ID NO:96; CDR-L1: SEQ ID NO:97; CDR-L2: SEQ ID NO:98; and, CDR-L3: SEQ ID NO:99. In some embodiments, the one or more amino acid changes comprises up to 1, 2, 3, 4, 5, or 6 amino acid changes for each of the CDRs.

In some embodiments (e.g., as for antibody SR-AB10, shown in Table 5), the antibody, or antigen-binding portion thereof, that selectively binds to a LTBP1-TGFβ complex and/or a LTBP3-TGFβ3 complex comprises: a CDRH1 comprising an amino acid sequence as set forth in SEQ ID NO: 94, a CDRH2 comprising an amino acid sequence as set forth in SEQ ID NO: 95, a CDRH3 comprising an amino acid sequence as set forth in SEQ ID NO: 96, a CDRL1 comprising an amino acid sequence as set forth in SEQ ID NO: 97, a CDRL2 comprising an amino acid sequence as set forth in SEQ ID NO: 98, and a CDRL3 comprising an amino acid sequence as set forth in SEQ ID NO: 99.

In some embodiments, the antibody, or antigen-binding portion thereof, comprises a heavy chain variable region comprising a complementarity determining region 3 (CDR3) having the amino acid sequence of SEQ ID NO: 96 and a light chain variable region comprising a CDR3 having the amino acid sequence of SEQ ID NO: 99. In some embodiments, the antibody, or antigen-binding portion thereof, comprises a heavy chain variable region comprising a complementarity determining region 2 (CDR2) having the amino acid sequence of SEQ ID NO: 95 and a light chain variable region comprising a CDR2 having the amino acid sequence of SEQ ID NO: 98. In some embodiments, the antibody, or antigen-binding portion thereof, comprises a heavy chain variable region comprising a complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 94 and a light chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 97.

The amino acid sequences of the heavy chain variable region (HCVR) and the light chain variable region (LCVR) of the antibody set forth in Table 5 (e.g., SR-AB10) are provided in Table 6.

In some embodiments, CDRH1 comprises a sequence as set forth in SEQ ID NO: 100. In some embodiments, CDRH2 comprises a sequence as set forth in SEQ ID NO: 101. In some embodiments, CDRH3 comprises a sequence as set forth in SEQ ID NO: 102. In some embodiments, CDRL1 comprises a sequence as set forth in SEQ ID NO: 103. In some embodiments, CDRL2 comprises a sequence as set forth in SEQ ID NO: 104. In some embodiments, CDRL3 comprises a sequence as set forth in SEQ ID NO: 105.

In one aspect, the invention provides an isolated antibody, or an antigen-binding fragment thereof, that specifically binds a human LTBP1-proTGFβ complex and/or a human LTBP3-proTGFβ complex and does not bind a human GARP-proTGFβ1 complex; wherein the antibody or the antigen-binding fragment thereof does not bind mature TGFβ1, mature TGFβ2 or mature TGFβ3; wherein the antibody or the antigen-binding fragment thereof is a fully human or humanized antibody or antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof comprises at least three CDRs selected from the following, optionally comprising one or more amino acid changes for each of the CDRs: CDR-H1: SEQ ID NO:100; CDR-H2: SEQ ID NO:101; CDR-H3: SEQ ID NO:102; CDR-L1: SEQ ID NO:103; CDR-L2: SEQ ID NO:104; and, CDR-L3: SEQ ID NO:105. In some embodiments, the one or more amino acid changes comprises up to 1, 2, 3, 4, 5, or 6 amino acid changes for each of the CDRs.

In some embodiments (e.g., as for antibody SR-AB13, shown in Table 5), the antibody, or antigen-binding portion thereof, that selectively binds to a LTBP1-TGFβ complex and/or a LTBP3-TGFβ complex comprises: a CDRH1 comprising an amino acid sequence as set forth in SEQ ID NO: 100, a CDRH2 comprising an amino acid sequence as set forth in SEQ ID NO: 101, a CDRH3 comprising an amino acid sequence as set forth in SEQ ID NO: 102, a CDRL1 comprising an amino acid sequence as set forth in SEQ ID NO: 103, a CDRL2 comprising an amino acid sequence as set forth in SEQ ID NO: 104, and a CDRL3 comprising an amino acid sequence as set forth in SEQ ID NO: 105.

In some embodiments, the antibody, or antigen-binding portion thereof, comprises a heavy chain variable region comprising a complementarity determining region 3 (CDR3) having the amino acid sequence of SEQ ID NO: 102 and a light chain variable region comprising a CDR3 having the amino acid sequence of SEQ ID NO: 105. In some embodiments, the antibody, or antigen-binding portion thereof, comprises a heavy chain variable region comprising a complementarity determining region 2 (CDR2) having the amino acid sequence of SEQ ID NO: 101 and a light chain variable region comprising a CDR2 having the amino acid sequence of SEQ ID NO: 104. In some embodiments, the antibody, or antigen-binding portion thereof, comprises a heavy chain variable region comprising a complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 100 and a light chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 103.

The amino acid sequences of the heavy chain variable region (HCVR) and the light chain variable region (LCVR) of the antibody set forth in Table 5 (e.g., SR-AB13) are provided in Table 6.

In some embodiments, CDRH1 comprises a sequence as set forth in SEQ ID NO: 124. In some embodiments, CDRH2 comprises a sequence as set forth in SEQ ID NO: 125. In some embodiments, CDRH3 comprises a sequence as set forth in SEQ ID NO: 126. In some embodiments, CDRL1 comprises a sequence as set forth in SEQ ID NO: 127. In some embodiments, CDRL2 comprises a sequence as set forth in SEQ ID NO: 128. In some embodiments, CDRL3 comprises a sequence as set forth in SEQ ID NO: 129.

In one aspect, the invention provides an isolated antibody, or an antigen-binding fragment thereof, that specifically binds a human LTBP1-proTGFβ complex and/or a human LTBP3-proTGFβ complex; wherein the antibody or the antigen-binding fragment thereof does not bind mature TGFβ1, mature TGFβ2 or mature TGFβ3; wherein the antibody or the antigen-binding fragment thereof is a fully human or humanized antibody or antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof comprises at least three (optionally all six) CDRs selected from the following, optionally comprising up to one or more amino acid changes for each of the CDRs: CDR-H1: SEQ ID NO:124; CDR-H2: SEQ ID NO:125; CDR-H3: SEQ ID NO:126; CDR-L1: SEQ ID NO:127; CDR-L2: SEQ ID NO:128; and, CDR-L3: SEQ ID NO:129. In some embodiments, the one or more amino acid changes comprises up to 1, 2, 3, 4, 5, or 6 amino acid changes for each of the CDRs.

In some embodiments (e.g., as for antibody SR-AB31, shown in Table 5), the antibody, or antigen-binding portion thereof, that selectively binds to a LTBP1-TGFβ complex and/or a LTBP3-TGFβ complex comprises: a CDRH1 comprising an amino acid sequence as set forth in SEQ ID NO: 124, a CDRH2 comprising an amino acid sequence as set forth in SEQ ID NO: 125, a CDRH3 comprising an amino acid sequence as set forth in SEQ ID NO: 126, a CDRL1 comprising an amino acid sequence as set forth in SEQ ID NO: 127, a CDRL2 comprising an amino acid sequence as set forth in SEQ ID NO: 128, and a CDRL3 comprising an amino acid sequence as set forth in SEQ ID NO: 129.

In some embodiments, the antibody, or antigen-binding portion thereof, comprises a heavy chain variable region comprising a complementarity determining region 3 (CDR3) having the amino acid sequence of SEQ ID NO: 126 and a light chain variable region comprising a CDR3 having the amino acid sequence of SEQ ID NO: 129. In some embodiments, the antibody, or antigen-binding portion thereof, comprises a heavy chain variable region comprising a complementarity determining region 2 (CDR2) having the amino acid sequence of SEQ ID NO: 125 and a light chain variable region comprising a CDR2 having the amino acid sequence of SEQ ID NO: 128. In some embodiments, the antibody, or antigen-binding portion thereof, comprises a heavy chain variable region comprising a complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 124 and a light chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 127.

The amino acid sequences of the HCVR and the LCVR of the antibody set forth in Table 5 (e.g., SR-AB31) are provided in Table 6.

In some embodiments, CDRH1 comprises a sequence as set forth in SEQ ID NO: 166. In some embodiments, CDRH2 comprises a sequence as set forth in SEQ ID NO: 167. In some embodiments, CDRH3 comprises a sequence as set forth in SEQ ID NO: 168. In some embodiments, CDRL1 comprises a sequence as set forth in SEQ ID NO: 169. In some embodiments, CDRL2 comprises a sequence as set forth in SEQ ID NO: 170. In some embodiments, CDRL3 comprises a sequence as set forth in SEQ ID NO: 171.

In one aspect, the invention provides an isolated antibody, or an antigen-binding fragment thereof, that specifically binds a human LTBP1-proTGFβ complex and/or a human LTBP3-proTGFβ complex; wherein the antibody or the antigen-binding fragment thereof does not bind mature TGFβ1, mature TGFβ2 or mature TGFβ3; wherein the antibody or the antigen-binding fragment thereof is a fully human or humanized antibody or antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof comprises at least three (optionally all six) CDRs selected from the following, optionally comprising up to one or more amino acid changes for each of the CDRs: CDR-H1: SEQ ID NO:166; CDR-H2: SEQ ID NO:167; CDR-H3: SEQ ID NO:168; CDR-L1: SEQ ID NO:169; CDR-L2: SEQ ID NO:170; and, CDR-L3: SEQ ID NO:171. In some embodiments, the one or more amino acid changes comprises up to 1, 2, 3, 4, 5, or 6 amino acid changes for each of the CDRs.

In some embodiments (e.g., as for antibody SR-AB42, shown in Table 5), the antibody, or antigen-binding portion thereof, that selectively binds to a LTBP1-TGFβ complex and/or a LTBP3-TGFβ complex comprises: a CDRH1 comprising an amino acid sequence as set forth in SEQ ID NO: 166, a CDRH2 comprising an amino acid sequence as set forth in SEQ ID NO: 167, a CDRH3 comprising an amino acid sequence as set forth in SEQ ID NO: 168, a CDRL1 comprising an amino acid sequence as set forth in SEQ ID NO: 169, a CDRL2 comprising an amino acid sequence as set forth in SEQ ID NO: 170, and a CDRL3 comprising an amino acid sequence as set forth in SEQ ID NO: 171.

In some embodiments, the antibody, or antigen-binding portion thereof, comprises a heavy chain variable region comprising a complementarity determining region 3 (CDR3) having the amino acid sequence of SEQ ID NO: 168 and a light chain variable region comprising a CDR3 having the amino acid sequence of SEQ ID NO: 171. In some embodiments, the antibody, or antigen-binding portion thereof, comprises a heavy chain variable region comprising a complementarity determining region 2 (CDR2) having the amino acid sequence of SEQ ID NO: 167 and a light chain variable region comprising a CDR2 having the amino acid sequence of SEQ ID NO: 170. In some embodiments, the antibody, or antigen-binding portion thereof, comprises a heavy chain variable region comprising a complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 166 and a light chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 169.

The amino acid sequences of the HCVR and the LCVR of the antibody set forth in Table 5 (e.g., SR-AB42) are provided in Table 6.

In some embodiments, CDRH1 comprises a sequence as set forth in SEQ ID NO: 292. In some embodiments, CDRH2 comprises a sequence as set forth in SEQ ID NO: 293. In some embodiments, CDRH3 comprises a sequence as set forth in SEQ ID NO: 294. In some embodiments, CDRL1 comprises a sequence as set forth in SEQ ID NO: 295. In some embodiments, CDRL2 comprises a sequence as set forth in SEQ ID NO: 296. In some embodiments, CDRL3 comprises a sequence as set forth in SEQ ID NO: 297.

In one aspect, the invention provides an isolated antibody, or an antigen-binding fragment thereof, that specifically binds a human LTBP1-proTGFβ complex and/or a human LTBP3-proTGFβ complex; wherein the antibody or the antigen-binding fragment thereof does not bind mature TGFβ1, mature TGFβ2 or mature TGFβ3; wherein the antibody or the antigen-binding fragment thereof is a fully human or humanized antibody or antigen-binding fragment thereof, wherein the antibody or the antigen-binding fragment thereof comprises at least three (optionally all six) CDRs selected from the following, optionally comprising up to one or more amino acid changes for each of the CDRs: CDR-H1: SEQ ID NO:292; CDR-H2: SEQ ID NO:293; CDR-H3: SEQ ID NO:294; CDR-L1: SEQ ID NO:295; CDR-L2: SEQ ID NO:296; and, CDR-L3: SEQ ID NO:297. In some embodiments, the one or more amino acid changes comprises up to 1, 2, 3, 4, 5, or 6 amino acid changes for each of the CDRs.

In some embodiments (e.g., as for antibody SR-AB63, shown in Table 5), the antibody, or antigen-binding portion thereof, that selectively binds to a LTBP1-TGFβ complex and/or a LTBP3-TGFβ complex comprises: a CDRH1 comprising an amino acid sequence as set forth in SEQ ID NO: 292, a CDRH2 comprising an amino acid sequence as set forth in SEQ ID NO: 293, a CDRH3 comprising an amino acid sequence as set forth in SEQ ID NO: 294, a CDRL1 comprising an amino acid sequence as set forth in SEQ ID NO: 295, a CDRL2 comprising an amino acid sequence as set forth in SEQ ID NO: 296, and a CDRL3 comprising an amino acid sequence as set forth in SEQ ID NO: 297.

In some embodiments, the antibody, or antigen-binding portion thereof, comprises a heavy chain variable region comprising a complementarity determining region 3 (CDR3) having the amino acid sequence of SEQ ID NO: 294 and a light chain variable region comprising a CDR3 having the amino acid sequence of SEQ ID NO: 297. In some embodiments, the antibody, or antigen-binding portion thereof, comprises a heavy chain variable region comprising a complementarity determining region 2 (CDR2) having the amino acid sequence of SEQ ID NO: 293 and a light chain variable region comprising a CDR2 having the amino acid sequence of SEQ ID NO: 296. In some embodiments, the antibody, or antigen-binding portion thereof, comprises a heavy chain variable region comprising a complementarity determining region 1 (CDR1) having the amino acid sequence of SEQ ID NO: 292 and a light chain variable region comprising a CDR1 having the amino acid sequence of SEQ ID NO: 295.

The amino acid sequences of the HCVR and the LCVR of the antibody set forth in Table 5 (e.g., SR-AB63) are provided in Table 6.

Ten additional antibodies (Ab3-Ab12) were developed that specifically bind to a LTBP1-TGFβ3 complex and/or a LTBP3-TGFβ complex, and inhibit release of mature TGFβ presented in the context of LTBP1/3. Table 6 also provides the HCVR and LCVR amino acid sequences of these additional LTBP context-specific antibodies, in addition to the HCVR and LCVR amino acid sequences of the antibodies referred to in Table 5.

Aspects of the invention relate to a monoclonal antibody, or antigen-binding portion thereof, that binds selectively to a LTBP1-TGFβ complex and/or a LTBP3-TGFβ complex, and that comprises a heavy chain variable region sequence and a light chain variable region sequence.

In one aspect, the invention provides an isolated antibody or an antigen-binding fragment thereof, that specifically binds a human LTBP1-proTGFβ complex and/or a human LTBP3-proTGFβ complex and does not bind a human GARP-proTGFβ1 complex; wherein the antibody or the antigen-binding fragment thereof does not bind mature TGFβ1, mature TGFβ2 or mature TGFβ3; wherein the antibody or the antigen-binding fragment thereof is a fully human or humanized antibody or an antigen-binding fragment thereof; wherein the antibody or the antigen-binding fragment thereof comprises a variable heavy chain having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of the variable region amino acid sequences set forth in Table 6.

In one embodiment, the antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 7, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, or SEQ ID NO: 106. In one embodiment, the antibody, or antigen-binding fragment thereof, comprises a light chain variable region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 8, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, or SEQ ID NO: 107.

In one embodiment, the antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 7 and/or a light chain variable region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 8. In one embodiment, the antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 7 or a light chain variable region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 8. In one embodiment, the antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 7 and a light chain variable region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 8.

In one embodiment, the antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 74 and/or a light chain variable region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 75. In one embodiment, the antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 74 or a light chain variable region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 75. In one embodiment, the antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 74 and a light chain variable region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 75.

In one embodiment, the antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 76 and/or a light chain variable region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 77. In one embodiment, the antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 76 or a light chain variable region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 77. In one embodiment, the antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 76 and a light chain variable region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 77.

In one embodiment, the antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 78 and/or a light chain variable region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 79. In one embodiment, the antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 78 or a light chain variable region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 79. In one embodiment, the antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 78 and a light chain variable region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 79.

In one embodiment, the antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 80 and/or a light chain variable region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 81. In one embodiment, the antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 80 or a light chain variable region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 81. In one embodiment, the antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 80 and a light chain variable region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 81.

In one embodiment, the antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 82 and/or a light chain variable region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 83. In one embodiment, the antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 82 or a light chain variable region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 83. In one embodiment, the antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 82 and a light chain variable region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 83.

In one embodiment, the antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 84 and/or a light chain variable region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 85. In one embodiment, the antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 84 or a light chain variable region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 85. In one embodiment, the antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 84 and a light chain variable region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 85.

In one embodiment, the antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 86 and/or a light chain variable region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 87. In one embodiment, the antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 86 or a light chain variable region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 87. In one embodiment, the antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 86 and a light chain variable region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 87.

In one embodiment, the antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 88 and/or a light chain variable region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 89. In one embodiment, the antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 88 or a light chain variable region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 89. In one embodiment, the antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 88 and a light chain variable region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 89.

In one embodiment, the antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 90 and/or a light chain variable region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 91. In one embodiment, the antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 90 or a light chain variable region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 91. In one embodiment, the antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 90 and a light chain variable region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 91.

In one embodiment, the antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 92 and/or a light chain variable region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 93. In one embodiment, the antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 92 or a light chain variable region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 93. In one embodiment, the antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 92 and a light chain variable region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 93.

In one embodiment, the antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 106 and/or a light chain variable region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 107. In one embodiment, the antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 106 or a light chain variable region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 107. In one embodiment, the antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 106 and a light chain variable region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 107.

In one aspect, the invention provides an isolated antibody or an antigen-binding fragment thereof, that specifically binds a human LTBP1-proTGFβ complex and/or a human LTBP3-proTGFβ complex. The antibody may selectively bind a human LTBP1-proTGFβ complex and/or a human LTBP3-proTGFβ complex. The antibody or the antigen-binding fragment thereof may not bind mature TGFβ1, mature TGFβ2 or mature TGFβ3. The antibody or the antigen-binding fragment thereof may be a fully human or humanized antibody or an antigen-binding fragment thereof. The antibody or the antigen-binding fragment thereof may comprises a variable heavy chain having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to any one of the variable region amino acid sequences set forth in Table 6. In some embodiments, the level of identity is at least 95% (optionally at least 98%).

Accordingly, in one embodiment, the antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 318 and/or a light chain variable region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 319. The antibody, or antigen-binding fragment thereof, may comprise a heavy chain variable region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 318 or a light chain variable region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 319. The antibody, or antigen-binding fragment thereof, may comprise a heavy chain variable region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 318 and a light chain variable region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 319.

In another embodiment, the antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 360 and/or a light chain variable region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 361. The antibody, or antigen-binding fragment thereof, may comprise a heavy chain variable region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 360 or a light chain variable region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 361. The antibody, or antigen-binding fragment thereof, may comprise a heavy chain variable region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 360 and a light chain variable region having an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 361.

In some embodiments, the heavy chain variable region and/or the light chain variable region sequences do not vary within any of the CDR sequences provided herein. For example, in some embodiments, the degree of sequence variation (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) may occur within a heavy chain variable and/or a light chain variable amino acid sequence excluding any of the CDR sequences provided herein. In some embodiments, the antibody, or antigen-binding portion thereof, comprises a heavy chain variable domain comprising an amino acid sequence set forth in SEQ ID NO: 7 and a light chain variable domain comprising an amino acid sequence set forth in SEQ ID NO: 8. In some embodiments, the antibody, or antigen-binding fragment thereof, comprising a heavy chain variable region having an amino acid sequence that is at least 90% identical to SEQ ID NO: 318 and/or a light chain variable region having an amino acid sequence that is at least 90% identical to SEQ ID NO: 319 does not vary within any of the CDR sequences of Ab42 provided herein. In some embodiments, the antibody, or antigen-binding fragment thereof, comprising a heavy chain variable region having an amino acid sequence that is at least 90% identical to SEQ ID NO: 360 and/or a light chain variable region having an amino acid sequence that is at least 90% identical to SEQ ID NO: 361 does not vary within any of the CDR sequences of Ab63 provided herein.

In some embodiments, the antibody, or antigen-binding portion thereof, comprises a heavy chain variable domain comprising an amino acid sequence set forth in Table 6, and/or a light chain variable domain comprising an amino acid sequence set forth in Table 6. For example, in some embodiments, the antibody, or antigen-binding portion thereof, comprises a heavy chain variable domain comprising an amino acid sequence set forth in SEQ ID NO: 6 and/or a light chain variable domain comprising an amino acid sequence set forth in SEQ ID NO: 7. In some embodiments, the antibody, or antigen-binding portion thereof, comprises a heavy chain variable domain comprising an amino acid sequence set forth in SEQ ID NO: 6 or a light chain variable domain comprising an amino acid sequence set forth in SEQ ID NO: 7. In some embodiments, the antibody, or antigen-binding portion thereof, comprises a heavy chain variable domain comprising an amino acid sequence set forth in SEQ ID NO: 6 and a light chain variable domain comprising an amino acid sequence set forth in SEQ ID NO: 7.

In some embodiments, the antibody, or antigen-binding portion thereof, comprises a heavy chain variable domain comprising an amino acid sequence set forth in SEQ ID NO: 74 and/or a light chain variable domain comprising an amino acid sequence set forth in SEQ ID NO: 75. In some embodiments, the antibody, or antigen-binding portion thereof, comprises a heavy chain variable domain comprising an amino acid sequence set forth in SEQ ID NO: 74 or a light chain variable domain comprising an amino acid sequence set forth in SEQ ID NO: 75. In some embodiments, the antibody, or antigen-binding portion thereof, comprises a heavy chain variable domain comprising an amino acid sequence set forth in SEQ ID NO: 74 and a light chain variable domain comprising an amino acid sequence set forth in SEQ ID NO: 75.

In some embodiments, the antibody, or antigen-binding portion thereof, comprises a heavy chain variable domain comprising an amino acid sequence set forth in SEQ ID NO: 76 and/or a light chain variable domain comprising an amino acid sequence set forth in SEQ ID NO: 77. In some embodiments, the antibody, or antigen-binding portion thereof, comprises a heavy chain variable domain comprising an amino acid sequence set forth in SEQ ID NO: 76 or a light chain variable domain comprising an amino acid sequence set forth in SEQ ID NO: 77. In some embodiments, the antibody, or antigen-binding portion thereof, comprises a heavy chain variable domain comprising an amino acid sequence set forth in SEQ ID NO: 76 and a light chain variable domain comprising an amino acid sequence set forth in SEQ ID NO: 77.

In some embodiments, the antibody, or antigen-binding portion thereof, comprises a heavy chain variable domain comprising an amino acid sequence set forth in SEQ ID NO: 78 and/or a light chain variable domain comprising an amino acid sequence set forth in SEQ ID NO: 79. In some embodiments, the antibody, or antigen-binding portion thereof, comprises a heavy chain variable domain comprising an amino acid sequence set forth in SEQ ID NO: 78 or a light chain variable domain comprising an amino acid sequence set forth in SEQ ID NO: 79. In some embodiments, the antibody, or antigen-binding portion thereof, comprises a heavy chain variable domain comprising an amino acid sequence set forth in SEQ ID NO: 78 and a light chain variable domain comprising an amino acid sequence set forth in SEQ ID NO: 79.

In some embodiments, the antibody, or antigen-binding portion thereof, comprises a heavy chain variable domain comprising an amino acid sequence set forth in SEQ ID NO: 80 and/or a light chain variable domain comprising an amino acid sequence set forth in SEQ ID NO: 81. In some embodiments, the antibody, or antigen-binding portion thereof, comprises a heavy chain variable domain comprising an amino acid sequence set forth in SEQ ID NO: 80 or a light chain variable domain comprising an amino acid sequence set forth in SEQ ID NO: 81. In some embodiments, the antibody, or antigen-binding portion thereof, comprises a heavy chain variable domain comprising an amino acid sequence set forth in SEQ ID NO: 80 and a light chain variable domain comprising an amino acid sequence set forth in SEQ ID NO: 81.

In some embodiments, the antibody, or antigen-binding portion thereof, comprises a heavy chain variable domain comprising an amino acid sequence set forth in SEQ ID NO: 82 and/or a light chain variable domain comprising an amino acid sequence set forth in SEQ ID NO: 83. In some embodiments, the antibody, or antigen-binding portion thereof, comprises a heavy chain variable domain comprising an amino acid sequence set forth in SEQ ID NO: 82 or a light chain variable domain comprising an amino acid sequence set forth in SEQ ID NO: 83. In some embodiments, the antibody, or antigen-binding portion thereof, comprises a heavy chain variable domain comprising an amino acid sequence set forth in SEQ ID NO: 82 and a light chain variable domain comprising an amino acid sequence set forth in SEQ ID NO: 83.

In some embodiments, the antibody, or antigen-binding portion thereof, comprises a heavy chain variable domain comprising an amino acid sequence set forth in SEQ ID NO: 84 and/or a light chain variable domain comprising an amino acid sequence set forth in SEQ ID NO: 85. In some embodiments, the antibody, or antigen-binding portion thereof, comprises a heavy chain variable domain comprising an amino acid sequence set forth in SEQ ID NO: 84 or a light chain variable domain comprising an amino acid sequence set forth in SEQ ID NO: 85. In some embodiments, the antibody, or antigen-binding portion thereof, comprises a heavy chain variable domain comprising an amino acid sequence set forth in SEQ ID NO: 84 and a light chain variable domain comprising an amino acid sequence set forth in SEQ ID NO: 85.

In some embodiments, the antibody, or antigen-binding portion thereof, comprises a heavy chain variable domain comprising an amino acid sequence set forth in SEQ ID NO: 86 and/or a light chain variable domain comprising an amino acid sequence set forth in SEQ ID NO: 87. In some embodiments, the antibody, or antigen-binding portion thereof, comprises a heavy chain variable domain comprising an amino acid sequence set forth in SEQ ID NO: 86 or a light chain variable domain comprising an amino acid sequence set forth in SEQ ID NO: 87. In some embodiments, the antibody, or antigen-binding portion thereof, comprises a heavy chain variable domain comprising an amino acid sequence set forth in SEQ ID NO: 86 and a light chain variable domain comprising an amino acid sequence set forth in SEQ ID NO: 87.

In some embodiments, the antibody, or antigen-binding portion thereof, comprises a heavy chain variable domain comprising an amino acid sequence set forth in SEQ ID NO: 88 and/or a light chain variable domain comprising an amino acid sequence set forth in SEQ ID NO: 89. In some embodiments, the antibody, or antigen-binding portion thereof, comprises a heavy chain variable domain comprising an amino acid sequence set forth in SEQ ID NO: 88 or a light chain variable domain comprising an amino acid sequence set forth in SEQ ID NO: 89. In some embodiments, the antibody, or antigen-binding portion thereof, comprises a heavy chain variable domain comprising an amino acid sequence set forth in SEQ ID NO: 88 and a light chain variable domain comprising an amino acid sequence set forth in SEQ ID NO: 89.

In some embodiments, the antibody, or antigen-binding portion thereof, comprises a heavy chain variable domain comprising an amino acid sequence set forth in SEQ ID NO: 90 and/or a light chain variable domain comprising an amino acid sequence set forth in SEQ ID NO: 91. In some embodiments, the antibody, or antigen-binding portion thereof, comprises a heavy chain variable domain comprising an amino acid sequence set forth in SEQ ID NO: 90 or a light chain variable domain comprising an amino acid sequence set forth in SEQ ID NO: 91. In some embodiments, the antibody, or antigen-binding portion thereof, comprises a heavy chain variable domain comprising an amino acid sequence set forth in SEQ ID NO: 90 and a light chain variable domain comprising an amino acid sequence set forth in SEQ ID NO: 91.

In some embodiments, the antibody, or antigen-binding portion thereof, comprises a heavy chain variable domain comprising an amino acid sequence set forth in SEQ ID NO: 92 and/or a light chain variable domain comprising an amino acid sequence set forth in SEQ ID NO: 93. In some embodiments, the antibody, or antigen-binding portion thereof, comprises a heavy chain variable domain comprising an amino acid sequence set forth in SEQ ID NO: 92 or a light chain variable domain comprising an amino acid sequence set forth in SEQ ID NO: 93. In some embodiments, the antibody, or antigen-binding portion thereof, comprises a heavy chain variable domain comprising an amino acid sequence set forth in SEQ ID NO: 92 and a light chain variable domain comprising an amino acid sequence set forth in SEQ ID NO: 93.

In some embodiments, the antibody, or antigen-binding portion thereof, comprises a heavy chain variable domain comprising an amino acid sequence set forth in SEQ ID NO: 106 and/or a light chain variable domain comprising an amino acid sequence set forth in SEQ ID NO: 107. In some embodiments, the antibody, or antigen-binding portion thereof, comprises a heavy chain variable domain comprising an amino acid sequence set forth in SEQ ID NO: 106 or a light chain variable domain comprising an amino acid sequence set forth in SEQ ID NO: 107. In some embodiments, the antibody, or antigen-binding portion thereof, comprises a heavy chain variable domain comprising an amino acid sequence set forth in SEQ ID NO: 106 and a light chain variable domain comprising an amino acid sequence set forth in SEQ ID NO: 107.

In some embodiments, the antibody, or antigen-binding portion thereof, comprises a heavy chain variable domain comprising an amino acid sequence set forth in SEQ ID NO: 318 and/or a light chain variable domain comprising an amino acid sequence set forth in SEQ ID NO: 319. In some embodiments, the antibody, or antigen-binding portion thereof, comprises a heavy chain variable domain comprising an amino acid sequence set forth in SEQ ID NO: 318 or a light chain variable domain comprising an amino acid sequence set forth in SEQ ID NO: 319. In some embodiments, the antibody, or antigen-binding portion thereof, comprises a heavy chain variable domain comprising an amino acid sequence set forth in SEQ ID NO: 318 and a light chain variable domain comprising an amino acid sequence set forth in SEQ ID NO: 319.

In some embodiments, the antibody, or antigen-binding portion thereof, comprises a heavy chain variable domain comprising an amino acid sequence set forth in SEQ ID NO: 360 and/or a light chain variable domain comprising an amino acid sequence set forth in SEQ ID NO: 361. In some embodiments, the antibody, or antigen-binding portion thereof, comprises a heavy chain variable domain comprising an amino acid sequence set forth in SEQ ID NO: 360 or a light chain variable domain comprising an amino acid sequence set forth in SEQ ID NO: 361. In some embodiments, the antibody, or antigen-binding portion thereof, comprises a heavy chain variable domain comprising an amino acid sequence set forth in SEQ ID NO: 360 and a light chain variable domain comprising an amino acid sequence set forth in SEQ ID NO: 361.

The amino acid sequences of the heavy chain variable region (HCVR) and the light chain variable region (LCVR) of the antibody SR-AB2 set forth in Table 5 are provided below.

SR-AB2 - Heavy chain variable region amino acid

sequence

(SEQ ID NO: 7)

SR-AB2 - Light chain variable region amino acid

sequence

(SEQ ID NO: 8)

The amino acid sequences of the heavy chain variable region (HCVR) and the light chain variable region (LCVR) of the antibody SR-AB10 set forth in Table 5 are provided below.

SR-AB10 - Heavy chain variable region amino acid

sequence

(SEQ ID NO: 88)

SR-AB10 - Light chain variable region amino acid

sequence

(SEQ ID NO: 89)

The amino acid sequences of the heavy chain variable region (HCVR) and the light chain variable region (LCVR) of the antibody SR-AB13 set forth in Table 5 are provided below.

SR-AB13 - Heavy chain variable region amino acid

sequence

(SEQ ID NO: 106)

SR-AB13 - Light chain variable region amino acid

sequence

(SEQ ID NO: 107)

The amino acid sequences of the heavy chain variable region (HCVR) and the light chain variable region (LCVR) of the antibody SR-AB42 set forth in Table 5 are provided below.

SR-AB42 - Heavy chain variable region amino acid

sequence

(SEQ ID NO: 318)

63 - Light chain variable region amino acid

sequence

(SEQ ID NO: 319)

The amino acid sequences of the heavy chain variable region (HCVR) and the light chain variable region (LCVR) of the antibody SR-AB63 set forth in Table 5 are provided below.

SR-AB63 - Heavy chain variable region amino acid

sequence

(SEQ ID NO: 360)

SR-AB63 - Light chain variable region amino acid

sequence

(SEQ ID NO: 361)

In some embodiments, antibodies, or antigen-binding portions thereof, of the invention that selectively bind to a LTBP1-TGFβ complex and/or a LTBP3-TGFβ complex have one or more CDR sequences substantially similar to CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and/or CDRL3. For example, the antibodies may include one or more CDR sequences as shown in Table 5 (SEQ ID NOs: 1-6, SEQ ID NOs: 94-99 or SEQ ID NOs: 100-105) containing up to 6, 5, 4, 3, 2, or 1 amino acid residue variations as compared to the corresponding CDR region in any one of SEQ ID NOs: 1-6, SEQ ID NOs: 94-99 or SEQ ID NOs: 100-105.

In one embodiment, the antibody, or antigen-binding fragment thereof, comprises at least three CDRs selected from the following, optionally comprising up to 6 amino acid changes, for example 1, 2, 3, 4, 5, or 6 amino acid changes, for each of the CDRs CDR-H1: SEQ ID NO: 1; CDR-H2: SEQ ID NO: 2; CDR-H3: SEQ ID NO: 3; CDR-L1: SEQ ID NO: 4; CDR-L2: SEQ ID NO: 5; and, CDR-L3: SEQ ID NO: 6. In one embodiment, the antibody, or antigen-binding fragment thereof, comprises at least three CDRs selected from the following, optionally comprising up to 6 amino acid changes, for example 1, 2, 3, 4, 5, or 6 amino acid changes, for each of the CDRs CDR-H1: SEQ ID NO: 94; CDR-H2: SEQ ID NO: 95; CDR-H3: SEQ ID NO: 96; CDR-L1: SEQ ID NO: 97; CDR-L2: SEQ ID NO: 98; and, CDR-L3: SEQ ID NO: 99. In one embodiment, the antibody, or antigen-binding fragment thereof, comprises at least three CDRs selected from the following, optionally comprising up to 6 amino acid changes, for example 1, 2, 3, 4, 5, or 6 amino acid changes, for each of the CDRs CDR-H1: SEQ ID NO: 100; CDR-H2: SEQ ID NO: 101; CDR-H3: SEQ ID NO: 102; CDR-L1: SEQ ID NO: 103; CDR-L2: SEQ ID NO: 104; and, CDR-L3: SEQ ID NO: 105. In one embodiment, the antibody, or antigen-binding fragment thereof, comprises at least three CDRs selected from the following, optionally comprising up to 6 amino acid changes, for example 1, 2, 3, 4, 5, or 6 amino acid changes, for each of the CDRs CDR-H1: SEQ ID NO: 166; CDR-H2: SEQ ID NO: 167; CDR-H3: SEQ ID NO: 168; CDR-L1: SEQ ID NO: 169; CDR-L2: SEQ ID NO: 170; and, CDR-L3: SEQ ID NO: 171. In one embodiment, the antibody, or antigen-binding fragment thereof, comprises at least three CDRs selected from the following, optionally comprising up to 6 amino acid changes, for example 1, 2, 3, 4, 5, or 6 amino acid changes, for each of the CDRs CDR-H1: SEQ ID NO: 292; CDR-H2: SEQ ID NO: 293; CDR-H3: SEQ ID NO: 294; CDR-L1: SEQ ID NO: 295; CDR-L2: SEQ ID NO: 296; and, CDR-L3: SEQ ID NO: 297.

In one aspect, the invention provides an antibody, or antigen-binding fragment thereof, comprising a heavy chain variable region comprising CDR-H1: SEQ ID NO: 1; CDR-H2: SEQ ID NO: 2; and CDR-H3: SEQ ID NO: 3; and a light chain variable region comprising CDR-L1: SEQ ID NO: 4; CDR-L2: SEQ ID NO: 5; and CDR-L3: SEQ ID NO: 6, optionally comprising one or more amino acid changes, for example 1, 2, 3, 4, 5, or 6 amino acid changes, for each of the CDRs.

In one aspect, the invention provides an antibody, or antigen-binding fragment thereof, comprising a heavy chain variable region comprising CDR-H1: SEQ ID NO: 94; CDR-H2: SEQ ID NO: 95; and CDR-H3: SEQ ID NO: 96; and a light chain variable region comprising CDR-L1: SEQ ID NO: 97; CDR-L2: SEQ ID NO: 98; and CDR-L3: SEQ ID NO: 99, optionally comprising one or more amino acid changes, for example 1, 2, 3, 4, 5, or 6 amino acid changes, for each of the CDRs.

In one aspect, the invention provides an antibody, or antigen-binding fragment thereof, comprising a heavy chain variable region comprising CDR-H1: SEQ ID NO: 100; CDR-H2: SEQ ID NO: 101; and CDR-H3: SEQ ID NO: 102; and a light chain variable region comprising CDR-L1: SEQ ID NO: 103; CDR-L2: SEQ ID NO: 104; and CDR-L3: SEQ ID NO: 105, optionally comprising one or more amino acid changes, for example 1, 2, 3, 4, 5, or 6 amino acid changes, for each of the CDRs.

In one aspect, the invention provides an antibody, or antigen-binding fragment thereof, comprising a heavy chain variable region comprising CDR-H1: SEQ ID NO: 166; CDR-H2: SEQ ID NO: 167; and CDR-H3: SEQ ID NO: 168; and a light chain variable region comprising CDR-L1: SEQ ID NO: 169; CDR-L2: SEQ ID NO: 170; and CDR-L3: SEQ ID NO: 171, optionally comprising one or more amino acid changes, for example 1, 2, 3, 4, 5, or 6 amino acid changes, for each of the CDRs. For instance, if there are changes within the CDRs, there may be up to 1 change per CDR. There may be no more than 2 changes across all 6 CDRs.

In one aspect, the invention provides an antibody, or antigen-binding fragment thereof, comprising a heavy chain variable region comprising CDR-H1: SEQ ID NO: 292; CDR-H2: SEQ ID NO: 293; and CDR-H3: SEQ ID NO: 294; and a light chain variable region comprising CDR-L1: SEQ ID NO: 295; CDR-L2: SEQ ID NO: 296; and CDR-L3: SEQ ID NO: 297, optionally comprising one or more amino acid changes, for example 1, 2, 3, 4, 5, or 6 amino acid changes (e.g., up to 2), for each of the CDRs. For instance, if there are changes within the CDRs, there may be up to 1 change per CDR. There may be no more than 2 changes across all 6 CDRs.

In one aspect, the invention provides an antibody, or antigen-binding fragment thereof comprising a heavy chain variable region comprising a CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and/or CDR-L3 having particular amino acid changes. As used herein, the phrase “amino acid changes” or “changes in amino acid residues” includes amino acid substitutions, additions, and/or deletions. In some embodiments, there are one or more changes to the amino acid residues with any one of the CDRs and/or variable regions described herein. For example, in some embodiments, the one or more amino acid changes comprises one amino acid change. In some embodiments, the one or more amino acid changes comprises up to two amino acid changes. In some embodiments, the one or more amino acid changes comprises up to three amino acid changes. In some embodiments, the one or more amino acid changes comprises up to four amino acid changes. In some embodiments, the one or more amino acid changes comprises up to five amino acid changes. In some embodiments, the one or more amino acid changes comprises up to six amino acid changes. In some embodiments, the one or more amino acid changes comprises up to seven amino acid changes.

For example, in some embodiments, the antibody, or antigen-binding fragment thereof, comprises a CDR-H1: SEQ ID NO:1, with the proviso that the threonine residue at position 4 of SEQ ID NO:1 may be substituted with a histidine, lysine, phenylalanine, or glycine. In some embodiments, the antibody, or antigen-binding fragment thereof, comprises a CDR-H1: SEQ ID NO:1, with the proviso that the serine residue at position 5 of SEQ ID NO:1 may be substituted with a leucine. In some embodiments, the antibody, or antigen-binding fragment thereof, comprises a CDR-H1: SEQ ID NO:1, with the proviso that the serine residue at position 9 of SEQ ID NO:1 may be substituted with an alanine.

In some embodiments, the antibody, or antigen-binding fragment thereof, comprises a CDR-H1: SEQ ID NO:1, with the proviso that (i) the threonine residue at position 4 of SEQ ID NO:1 may be substituted with a histidine, lysine, phenylalanine, or glycine; (ii) the serine residue at position 5 of SEQ ID NO:1 may be substituted with a leucine; and/or, (iii) the serine residue at position 9 of SEQ ID NO:1 may be substituted with an alanine.

In some embodiments, the antibody, or antigen-binding fragment thereof, comprises a CDR-H2: SEQ ID NO:2, with the proviso that the serine residue at position 3 of SEQ ID NO:2 may be substituted with an aspartate or asparagine. In some embodiments, the antibody, or antigen-binding fragment thereof, comprises a CDR-H2: SEQ ID NO:2, with the proviso that the tyrosine residue at position 5 of SEQ ID NO:2 may be substituted with a histidine. In some embodiments, the antibody, or antigen-binding fragment thereof, comprises a CDR-H2: SEQ ID NO:2, with the proviso that the asparagine residue at position 6 of SEQ ID NO:2 may be substituted with a serine. In some embodiments, the antibody, or antigen-binding fragment thereof, comprises a CDR-H2: SEQ ID NO:2, with the proviso that the asparagine residue at position 8 of SEQ ID NO:2 may be substituted with a phenylalanine, leucine, alanine, tyrosine, aspartate, or serine. In some embodiments, the antibody, or antigen-binding fragment thereof, comprises a CDR-H2: SEQ ID NO:2, with the proviso that the asparagine residue at position 10 of SEQ ID NO:2 may be substituted with an aspartate or alanine.

In some embodiments, the antibody, or antigen-binding fragment thereof, comprises a CDR-H2: SEQ ID NO:2, with the proviso that (i) the serine residue at position 3 of SEQ ID NO:2 may be substituted with an aspartate or asparagine; (ii) the tyrosine residue at position 5 of SEQ ID NO:2 may be substituted with a histidine; (iii) the asparagine residue at position 6 of SEQ ID NO:2 may be substituted with a serine; (iv) the asparagine residue at position 8 of SEQ ID NO:2 may be substituted with a phenylalanine, leucine, alanine, tyrosine, aspartate, or serine; and/or, (v) the asparagine residue at position 10 of SEQ ID NO:2 may be substituted with a aspartate or alanine.

In some embodiments, the antibody, or antigen-binding fragment thereof, comprises three heavy chain CDRs and three light chain CDRs, wherein the heavy chain CDRs comprise:

a) CDR-H1: SEQ ID NO:1, with the proviso that:

i. the threonine residue at position 4 of SEQ ID NO:1 may be substituted with a histidine, lysine, phenylalanine, or glycine; ii. the serine residue at position 5 of SEQ ID NO:1 may be substituted with a leucine; and/or, iii. the serine residue at position 9 of SEQ ID NO:1 may be substituted with an alanine;

b) CDR-H2: SEQ ID NO:2, with the proviso that:

i. the serine residue at position 3 of SEQ ID NO:2 may be substituted with an aspartate or asparagine; ii. the tyrosine residue at position 5 of SEQ ID NO:2 may be substituted with a histidine; iii. the asparagine residue at position 6 of SEQ ID NO:2 may be substituted with a serine; iv. the asparagine residue at position 8 of SEQ ID NO:2 may be substituted with a phenylalanine, leucine, alanine, tyrosine, aspartate, or serine; and/or, v. the asparagine residue at position 10 of SEQ ID NO:2 may be substituted with a aspartate or alanine;

c) CDR-H3: SEQ ID NO:3, optionally comprising one or more amino acid changes.

In some embodiments, the antibody, or antigen-binding fragment thereof, further comprises a CDR-L1 as set forth in SEQ ID NO:4, optionally comprising one or more amino acid changes. In some embodiments, the antibody, or antigen-binding fragment thereof, further comprises a CDR-L2 as set forth in SEQ ID NO:5, optionally comprising one or more amino acid changes. In some embodiments, the antibody, or antigen-binding fragment thereof, further comprises a CDR-L3 as set forth in SEQ ID NO:6, optionally comprising one or more amino acid changes.

In some embodiments, the antibody, or antigen-binding fragment thereof, comprises at least three of the following six CDRs:

a) CDR-H1: SEQ ID NO:1, with the proviso that:

i. the threonine residue at position 4 of SEQ ID NO:1 may be substituted with a histidine, lysine, phenylalanine, or glycine; ii. the serine residue at position 5 of SEQ ID NO:1 may be substituted with an leucine; and/or, iii. the serine residue at position 9 of SEQ ID NO:1 may be substituted with an alanine;

b) CDR-H2: SEQ ID NO:2, with the proviso that:

i. the serine residue at position 3 of SEQ ID NO:2 may be substituted with an aspartate or asparagine; ii. the tyrosine residue at position 5 of SEQ ID NO:2 may be substituted with a histidine; iii. the asparagine residue at position 6 of SEQ ID NO:2 may be substituted with a serine; iv. the asparagine residue at position 8 of SEQ ID NO:2 may be substituted with a phenylalanine, leucine, alanine, tyrosine, aspartate, or serine; and/or, v. the asparagine residue at position 10 of SEQ ID NO:2 may be substituted with a aspartate or alanine;

c) CDR-H3: SEQ ID NO:3, optionally comprising one or more amino acid changes; d) CDR-L1: SEQ ID NO:4, optionally comprising one or more amino acid changes; e) CDR-L2: SEQ ID NO:5, optionally comprising one or more amino acid changes; and, f) CDR-L3: SEQ ID NO:6, optionally comprising one or more amino acid changes.

In some embodiments, the antibody, or antigen-binding fragment thereof, specifically binds a human LTBP1-proTGFβ1 complex and/or a human LTBP3-proTGFβ1 complex, and does not bind a human GARP-proTGFβ1 complex. In some embodiments, the antibody, or antigen-binding fragment thereof, does not bind a human GARP-proTGFβ1 complex. In some embodiments, the antibody, or antigen-binding fragment thereof, does not bind mature TGFβ1, mature TGFβ2 or mature TGFβ3.

In a particular embodiment, the invention provides an isolated antibody that specifically binds a human LTBP1-proTGFβ1 complex and/or a human LTBP3-proTGFβ1 complex, and does not bind a human GARP-proTGFβ1 complex or a human LRRC33-proTGFβ1 complex; wherein the antibody does not bind mature TGFβ1, mature TGFβ2 or mature TGFβ33; wherein the antibody is a fully human or humanized antibody or a fragment thereof, and wherein the antibody comprises at least three of the following six CDRs:

a) CDR-H1: SEQ ID NO:1, with the proviso that:

i. the threonine residue at position 4 of SEQ ID NO:1 may be substituted with a histidine, lysine, phenylalanine, or glycine; ii. the serine residue at position 5 of SEQ ID NO:1 may be substituted with an leucine; and/or, iii. the serine residue at position 9 of SEQ ID NO:1 may be substituted with an alanine;

b) CDR-H2: SEQ ID NO:2, with the proviso that:

i. the serine residue at position 3 of SEQ ID NO:2 may be substituted with an aspartate or asparagine; ii. the tyrosine residue at position 5 of SEQ ID NO:2 may be substituted with a histidine; iii. the asparagine residue at position 6 of SEQ ID NO:2 may be substituted with a serine; iv. the asparagine residue at position 8 of SEQ ID NO:2 may be substituted with a phenylalanine, leucine, alanine, tyrosine, aspartate, or serine; and/or, v. the asparagine residue at position 10 of SEQ ID NO:2 may be substituted with a aspartate or alanine;

c) CDR-H3: SEQ ID NO:3, optionally comprising one or more amino acid changes; d) CDR-L1: SEQ ID NO:4, optionally comprising one or more amino acid changes; e) CDR-L2: SEQ ID NO:5, optionally comprising one or more amino acid changes; and, f) CDR-L3: SEQ ID NO:6, optionally comprising one or more amino acid changes.

In some embodiments, such antibody binds a human LTBP1-proTGFβ1 complex with a K D of <100 nM as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI); and/or, such antibody binds a human LTBP3-proTGFβ1 complex with a K D of <100 nM as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI). In some embodiments, such antibody binds a human LTBP1-proTGFβ1 complex with a K D of <50 nM as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI); and/or, such antibody binds a human LTBP3-proTGFβ1 complex with a K D of <50 nM as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI). In some embodiments, such antibody binds a human LTBP1-proTGFβ1 complex with a K D of <25 nM as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI); and/or, such antibody binds a human LTBP3-proTGFβ1 complex with a K D of <25 nM as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI). In some embodiments, such antibody binds a human LTBP1-proTGFβ1 complex with a K D of <10 nM as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI); and/or, such antibody binds a human LTBP3-proTGFβ1 complex with a K D of <10 nM as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI).

In another embodiment, such antibody is cross-reactive with mouse LTBP1-proTGFβ1. In some embodiments, such antibody is also cross-reactive with mouse LTBP3-proTGFβ1. In some embodiments, such antibody binds a mouse LTBP1-proTGFβ1 complex with a K D of <100 nM as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI); and/or, the antibody binds a mouse LTBP3-proTGFβ1 complex with a K D of <100 nM as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI). In some embodiments, such antibody binds a mouse LTBP1-proTGFβ1 complex with a K D of <50 nM as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI); and/or, the antibody binds a mouse LTBP3-proTGFβ1 complex with a K D of <50 nM as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI). In some embodiments, such antibody binds a mouse LTBP1-proTGFβ1 complex with a K D of <25 nM as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI); and/or, the antibody binds a mouse LTBP3-proTGFβ1 complex with a K D of <25 nM as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI). In some embodiments, such antibody binds a mouse LTBP1-proTGFβ1 complex with a K D of <10 nM as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI); and/or, the antibody binds a mouse LTBP3-proTGFβ1 complex with a K D of <10 nM as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI).

In another embodiment, such antibody does not bind to human GARP-proTGFβ1. In preferred embodiments, such context-selective antibody is isoform-specific in that it selectively binds and inhibits the activation of TGFβ1 associated with LTBP1/3 and does not bind to human GARP-proTGFβ1.

In another aspect, the antibody, or antigen-binding fragment thereof, comprises a CDR-H1: SEQ ID NO:94, with the proviso that the threonine residue at position 2 of SEQ ID NO:94 may be substituted with an alanine. In some embodiments, the antibody, or antigen-binding fragment thereof, comprises a CDR-H1: SEQ ID NO:94, with the proviso that the asparagine residue at position 4 of SEQ ID NO:94 may be substituted with an alanine, tyrosine, aspartate, serine, arginine, or histidine. In some embodiments, the antibody, or antigen-binding fragment thereof, comprises a CDR-H1: SEQ ID NO:94, with the proviso that the asparagine residue at position 5 of SEQ ID NO:94 may be substituted with a glutamine, serine, glycine, lysine, glutamate, arginine, or histidine. In some embodiments, the antibody, or antigen-binding fragment thereof, comprises a CDR-H1: SEQ ID NO:94, with the proviso that the tyrosine residue at position 6 of SEQ ID NO:94 may be substituted with a arginine. In some embodiments, the antibody, or antigen-binding fragment thereof, comprises a CDR-H1: SEQ ID NO:94, with the proviso that the proline residue at position 7 of SEQ ID NO:94 may be substituted with a glycine, alanine, leucine, serine, asparagine, valine, aspartate, or glutamine. In some embodiments, the antibody, or antigen-binding fragment thereof, comprises a CDR-H1: SEQ ID NO:94, with the proviso that the isoleucine residue at position 8 of SEQ ID NO:94 may be substituted with a methionine or leucine. In some embodiments, the antibody, or antigen-binding fragment thereof, comprises a CDR-H1: SEQ ID NO:94, with the proviso that the histidine residue at position 9 of SEQ ID NO:94 may be substituted with a phenylalanine, tyrosine, asparagine, or serine. SEQ ID NO: 94 comprising these substitutions is disclosed as SEQ ID NO: 399.

In some embodiments, the antibody, or antigen-binding fragment thereof, comprises a CDR-H1: SEQ ID NO:94, with the proviso that (i) the threonine residue at position 2 of SEQ ID NO:94 may be substituted with an alanine; (ii) the asparagine residue at position 4 of SEQ ID NO:94 may be substituted with an alanine, tyrosine, aspartate, serine, arginine, or histidine; (iii) the asparagine residue at position 5 of SEQ ID NO:94 may be substituted with a glutamine, serine, glycine, lysine, glutamate, arginine, or histidine; (iv) the tyrosine residue at position 6 of SEQ ID NO:94 may be substituted with a arginine; (v) the proline residue at position 7 of SEQ ID NO:94 may be substituted with a glycine, alanine, leucine, serine, asparagine, valine, aspartate, or glutamine; (vi) the isoleucine residue at position 8 of SEQ ID NO:94 may be substituted with a methionine or leucine; and/or, (vii) the histidine residue at position 9 of SEQ ID NO:94 may be substituted with a phenylalanine, tyrosine, asparagine, or serine. SEQ ID NO: 94 comprising these substitutions is disclosed as SEQ ID NO: 399.

In some embodiments, the antibody, or antigen-binding fragment thereof, comprises three heavy chain CDRs and three light chain CDRs, wherein the heavy chain CDRs comprise:

a) CDR-H1: SEQ ID NO:94 (SEQ ID NO: 94 comprising these substitutions is disclosed as SEQ ID NO: 399), with the proviso that:

i. the threonine residue at position 2 of SEQ ID NO:94 may be substituted with an alanine; ii. the asparagine residue at position 4 of SEQ ID NO:94 may be substituted with an alanine, tyrosine, aspartate, serine, arginine, or histidine; iii. the asparagine residue at position 5 of SEQ ID NO:94 may be substituted with a glutamine, serine, glycine, lysine, glutamate, arginine, or histidine; iv. the tyrosine residue at position 6 of SEQ ID NO:94 may be substituted with a arginine; v. the proline residue at position 7 of SEQ ID NO:94 may be substituted with a glycine, alanine, leucine, serine, asparagine, valine, aspartate, or glutamine; vi. the isoleucine residue at position 8 of SEQ ID NO:94 may be substituted with a methionine or leucine; and/or, vii. the histidine residue at position 9 of SEQ ID NO:94 may be substituted with a phenylalanine, tyrosine, asparagine, or serine;

b) CDR-H2: SEQ ID NO:95, optionally comprising one or more amino acid changes; and c) CDR-H3: SEQ ID NO:96, optionally comprising one or more amino acid changes.

In some embodiments, the antibody, or antigen-binding fragment thereof, further comprises a CDR-L1 as set forth in SEQ ID NO:97, optionally comprising one or more amino acid changes. In some embodiments, the antibody, or antigen-binding fragment thereof, further comprises a CDR-L2 as set forth in SEQ ID NO:98, optionally comprising one or more amino acid changes. In some embodiments, the antibody, or antigen-binding fragment thereof, further comprises a CDR-L3 as set forth in SEQ ID NO:99, optionally comprising one or more amino acid changes.

In some embodiments, the antibody, or antigen-binding fragment thereof, comprises at least three of the following six CDRs:

a) CDR-H1: SEQ ID NO:94 (SEQ ID NO: 94 comprising these substitutions is disclosed as SEQ ID NO: 399), with the proviso that:

i. the threonine residue at position 2 of SEQ ID NO:94 may be substituted with an alanine; ii. the asparagine residue at position 4 of SEQ ID NO:94 may be substituted with an alanine, tyrosine, aspartate, serine, arginine, or histidine; iii. the asparagine residue at position 5 of SEQ ID NO:94 may be substituted with a glutamine, serine, glycine, lysine, glutamate, arginine, or histidine; iv. the tyrosine residue at position 6 of SEQ ID NO:94 may be substituted with a arginine; v. the proline residue at position 7 of SEQ ID NO:94 may be substituted with a glycine, alanine, leucine, serine, asparagine, valine, aspartate, or glutamine; vi. the isoleucine residue at position 8 of SEQ ID NO:94 may be substituted with a methionine or leucine; and/or, vii. the histidine residue at position 9 of SEQ ID NO:94 may be substituted with a phenylalanine, tyrosine, asparagine, or serine;

b) CDR-H2: SEQ ID NO:95, optionally comprising one or more amino acid changes; c) CDR-H3: SEQ ID NO:96, optionally comprising one or more amino acid changes; d) CDR-L1: SEQ ID NO:97, optionally comprising one or more amino acid changes; e) CDR-L2: SEQ ID NO:98, optionally comprising one or more amino acid changes; and, f) CDR-L3: SEQ ID NO:99, optionally comprising one or more amino acid changes.

In some embodiments, the antibody, or antigen-binding fragment thereof, specifically binds a human LTBP1-proTGFβ1 complex and/or a human LTBP3-proTGFβ1 complex, and does not bind a human GARP-proTGFβ1 complex. In some embodiments, the antibody, or antigen-binding fragment thereof, does not bind a human GARP-proTGFβ1 complex. In some embodiments, the antibody, or antigen-binding fragment thereof, does not bind mature TGFβ1, mature TGFβ2 or mature TGFβ3.

In a particular embodiment, the antibody, or antigen-binding fragment thereof, specifically binds a human LTBP1-proTGFβ1 complex and/or a human LTBP3-proTGFβ1 complex, and does not bind a human GARP-proTGFβ1 complex; wherein the antibody does not bind mature TGFβ1, mature TGFβ2 or mature TGFβ33; wherein the antibody is a fully human or humanized antibody or a fragment thereof, and wherein the antibody comprises at least three of the following six CDRs:

a) CDR-H1: SEQ ID NO:94 (SEQ ID NO: 94 comprising these substitutions is disclosed as SEQ ID NO: 399), with the proviso that:

i. the threonine residue at position 2 of SEQ ID NO:94 may be substituted with an alanine; ii. the asparagine residue at position 4 of SEQ ID NO:94 may be substituted with an alanine, tyrosine, aspartate, serine, arginine, or histidine; iii. the asparagine residue at position 5 of SEQ ID NO:94 may be substituted with a glutamine, serine, glycine, lysine, glutamate, arginine, or histidine; iv. the tyrosine residue at position 6 of SEQ ID NO:94 may be substituted with a arginine; v. the proline residue at position 7 of SEQ ID NO:94 may be substituted with a glycine, alanine, leucine, serine, asparagine, valine, aspartate, or glutamine; vi. the isoleucine residue at position 8 of SEQ ID NO:94 may be substituted with a methionine or leucine; and/or, vii. the histidine residue at position 9 of SEQ ID NO:94 may be substituted with a phenylalanine, tyrosine, asparagine, or serine;

b) CDR-H2: SEQ ID NO:95, optionally comprising one or more amino acid changes; c) CDR-H3: SEQ ID NO:96, optionally comprising one or more amino acid changes; d) CDR-L1: SEQ ID NO:97, optionally comprising one or more amino acid changes; e) CDR-L2: SEQ ID NO:98, optionally comprising one or more amino acid changes; and, f) CDR-L3: SEQ ID NO:99, optionally comprising one or more amino acid changes.

In some embodiments, such antibody binds a human LTBP1-proTGFβ1 complex with a K D of <100 nM as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI); and/or, such antibody binds a human LTBP3-proTGFβ1 complex with a K D of <100 nM as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI). In some embodiments, such antibody binds a human LTBP1-proTGFβ1 complex with a K D of <50 nM as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI); and/or, such antibody binds a human LTBP3-proTGFβ1 complex with a K D of <50 nM as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI). In some embodiments, such antibody binds a human LTBP1-proTGFβ1 complex with a K D of <25 nM as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI); and/or, such antibody binds a human LTBP3-proTGFβ1 complex with a K D of <25 nM as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI). In some embodiments, such antibody binds a human LTBP1-proTGFβ1 complex with a K D of <10 nM as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI); and/or, such antibody binds a human LTBP3-proTGFβ1 complex with a K D of <10 nM as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI).

In another embodiment, such antibody is cross-reactive with mouse LTBP1-proTGFβ1. In some embodiments, such antibody is also cross-reactive with mouse LTBP3-proTGFβ1. In some embodiments, such antibody binds a mouse LTBP1-proTGFβ1 complex with a K D of <100 nM as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI); and/or, the antibody binds a mouse LTBP3-proTGFβ1 complex with a K D of <100 nM as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI). In some embodiments, such antibody binds a mouse LTBP1-proTGFβ1 complex with a K D of <50 nM as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI); and/or, the antibody binds a mouse LTBP3-proTGFβ1 complex with a K D of <50 nM as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI). In some embodiments, such antibody binds a mouse LTBP1-proTGFβ1 complex with a K D of <25 nM as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI); and/or, the antibody binds a mouse LTBP3-proTGFβ1 complex with a K D of <25 nM as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI). In some embodiments, such antibody binds a mouse LTBP1-proTGFβ1 complex with a K D of <10 nM as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI); and/or, the antibody binds a mouse LTBP3-proTGFβ1 complex with a K D of <10 nM as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI).

In another embodiment, such antibody does not bind to human GARP-proTGFβ1. In preferred embodiments, such context-selective antibody is also isoform-specific in that it selectively binds and inhibits the activation of TGFβ1 associated with LTBP1/3.

In another aspect, the antibody, or antigen-binding fragment thereof, comprises a CDR-H1 comprising the amino acid sequence FTF(X 1 )(X 2 )YVMH, wherein, optionally: X 1 is S or R; and X 2 is G or S (SEQ ID NO: 392). In some embodiments, X 1 is S. In some embodiments, X 1 is R. In some embodiments, X 2 is G. In some embodiments, X 2 is S.

In some embodiments, the antibody, or antigen-binding fragment thereof, comprises a CDR-H2 comprising the amino acid sequence (X 1 )ISHEG(X 2 )(X 3 )KYYADSVKG, wherein, optionally: X 1 is V or S; X 2 is S or G; and X 3 is F or L (SEQ ID NO: 393). In some embodiments, X 1 is a V. In some embodiment, X 1 is a S. In some embodiments, X 2 is S. In some embodiments, X 2 is G. In some embodiments, X 3 is F. In some embodiments, X 3 is L.

In some embodiments, the antibody, or antigen-binding fragment thereof, comprises a CDR-H3 comprising the amino acid sequence (X 1 )(X 2 )P(X 3 )(X 4 )(X 5 )(X 6 )RRGG(X 7 )(X 8 )(X 9 ), wherein, optionally: X 1 is A or V; X 2 is R, V, G or K; X 3 is R, H or L; X 4 is I, V or G; X 5 is A, S, or L; X 6 is A or V; X 7 is F or Y; X 8 is D, G, R, or S; and, X 9 is Y, G, R, L, V, A or K (SEQ ID NO: 394). In some embodiments, X 1 is A. In some embodiments, X 1 is V. In some embodiments, X 2 is R. In some embodiments, X 2 is V. In some embodiments, X 2 is G. In some embodiments, X 2 is K. In some embodiments, X 3 is R. In some embodiments, X 3 is H. In some embodiments, X 3 is L. In some embodiments, X 4 is I. In some embodiments, X 4 is V. In some embodiments, X 4 is G. In some embodiments, X 5 is A. In some embodiments, X 5 is S. In some embodiments, X 5 is L. In some embodiments, X 6 is A. In some embodiments, X 6 is V. In some embodiments, X 7 is F. In some embodiments, X 7 is Y. In some embodiments, X 8 is D. In some embodiments, X 8 is G. In some embodiments, X 8 is R. In some embodiments, X 8 is S. In some embodiments, X 9 is Y. In some embodiments, X 9 is G. In some embodiments, X 9 is R. In some embodiments, X 9 is L. In some embodiments, X 9 is V. In some embodiments, X 9 is A. In some embodiments, X 9 is K.

In some embodiments, the antibody, or antigen-binding fragment thereof, comprises three heavy chain CDRs and three light chain CDRs, wherein the heavy chain CDRs comprise:

a) CDR-H1 comprising the amino acid sequence FTF(X 1 )(X 2 )YVMH, wherein, optionally: X 1 is S or R; and X 2 is G or S (SEQ ID NO: 392); b) CDR-H2 comprising the amino acid sequence (X 1 )ISHEG(X 2 )(X 3 )KYYADSVKG, wherein, optionally: X 1 is V or S; X 2 is S or G; and X 3 is F or L (SEQ ID NO: 393); and c) CDR-H3 comprising the amino acid sequence (X 1 )(X 2 )P(X 3 )(X 4 )(X 5 )(X 6 )RRGG(X 7 ) (X 8 )(X 9 ), wherein, optionally: X 1 is A or V; X 2 is R, V, G or K; X 3 is R, H or L; X 4 is I, V or G; X 5 is A, S, or L; X 6 is A or V; X 7 is F or Y; X 8 is D, G, R, or S; and, X 9 is Y, G, R, L, V, A or K (SEQ ID NO: 394).

In some embodiments, the antibody, or antigen-binding fragment thereof, further comprises a CDR-L1 as set forth in SEQ ID NO:97, optionally comprising one or more amino acid changes. In some embodiments, the antibody, or antigen-binding fragment thereof, further comprises a CDR-L2 as set forth in SEQ ID NO:98, optionally comprising one or more amino acid changes. In some embodiments, the antibody, or antigen-binding fragment thereof, further comprises a CDR-L3 as set forth in SEQ ID NO:99, optionally comprising one or more amino acid changes.

In some embodiments, the antibody, or antigen-binding fragment, comprises at least three of the following six CDRs:

a) CDR-H1 comprising the amino acid sequence FTF(X 1 )(X 2 )YVMH, wherein, optionally: X 1 is S or R; and X 2 is G or S (SEQ ID NO: 392); b) CDR-H2 comprising the amino acid sequence (X 1 )ISHEG(X 2 )(X 3 )KYYADSVKG, wherein, optionally: X 1 is V or S; X 2 is S or G; and X 3 is F or L (SEQ ID NO: 393); c) CDR-H3 comprising the amino acid sequence (X 1 )(X 2 )P(X 3 )(X 4 )(X 5 )(X 6 )RRGG(X 7 ) (X 8 )(X 9 ), wherein, optionally: X 1 is A or V; X 2 is R, V, G or K; X 3 is R, H or L; X 4 is I, V or G; X 5 is A, S, or L; X 6 is A or V; X 7 is F or Y; X 8 is D, G, R, or S; and, X 9 is Y, G, R, L, V, A or K (SEQ ID NO: 394); d) CDR-L1 as set forth in SEQ ID NO:97, optionally comprising one or more amino acid changes; e) CDR-L2 as set forth in SEQ ID NO:98, optionally comprising one or more amino acid changes; and f) CDR-L3 as set forth in SEQ ID NO:99, optionally comprising one or more amino acid changes.

In some embodiments, the antibody, or antigen-binding fragment thereof, specifically binds a human LTBP1-proTGFβ1 complex and/or a human LTBP3-proTGFβ1 complex, and does not bind a human GARP-proTGFβ1 complex. In some embodiments, the antibody, or antigen-binding fragment thereof, does not bind a human GARP-proTGFβ1 complex. In some embodiments, the antibody, or antigen-binding fragment thereof, does not bind mature TGFβ1, mature TGFβ2 or mature TGFβ3.

In a particular embodiment, the antibody, or antigen-binding fragment, specifically binds a human LTBP1-proTGFβ1 complex and/or a human LTBP3-proTGFβ1 complex, and does not bind a human GARP-proTGFβ1 complex; wherein the antibody does not bind mature TGFβ1, mature TGFβ2 or mature TGFβ33; wherein the antibody is a fully human or humanized antibody or a fragment thereof, wherein the antibody comprises at least three of the following six CDRs:

a) CDR-H1 comprising the amino acid sequence FTF(X 1 )(X 2 )YVMH, wherein, optionally: X 1 is S or R; and X 2 is G or S (SEQ ID NO: 392); b) CDR-H2 comprising the amino acid sequence (X 1 )ISHEG(X 2 )(X 3 )KYYADSVKG, wherein, optionally: X 1 is V or S; X 2 is S or G; and X 3 is F or L (SEQ ID NO: 393); c) CDR-H3 comprising the amino acid sequence (X 1 )(X 2 )P(X 3 )(X 4 )(X 5 )(X 6 )RRGG(X 7 ) (X 8 )(X 9 ), wherein, optionally: X 1 is A or V; X 2 is R, V, G or K; X 3 is R, H or L; X 4 is I, V or G; X 5 is A, S, or L; X 6 is A or V; X 7 is F or Y; X 8 is D, G, R, or S; and, X 9 is Y, G, R, L, V, A or K (SEQ ID NO: 394); d) CDR-L1 as set forth in SEQ ID NO:97, optionally comprising one or more amino acid changes; e) CDR-L2 as set forth in SEQ ID NO:98, optionally comprising one or more amino acid changes; and f) CDR-L3 as set forth in SEQ ID NO:99, optionally comprising one or more amino acid changes.

In some embodiments, such antibody binds a human LTBP1-proTGFβ1 complex with a K D of <100 nM as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI); and/or, such antibody binds a human LTBP3-proTGFβ1 complex with a K D of <100 nM as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI). In some embodiments, such antibody binds a human LTBP1-proTGFβ1 complex with a K D of <50 nM as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI); and/or, such antibody binds a human LTBP3-proTGFβ1 complex with a K D of <50 nM as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI). In some embodiments, such antibody binds a human LTBP1-proTGFβ1 complex with a K D of <25 nM as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI); and/or, such antibody binds a human LTBP3-proTGFβ1 complex with a K D of <25 nM as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI). In some embodiments, such antibody binds a human LTBP1-proTGFβ1 complex with a K D of <10 nM as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI); and/or, such antibody binds a human LTBP3-proTGFβ1 complex with a K D of <10 nM as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI).

In another embodiment, such antibody is cross-reactive with mouse LTBP1-proTGFβ1. In some embodiments, such antibody is also cross-reactive with mouse LTBP3-proTGFβ1. In some embodiments, such antibody binds a mouse LTBP1-proTGFβ1 complex with a K D of <100 nM as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI); and/or, the antibody binds a mouse LTBP3-proTGFβ1 complex with a K D of <100 nM as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI). In some embodiments, such antibody binds a mouse LTBP1-proTGFβ1 complex with a K D of <50 nM as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI); and/or, the antibody binds a mouse LTBP3-proTGFβ1 complex with a K D of <50 nM as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI). In some embodiments, such antibody binds a mouse LTBP1-proTGFβ1 complex with a K D of <25 nM as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI); and/or, the antibody binds a mouse LTBP3-proTGFβ1 complex with a K D of <25 nM as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI). In some embodiments, such antibody binds a mouse LTBP1-proTGFβ1 complex with a K D of <10 nM as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI); and/or, the antibody binds a mouse LTBP3-proTGFβ1 complex with a K D of <10 nM as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI).

In another embodiment, such antibody does not bind to human GARP-proTGFβ1. In preferred embodiments, such context-selective antibody is also isoform-specific in that it selectively binds and inhibits the activation of TGFβ1 associated with LTBP1/3.

In another aspect, the antibody, or antigen-binding fragment thereof, comprises a CDR-H1 comprising the amino acid sequence FTF(X 1 )(X 2 )YVMH, wherein, optionally: X 1 is S or R; and X 2 is G or S (SEQ ID NO: 392). In some embodiments, X 1 is S. In some embodiments, X 1 is R. In some embodiments, X 2 is G. In some embodiments, X 2 is S.

In some embodiments, the antibody, or antigen-binding fragment thereof, comprises a CDR-H2 comprising the amino acid sequence (X 1 )ISHEGS(X 2 )KYYADSVKG, wherein, optionally: X 1 is V or S; and, X 2 is F or L (SEQ ID NO: 382). In some embodiments, X 1 is a V. In some embodiment, X 1 is a S. In some embodiments, X 3 is F. In some embodiments, X 3 is L.

In some embodiments, the antibody, or antigen-binding fragment thereof, comprises a CDR-H3 comprising the amino acid sequence A(X 1 )PRI(X 2 )ARRGGFGY, wherein, optionally: X 1 is R or V; X 2 is A or L (SEQ ID NO: 383). In some embodiments, X 1 is R. In some embodiments, X 1 is V. In some embodiments, X 2 is A. In some embodiments, X 2 is L.

In some embodiments, the antibody, or antigen-binding fragment thereof, comprises three heavy chain CDRs and three light chain CDRs, wherein the heavy chain CDRs comprise:

a) CDR-H1 comprising the amino acid sequence FTF(X 1 )(X 2 )YVMH, wherein, optionally: X 1 is S or R; and X 2 is G or S (SEQ ID NO: 392); b) CDR-H2 comprising the amino acid sequence (X 1 )ISHEGS(X 2 )KYYADSVKG, wherein, optionally: X 1 is V or S; and, X 2 is F or L (SEQ ID NO: 382); and c) CDR-H3 comprising the amino acid sequence A(X 1 )PRI(X 2 )ARRGGFGY, wherein, optionally: X 1 is R or V; X 2 is A or L (SEQ ID NO: 383).

In some embodiments, the antibody, or antigen-binding fragment thereof, further comprises a CDR-L1 as set forth in SEQ ID NO:97, optionally comprising one or more amino acid changes. In some embodiment, the antibody, or antigen-binding fragment thereof, further comprises a CDR-L2 as set forth in SEQ ID NO:98, optionally comprising one or more amino acid changes. In some embodiments, the antibody, or antigen-binding fragment thereof, further comprises a CDR-L3 as set forth in SEQ ID NO:99, optionally comprising one or more amino acid changes.

In some embodiments, the antibody, or antigen-binding fragment, comprises at least three of the following six CDRs:

a) CDR-H1 comprising the amino acid sequence FTF(X 1 )(X 2 )YVMH, wherein, optionally: X 1 is S or R; and X 2 is G or S (SEQ ID NO: 392); b) CDR-H2 comprising the amino acid sequence (X 1 )ISHEGS(X 2 )KYYADSVKG, wherein, optionally: X 1 is V or S; and, X 2 is F or L (SEQ ID NO: 382); c) CDR- comprising the amino acid sequence A(X 1 )PRI(X 2 )ARRGGFGY, wherein, optionally: X 1 is R or V; X 2 is A or L (SEQ ID NO: 383); d) CDR-L1 as set forth in SEQ ID NO:97, optionally comprising one or more amino acid changes; e) CDR-L2 as set forth in SEQ ID NO:98, optionally comprising one or more amino acid changes; and f) CDR-L3 as set forth in SEQ ID NO:99, optionally comprising one or more amino acid changes.

In some embodiments, the antibody, or antigen-binding fragment thereof, specifically binds a human LTBP1-proTGFβ1 complex and/or a human LTBP3-proTGFβ1 complex, and does not bind a human GARP-proTGFβ1 complex. In some embodiments, the antibody, or antigen-binding fragment thereof, does not bind a human GARP-proTGFβ1 complex. In some embodiments, the antibody, or antigen-binding fragment thereof, does not bind mature TGFβ1, mature TGFβ2 or mature TGFβ3.

In a particular embodiment, the antibody, or antigen-binding fragment thereof, specifically binds a human LTBP1-proTGFβ1 complex and/or a human LTBP3-proTGFβ1 complex, and does not bind a human GARP-proTGFβ1 complex; wherein the antibody does not bind mature TGFβ1, mature TGFβ2 or mature TGFβ33; wherein the antibody is a fully human or humanized antibody or a fragment thereof, wherein the antibody comprises at least three of the following six CDRs:

a) CDR-H1 comprising the amino acid sequence FTF(X 1 )(X 2 )YVMH, wherein, optionally: X 1 is S or R; and X 2 is G or S (SEQ ID NO: 392); b) CDR-H2 comprising the amino acid sequence (X 1 )ISHEGS(X 2 )KYYADSVKG, wherein: X 1 is V or S; and, X 2 is F or L (SEQ ID NO: 382); c) CDR-H3 comprising the amino acid sequence A(X 1 )PRI(X 2 )ARRGGFGY, wherein, optionally: X 1 is R or V; X 2 is A or L (SEQ ID NO: 383); d) CDR-L1 as set forth in SEQ ID NO:97, optionally comprising one or more amino acid changes; e) CDR-L2 as set forth in SEQ ID NO:98, optionally comprising one or more amino acid changes; and f) CDR-L3 as set forth in SEQ ID NO:99, optionally comprising one or more amino acid changes.

In some embodiments, such antibody binds a human LTBP1-proTGFβ1 complex with a K D of <100 nM as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI); and/or, such antibody binds a human LTBP3-proTGFβ1 complex with a K D of <100 nM as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI). In some embodiments, such antibody binds a human LTBP1-proTGFβ1 complex with a K D of <50 nM as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI); and/or, such antibody binds a human LTBP3-proTGFβ1 complex with a K D of <50 nM as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI). In some embodiments, such antibody binds a human LTBP1-proTGFβ1 complex with a K D of <25 nM as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI); and/or, such antibody binds a human LTBP3-proTGFβ1 complex with a K D of <25 nM as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI). In some embodiments, such antibody binds a human LTBP1-proTGFβ1 complex with a K D of <10 nM as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI); and/or, such antibody binds a human LTBP3-proTGFβ1 complex with a K D of <10 nM as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI).

In another embodiment, such antibody is cross-reactive with mouse LTBP1-proTGFβ1. In some embodiments, such antibody is also cross-reactive with mouse LTBP3-proTGFβ1. In some embodiments, such antibody binds a mouse LTBP1-proTGFβ1 complex with a K D of <100 nM as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI); and/or, the antibody binds a mouse LTBP3-proTGFβ1 complex with a K D of <100 nM as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI). In some embodiments, such antibody binds a mouse LTBP1-proTGFβ1 complex with a K D of <50 nM as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI); and/or, the antibody binds a mouse LTBP3-proTGFβ1 complex with a K D of <50 nM as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI). In some embodiments, such antibody binds a mouse LTBP1-proTGFβ1 complex with a K D of <25 nM as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI); and/or, the antibody binds a mouse LTBP3-proTGFβ1 complex with a K D of <25 nM as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI). In some embodiments, such antibody binds a mouse LTBP1-proTGFβ1 complex with a K D of <10 nM as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI); and/or, the antibody binds a mouse LTBP3-proTGFβ1 complex with a K D of <10 nM as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI).

In another embodiment, such antibody does not bind to human GARP-proTGFβ1. In preferred embodiments, such context-selective antibody is also isoform-specific in that it selectively binds and inhibits the activation of TGFβ1 associated with LTBP1/3.

In another aspect, the antibody, or antigen-binding fragment thereof, comprises a CDR-H1: SEQ ID NO:100, with the proviso that the serine residue at position 4 of SEQ ID NO:100 may be substituted with a histidine. In some embodiments, the antibody, or antigen-binding fragment thereof, comprises a CDR-H1: SEQ ID NO:100, with the proviso that the serine residue at position 7 of SEQ ID NO:100 may be substituted with an alanine or glycine. In some embodiments, the antibody, or antigen-binding fragment thereof, comprises a CDR-H1: SEQ ID NO:100, with the proviso that the glycine residue at position 11 of SEQ ID NO:100 may be substituted with a threonine, serine, histidine, leucine, isoleucine, asparagine, valine, or alanine. SEQ ID NO: 100 comprising these substitutions is disclosed as SEQ ID NO: 400.

In some embodiments, the antibody, or antigen-binding fragment thereof, comprises a CDR-H1: SEQ ID NO:100, with the proviso that (i) the serine residue at position 4 of SEQ ID NO:100 may be substituted with a histidine; (ii) the serine residue at position 7 of SEQ ID NO:100 may be substituted with an alanine or glycine; and/or, (iii) the glycine residue at position 11 of SEQ ID NO:100 may be substituted with a threonine, serine, histidine, leucine, isoleucine, asparagine, valine, or alanine. SEQ ID NO: 100 comprising these substitutions is disclosed as SEQ ID NO: 400.

In some embodiments, the antibody, or antigen-binding fragment thereof, comprises a CDR-H1: SEQ ID NO:101, with the proviso that the serine residue at position 3 of SEQ ID NO:101 may be substituted with an alanine. In some embodiments, the antibody, or antigen-binding fragment thereof, comprises a CDR-H1: SEQ ID NO:101, with the proviso that the glycine residue at position 6 of SEQ ID NO:101 may be substituted with an alanine or serine. In some embodiments, the antibody, or antigen-binding fragment thereof, comprises a CDR-H1: SEQ ID NO:101, with the proviso that the serine residue at position 7 of SEQ ID NO:101 may be substituted with a threonine. SEQ ID NO: 101 comprising these substitutions is disclosed as SEQ ID NO: 401.

In some embodiments, the antibody, or antigen-binding fragment thereof, comprises a CDR-H1: SEQ ID NO:101, with the proviso that (i) the serine residue at position 3 of SEQ ID NO:101 may be substituted with an alanine; (ii) the glycine residue at position 6 of SEQ ID NO:101 may be substituted with an alanine or serine; and/or, (iii) the serine residue at position 7 of SEQ ID NO:101 may be substituted with a threonine. SEQ ID NO: 101 comprising these substitutions is disclosed as SEQ ID NO: 401.

In some embodiments, the antibody, or antigen-binding fragment thereof, comprises three heavy chain CDRs and three light chain CDRs, wherein the heavy chain CDRs comprise:

a) CDR-H1: SEQ ID NO:100 (SEQ ID NO: 100 comprising these substitutions is disclosed as SEQ ID NO: 400), with the proviso that:

i. the serine residue at position 4 of SEQ ID NO:100 may be substituted with a histidine; ii. the serine residue at position 7 of SEQ ID NO:100 may be substituted with an alanine or glycine; and/or, iii. the glycine residue at position 11 of SEQ ID NO:100 may be substituted with a threonine, serine, histidine, leucine, isoleucine, asparagine, valine, or alanine;

b) CDR-H2: SEQ ID NO:101 (SEQ ID NO: 101 comprising these substitutions is disclosed as SEQ ID NO: 401), with the proviso that:

i. the serine residue at position 3 of SEQ ID NO:101 may be substituted with an alanine; ii. the glycine residue at position 6 of SEQ ID NO:101 may be substituted with an alanine or serine; and/or, iii. the serine residue at position 7 of SEQ ID NO:101 may be substituted with a threonine; and

c) CDR-H3: SEQ ID NO:102, optionally comprising up to three, four, five or six amino acid changes.

In some embodiments, the antibody, or antigen-binding fragment thereof, further comprises a CDR-L1 as set forth in SEQ ID NO:103, optionally comprising one or more amino acid changes. In some embodiments, the antibody, or antigen-binding fragment thereof, further comprises a CDR-L2 as set forth in SEQ ID NO:104, optionally comprising one or more amino acid changes. In some embodiments, the antibody, or antigen-binding fragment thereof, further comprises a CDR-L3 as set forth in SEQ ID NO:105, optionally comprising one or more amino acid changes.

In some embodiments, the antibody, or antigen-binding fragment thereof, comprises at least three of the following six CDRs:

a) CDR-H1: SEQ ID NO:100 (SEQ ID NO: 100 comprising these substitutions is disclosed as SEQ ID NO: 400), with the proviso that:

i. the serine residue at position 4 of SEQ ID NO:100 may be substituted with a histidine; ii. the serine residue at position 7 of SEQ ID NO:100 may be substituted with an alanine or glycine; and/or, iii. the glycine residue at position 11 of SEQ ID NO:100 may be substituted with a threonine, serine, histidine, leucine, isoleucine, asparagine, valine, or alanine;

b) CDR-H2: SEQ ID NO:101 (SEQ ID NO: 101 comprising these substitutions is disclosed as SEQ ID NO: 401), with the proviso that:

i. the serine residue at position 3 of SEQ ID NO:101 may be substituted with an alanine; ii. the glycine residue at position 6 of SEQ ID NO:101 may be substituted with an alanine or serine; and/or, iii. the serine residue at position 7 of SEQ ID NO:101 may be substituted with a threonine; and

c) CDR-H3: SEQ ID NO:102, optionally comprising one or more amino acid changes. d) CDR-L1: SEQ ID NO:103, optionally comprising one or more amino acid changes; e) CDR-L2: SEQ ID NO:104, optionally comprising one or more amino acid changes; and, f) CDR-L3: SEQ ID NO:105, optionally comprising one or more amino acid changes.

In some embodiments, the antibody, or antigen-binding fragment, specifically binds a human LTBP1-proTGFβ1 complex and/or a human LTBP3-proTGFβ1 complex, and does not bind a human GARP-proTGFβ1 complex. In some embodiments, the antibody, or antigen-binding fragment thereof, does not bind a human GARP-proTGFβ1 complex. In some embodiments, the antibody, or antigen-binding fragment thereof, does not bind mature TGFβ1, mature TGFβ2 or mature TGFβ3.

In a particular embodiment, the antibody, or antigen-binding fragment, specifically binds a human LTBP1-proTGFβ1 complex and/or a human LTBP3-proTGFβ1 complex, and does not bind a human GARP-proTGFβ1 complex; wherein the antibody does not bind mature TGFβ1, mature TGFβ2 or mature TGFβ33; wherein the antibody is a fully human or humanized antibody or a fragment thereof, wherein the antibody comprises at least three of the following six CDRs:

a) CDR-H1: SEQ ID NO:100 (SEQ ID NO: 100 comprising these substitutions is disclosed as SEQ ID NO: 400), with the proviso that:

i. the serine residue at position 4 of SEQ ID NO:100 may be substituted with a histidine; ii. the serine residue at position 7 of SEQ ID NO:100 may be substituted with an alanine or glycine; and/or, iii. the glycine residue at position 11 of SEQ ID NO:100 may be substituted with a threonine, serine, histidine, leucine, isoleucine, asparagine, valine, or alanine;

b) CDR-H2: SEQ ID NO:101 (SEQ ID NO: 101 comprising these substitutions is disclosed as SEQ ID NO: 401), with the proviso that:

i. the serine residue at position 3 of SEQ ID NO:101 may be substituted with an alanine; ii. the glycine residue at position 6 of SEQ ID NO:101 may be substituted with an alanine or serine; and/or, iii. the serine residue at position 7 of SEQ ID NO:101 may be substituted with a threonine;

c) CDR-H3: SEQ ID NO:102, optionally comprising one or more amino acid changes; d) CDR-L1: SEQ ID NO:103, optionally comprising one or more amino acid changes; e) CDR-L2: SEQ ID NO:104, optionally comprising one or more amino acid changes; and, f) CDR-L3: SEQ ID NO:105, optionally comprising one or more amino acid changes.

In some embodiments, such antibody binds a human LTBP1-proTGFβ1 complex with a KD of <100 nM as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI); and/or, such antibody binds a human LTBP3-proTGFβ1 complex with a KD of <100 nM as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI). In some embodiments, such antibody binds a human LTBP1-proTGFβ1 complex with a KD of <50 nM as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI); and/or, such antibody binds a human LTBP3-proTGFβ1 complex with a KD of <50 nM as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI). In some embodiments, such antibody binds a human LTBP1-proTGFβ1 complex with a KD of <25 nM as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI); and/or, such antibody binds a human LTBP3-proTGFβ1 complex with a KD of <25 nM as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI). In some embodiments, such antibody binds a human LTBP1-proTGFβ1 complex with a KD of <10 nM as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI); and/or, such antibody binds a human LTBP3-proTGFβ1 complex with a KD of <10 nM as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI).

In another embodiment, such antibody is cross-reactive with mouse LTBP1-proTGFβ1. In some embodiments, such antibody is also cross-reactive with mouse LTBP3-proTGFβ1. In some embodiments, such antibody binds a mouse LTBP1-proTGFβ1 complex with a KD of <100 nM as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI); and/or, the antibody binds a mouse LTBP3-proTGFβ1 complex with a KD of <100 nM as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI). In some embodiments, such antibody binds a mouse LTBP1-proTGFβ1 complex with a KD of <50 nM as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI); and/or, the antibody binds a mouse LTBP3-proTGFβ1 complex with a KD of <50 nM as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI). In some embodiments, such antibody binds a mouse LTBP1-proTGFβ1 complex with a KD of <25 nM as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI); and/or, the antibody binds a mouse LTBP3-proTGFβ1 complex with a KD of <25 nM as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI). In some embodiments, such antibody binds a mouse LTBP1-proTGFβ1 complex with a KD of <10 nM as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI); and/or, the antibody binds a mouse LTBP3-proTGFβ1 complex with a KD of <10 nM as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI).

In another embodiment, such antibody does not bind to human GARP-proTGFβ1. In preferred embodiments, such context-selective antibody is also isoform-specific in that it selectively binds and inhibits the activation of TGFβ1 associated with LTBP1/3.

In another aspect, the antibody, or antigen-binding fragment thereof, comprises a CDR-H1 comprising the amino acid sequence G(X 1 )I(X 2 )S(X 3 )SYYW(X 4 ), wherein, optionally: X 1 is S or P; X 2 is S, H or R; X 3 is S or G; and, X 4 is G, I, N or V (SEQ ID NO: 395). In some embodiments, X 1 is a S. In some embodiments, X 1 is P. In some embodiments, X 2 is S. In some embodiments, X 2 is H. In some embodiments, X 2 is R. In some embodiments, X 3 is S. In some embodiments, X 3 is G. In some embodiments, X 4 is G. In some embodiments, X 4 is I. In some embodiments, X 4 is N. In some embodiments, X 4 is V.

In some embodiments, the antibody, or antigen-binding fragment thereof, comprises a CDR-H2 comprising the amino acid sequence SISYSA(X 1 )TYYNPSLKS, wherein, optionally, X 1 is S or T (SEQ ID NO: 396). In some embodiments, X 1 is a S. In some embodiment, X 1 is a T.

In some embodiments, the antibody, or antigen-binding fragment thereof, comprises a CDR-H3 comprising the amino acid sequence (X 1 )(X 2 )D(X 3 )(X 4 )Y(X 5 )(X 6 )(X 7 )(X 8 )G(X 9 )(X 10 )(X 11 ), wherein, optionally: X 1 is A or V; X 2 is R, S or G, X 3 is P, Y, R, V, I, H, T or E; X 4 is S, D, E or N; X 5 is D, A or T; X 6 is S, G, T or A; X 7 is I, A, R, Q, or V; X 8 is A, E, K, G or T; X 9 is M or I; X 10 is D, L, Q, V, N or G; and, X 11 is V, R, N, E or K (SEQ ID NO: 397). In some embodiments, X 1 is A. In some embodiments, X 1 is V. In some embodiments, X 2 is R. In some embodiments, X 2 is S. In some embodiments, X 2 is G. In some embodiments, X 3 is P. In some embodiments, X 3 is Y. In some embodiments, X 3 is R. In some embodiments, X 3 is V. In some embodiments, X 3 is I. In some embodiments, X 3 is H. In some embodiments, X 3 is T. In some embodiments, X 3 is E. In some embodiments, X 4 is S. In some embodiments, X 4 is D. In some embodiments, X 4 is E. In some embodiments, X 4 is N. In some embodiments, X 5 is D. In some embodiments, X 5 is A. In some embodiments, X 5 is T. In some embodiments, X 6 is S. In some embodiments, X 6 is G. In some embodiments, X 6 is T. In some embodiments, X 6 is A. In some embodiments, X 7 is I. In some embodiments, X 7 is A. In some embodiments, X 7 is R. In some embodiments, X 7 is Q. In some embodiments, X 7 is V. In some embodiments, X 8 is A. In some embodiments, X 8 is E. In some embodiments, X 8 is K. In some embodiments, X 8 is G. In some embodiments, X 8 is T. In some embodiments, X 9 is M. In some embodiments, X 9 is I. In some embodiments, X 10 is D. In some embodiments, X 10 is L. In some embodiments, X 10 is Q. In some embodiments, X 10 is V. In some embodiments, X 10 is N. In some embodiments, X 10 is G. In some embodiments, X 11 is V. In some embodiments, X 11 is R. In some embodiments, X 11 is N. In some embodiments, X 11 is E. In some embodiments, X 11 is K.

In some embodiments, the antibody, or antigen-binding fragment thereof, comprises three heavy chain CDRs and three light chain CDRs, wherein the heavy chain CDRs comprise:

a) CDR-H1 comprising the amino acid sequence G(X 1 )I(X 2 )S(X 3 )SYYW(X 4 ), wherein, optionally: X 1 is S or P; X 2 is S, H or R; X 3 is S or G; and, X 4 is G, I, N or V (SEQ ID NO: 395); b) CDR-H2 comprising the amino acid sequence SISYSA(X 1 )TYYNPSLKS, wherein, optionally, X 1 is S or T (SEQ ID NO: 396); and c) CDR-H3 comprising the amino acid sequence

(X 1 )(X 2 )D(X 3 )(X 4 )Y(X 5 )(X 6 )(X 7 )(X 8 )G(X 9 )(X 10 )(X 11 ), wherein, optionally: X 1 is A or V; X 2 is R, S or G, X 3 is P, Y, R, V, I, H, T or E; X 4 is S, D, E or N; X 5 is D, A or T; X 6 is S, G, T or A; X 7 is I, A, R, Q, or V; X 8 is A, E, K, G or T; X 9 is M or I; X 10 is D, L, Q, V, N or G; and, X 11 is V, R, N, E or K (SEQ ID NO: 397).

In some embodiments, the antibody, or antigen-binding fragment thereof, further comprises a CDR-L1 as set forth in SEQ ID NO:103, optionally comprising one or more amino acid changes. In some embodiment, the antibody, or antigen-binding fragment thereof, further comprises a CDR-L2 as set forth in SEQ ID NO:104, optionally comprising one or more amino acid changes. In some embodiments, the antibody, or antigen-binding fragment thereof, further comprises a CDR-L3 as set forth in SEQ ID NO:105, optionally comprising one or more amino acid changes.

In some embodiments, the antibody, or antigen-binding fragment, comprises at least three of the following six CDRs:

a) CDR-H1 comprising the amino acid sequence G(X 1 )I(X 2 )S(X 3 )SYYW(X 4 ), wherein, optionally: X 1 is S or P; X 2 is S, H or R; X 3 is S or G; and, X 4 is G, I, N or V (SEQ ID NO: 395); b) CDR-H2 comprising the amino acid sequence SISYSA(X 1 )TYYNPSLKS, wherein, optionally, X 1 is S or T (SEQ ID NO: 396); c) CDR-H3 comprising the amino acid sequence

(X 1 )(X 2 )D(X 3 )(X 4 )Y(X 5 )(X 6 )(X 7 )(X 8 )G(X 9 )(X 10 )(X 11 ), wherein, optionally: X 1 is A or V; X 2 is R, S or G, X 3 is P, Y, R, V, I, H, T or E; X 4 is S, D, E or N; X 5 is D, A or T; X 6 is S, G, T or A; X 7 is I, A, R, Q, or V; X 8 is A, E, K, G or T; X 9 is M or I; X 10 is D, L, Q, V, N or G; and, X 11 is V, R, N, E or K (SEQ ID NO: 397);

d) CDR-L1: SEQ ID NO:103, optionally comprising one or more amino acid changes; e) CDR-L2: SEQ ID NO: 104, optionally comprising one or more amino acid changes; and f) CDR-L3: SEQ ID NO: 105, optionally comprising one or more amino acid changes.

In some embodiments, the antibody, or antigen-binding fragment thereof, specifically binds a human LTBP1-proTGFβ1 complex and/or a human LTBP3-proTGFβ1 complex, and does not bind a human GARP-proTGFβ1 complex. In some embodiments, the antibody, or antigen-binding fragment thereof, does not bind a human GARP-proTGFβ1 complex. In some embodiments, the antibody, or antigen-binding fragment thereof, does not bind mature TGFβ1, mature TGFβ2 or mature TGFβ3.

In a particular embodiment, the antibody, or antigen-binding fragment, specifically binds a human LTBP1-proTGFβ1 complex and/or a human LTBP3-proTGFβ1 complex, and does not bind a human GARP-proTGFβ1 complex; wherein the antibody does not bind mature TGFβ1, mature TGFβ2 or mature TGFβ33; wherein the antibody is a fully human or humanized antibody or a fragment thereof, wherein the antibody comprises at least three of the following six CDRs:

a) CDR-H1 comprising the amino acid sequence G(X 1 )I(X 2 )S(X 3 )SYYW(X 4 ), wherein, optionally: X 1 is S or P; X 2 is S, H or R; X 3 is S or G; and, X 4 is G, I, N or V (SEQ ID NO: 395); b) CDR-H2 comprising the amino acid sequence SISYSA(X 1 )TYYNPSLKS, wherein, optionally, X 1 is S or T (SEQ ID NO: 396); c) CDR-H3 comprising the amino acid sequence

(X 1 )(X 2 )D(X 3 )(X 4 )Y(X 5 )(X 6 )(X 7 )(X 8 )G(X 9 ) (X 10 )(X 11 ), wherein, optionally: X 1 is A or V; X 2 is R, S or G, X 3 is P, Y, R, V, I, H, T or E; X 4 is S, D, E or N; X 5 is D, A or T; X 6 is S, G, T or A; X 7 is I, A, R, Q, or V; X 8 is A, E, K, G or T; X 9 is M or I; X 10 is D, L, Q, V, N or G; and, X 11 is V, R, N, E or K (SEQ ID NO: 397);

d) CDR-L1 as set forth in SEQ ID NO:103, optionally comprising one or more amino acid changes; e) CDR-L2 as set forth in SEQ ID NO: 104, optionally comprising one or more amino acid changes; and f) CDR-L3 as set forth in SEQ ID NO: 105, optionally comprising one or more amino acid changes.

In some embodiments, such antibody binds a human LTBP1-proTGFβ1 complex with a KD of <100 nM as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI); and/or, such antibody binds a human LTBP3-proTGFβ1 complex with a KD of <100 nM as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI). In some embodiments, such antibody binds a human LTBP1-proTGFβ1 complex with a KD of <50 nM as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI); and/or, such antibody binds a human LTBP3-proTGFβ1 complex with a KD of <50 nM as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI). In some embodiments, such antibody binds a human LTBP1-proTGFβ1 complex with a KD of <25 nM as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI); and/or, such antibody binds a human LTBP3-proTGFβ1 complex with a KD of <25 nM as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI). In some embodiments, such antibody binds a human LTBP1-proTGFβ1 complex with a KD of <10 nM as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI); and/or, such antibody binds a human LTBP3-proTGFβ1 complex with a KD of <10 nM as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI).

In another embodiment, such antibody is cross-reactive with mouse LTBP1-proTGFβ1. In some embodiments, such antibody is also cross-reactive with mouse LTBP3-proTGFβ1. In some embodiments, such antibody binds a mouse LTBP1-proTGFβ1 complex with a KD of <100 nM as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI); and/or, the antibody binds a mouse LTBP3-proTGFβ1 complex with a KD of <100 nM as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI). In some embodiments, such antibody binds a mouse LTBP1-proTGFβ1 complex with a KD of <50 nM as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI); and/or, the antibody binds a mouse LTBP3-proTGFβ1 complex with a KD of <50 nM as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI). In some embodiments, such antibody binds a mouse LTBP1-proTGFβ1 complex with a KD of <25 nM as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI); and/or, the antibody binds a mouse LTBP3-proTGFβ1 complex with a KD of <25 nM as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI). In some embodiments, such antibody binds a mouse LTBP1-proTGFβ1 complex with a KD of <10 nM as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI); and/or, the antibody binds a mouse LTBP3-proTGFβ1 complex with a KD of <10 nM as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI).

In another embodiment, such antibody does not bind to human GARP-proTGFβ1. In preferred embodiments, such context-selective antibody is also isoform-specific in that it selectively binds and inhibits the activation of TGFβ1 associated with LTBP1/3.

In another aspect, the antibody, or antigen-binding fragment thereof, comprises a CDR-H1 comprising the amino acid G(X 1 )I(X 2 )SSSYYW(X 3 ), wherein, optionally: X 1 is S or P; X 2 is H or R; and, X 3 is G, I or N (SEQ ID NO: 384). In some embodiments, X 1 is a S. In some embodiments, X 1 is a P. In some embodiments, X 2 is a H. In some embodiments, X 2 is an R. In some embodiments, X 3 is a G. In some embodiments, X 3 is an I. In some embodiments, X 3 is an N.

In some embodiments, the antibody, or antigen-binding fragment thereof, comprises a CDR-H2 comprising the amino acid sequence SISYSA(X 1 )TYYNPSLKS, wherein, optionally, X 1 is S or T (SEQ ID NO: 396). In some embodiments, X 1 is a S. In some embodiment, X 1 is a T.

In some embodiments, the antibody, or antigen-binding fragment thereof, comprises a CDR-H3 comprising the amino acid sequence A(X 1 )D(X 2 )SYD(X 3 )(X 4 )AGM(X 5 )(X 6 ), wherein, optionally: X 1 is R, S or G, X 2 is P or V; X 3 is S or A; X 4 is A, R, I or V; X 5 D, Q, or G; and, X 6 is V or R (SEQ ID NO: 385). In some embodiments, X 1 is R. In some embodiments, X 1 is S. In some embodiments, X 1 is G. In some embodiments, X 2 is P. In some embodiments, X 2 is V. In some embodiments, X 3 is S. In some embodiments, X 3 is A. In some embodiments, X 4 is A. In some embodiments, X 4 is R. In some embodiments, X 4 is I. In some embodiments, X 4 is V. In some embodiments, X 5 is D. In some embodiments, X 5 is Q. In some embodiments, X 5 is G. In some embodiments, X 6 is V. In some embodiments, X 6 is R.

In some embodiments, the antibody, or antigen-binding fragment thereof, comprises three heavy chain CDRs and three light chain CDRs, wherein the heavy chain CDRs comprise:

a) CDR-H1 comprising the amino acid sequence G(X 1 )I(X 2 )SSSYYW(X 3 ), wherein, optionally: X 1 is S or P; X 2 is H or R; and, X 3 is G, I or N (SEQ ID NO: 384); b) CDR-H2 comprising the amino acid sequence SISYSA(X 1 )TYYNPSLKS, wherein, optionally, X 1 is S or T (SEQ ID NO: 396); and c) CDR-H3 comprising the amino acid sequence A(X 1 )D(X 2 )SYD(X 3 )(X 4 )AGM(X 5 )(X 6 ), wherein, optionally: X 1 is R, S or G, X 2 is P or V; X 3 is S or A; X 4 is A, R, I or V; X 5 D, Q, or G; and, X 6 is V or R (SEQ ID NO: 385).

In some embodiments, the antibody, or antigen-binding fragment thereof, further comprises a CDR-L1 as set forth in SEQ ID NO:103, optionally comprising one or more amino acid changes. In some embodiment, the antibody, or antigen-binding fragment thereof, further comprises a CDR-L2 as set forth in SEQ ID NO:104, optionally comprising one or more amino acid changes. In some embodiments, the antibody, or antigen-binding fragment thereof, further comprises a CDR-L3 as set forth in SEQ ID NO:105, optionally comprising one or more amino acid changes.

In some embodiments, the antibody, or antigen-binding fragment, comprises at least three of the following six CDRs:

a) CDR-H1 comprising the amino acid sequence G(X 1 )I(X 2 )SSSYYW(X 3 ), wherein, optionally: X 1 is S or P; X 2 is H or R; and, X 3 is G, I or N (SEQ ID NO: 384); b) CDR-H2 comprising the amino acid sequence SISYSA(X 1 )TYYNPSLKS, wherein, optionally, X 1 is S or T (SEQ ID NO: 396); c) CDR-H3 comprising the amino acid sequence A(X 1 )D(X 2 )SYD(X 3 )(X 4 )AGM(X 5 )(X 6 ), wherein, optionally: X 1 is R, S or G, X 2 is P or V; X 3 is S or A; X 4 is A, R, I or V; X 5 D, Q, or G; and, X 6 is V or R (SEQ ID NO: 385); d) CDR-L1: SEQ ID NO:103, optionally comprising one or more amino acid changes; e) CDR-L2: SEQ ID NO: 104, optionally comprising one or more amino acid changes; and f) CDR-L3: SEQ ID NO: 105, optionally comprising one or more amino acid changes.

In some embodiments, the antibody, or antigen-binding fragment thereof, specifically binds a human LTBP1-proTGFβ1 complex and/or a human LTBP3-proTGFβ1 complex, and does not bind a human GARP-proTGFβ1 complex. In some embodiments, the antibody, or antigen-binding fragment thereof, does not bind a human GARP-proTGFβ1 complex. In some embodiments, the antibody, or antigen-binding fragment thereof, does not bind mature TGFβ1, mature TGFβ2 or mature TGFβ3.

In a particular embodiment, the antibody, or antigen-binding fragment thereof, specifically binds a human LTBP1-proTGFβ1 complex and/or a human LTBP3-proTGFβ1 complex, and does not bind a human GARP-proTGFβ1 complex; wherein the antibody does not bind mature TGFβ1, mature TGFβ2 or mature TGFβ33; wherein the antibody is a fully human or humanized antibody or a fragment thereof, wherein the antibody comprises at least three of the following six CDRs:

a) CDR-H1 comprising the amino acid sequence G(X 1 )I(X 2 )SSSYYW(X 3 ), wherein, optionally: X 1 is S or P; X 2 is H or R; and, X 3 is G, I or N (SEQ ID NO: 384); b) CDR-H2 comprising the amino acid sequence SISYSA(X 1 )TYYNPSLKS, wherein, optionally, X 1 is S or T (SEQ ID NO: 396); c) CDR-H3 comprising the amino acid sequence A(X 1 )D(X 2 )SYD(X 3 )(X 4 )AGM(X 5 )(X 6 ), wherein, optionally: X 1 is R, S or G, X 2 is P or V; X 3 is S or A; X 4 is A, R, I or V; X 5 D, Q, or G; and, X 6 is V or R (SEQ ID NO: 385); d) CDR-L1: SEQ ID NO:103, optionally comprising one or more amino acid changes; e) CDR-L2: SEQ ID NO: 104, optionally comprising one or more amino acid changes; and f) CDR-L3: SEQ ID NO: 105, optionally comprising one or more amino acid changes.

In some embodiments, such antibody binds a human LTBP1-proTGFβ1 complex with a KD of <100 nM as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI); and/or, such antibody binds a human LTBP3-proTGFβ1 complex with a KD of <100 nM as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI). In some embodiments, such antibody binds a human LTBP1-proTGFβ1 complex with a KD of <50 nM as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI); and/or, such antibody binds a human LTBP3-proTGFβ1 complex with a KD of <50 nM as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI). In some embodiments, such antibody binds a human LTBP1-proTGFβ1 complex with a KD of <25 nM as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI); and/or, such antibody binds a human LTBP3-proTGFβ1 complex with a KD of <25 nM as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI). In some embodiments, such antibody binds a human LTBP1-proTGFβ1 complex with a KD of <10 nM as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI); and/or, such antibody binds a human LTBP3-proTGFβ1 complex with a KD of <10 nM as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI).

In another embodiment, such antibody is cross-reactive with mouse LTBP1-proTGFβ1. In some embodiments, such antibody is also cross-reactive with mouse LTBP3-proTGFβ1. In some embodiments, such antibody binds a mouse LTBP1-proTGFβ1 complex with a KD of <100 nM as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI); and/or, the antibody binds a mouse LTBP3-proTGFβ1 complex with a KD of <100 nM as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI). In some embodiments, such antibody binds a mouse LTBP1-proTGFβ1 complex with a KD of <50 nM as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI); and/or, the antibody binds a mouse LTBP3-proTGFβ1 complex with a KD of <50 nM as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI). In some embodiments, such antibody binds a mouse LTBP1-proTGFβ1 complex with a KD of <25 nM as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI); and/or, the antibody binds a mouse LTBP3-proTGFβ1 complex with a KD of <25 nM as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI). In some embodiments, such antibody binds a mouse LTBP1-proTGFβ1 complex with a KD of <10 nM as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI); and/or, the antibody binds a mouse LTBP3-proTGFβ1 complex with a KD of <10 nM as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI).

In another embodiment, such antibody does not bind to human GARP-proTGFβ1. In preferred embodiments, such context-selective antibody is also isoform-specific in that it selectively binds and inhibits the activation of TGFβ1 associated with LTBP1/3.

Also provided herein are antibodies, or antigen-binding fragments thereof, which binds a human LTBP1-proTGFβ1 complex and/or a human LTBP3-TGFβ1 complex with yet higher affinities and further advantageous combinations of binding properties.

Accordingly, in one aspect, the antibody, or antigen-binding fragment thereof, comprises the following six CDRs:

a) CDR-H1 comprising the amino acid sequence FTFRSYVMH (SEQ ID NO: 166); b) CDR-H2 comprising the amino acid sequence VISHEGS(X 1 )KYYADSVKG, wherein: X 1 is L or G (SEQ ID NO: 366); and c) CDR-H3 comprising the amino acid sequence A(X 1 )PRIAARRGGFG(X 2 ), wherein: X 1 is V, R or L; and X 2 is Y, S or T (SEQ ID NO: 367); d) CDR-L1 comprising the amino acid sequence TRS(X 1 )G(X 2 )ID(X 3 )NYVQ, wherein, X 1 is S or H; X 2 is N, L, S or A; and X 3 is N, D or Y (SEQ ID NO: 368); e) CDR-L2 comprising the amino acid sequence ED(X 1 )(X 2 )RPS, wherein: X 1 is N, F or A; and X 2 is Q, I or V (SEQ ID NO: 369); and f) CDR-L3 comprising the amino acid sequence Q(X 1 )YD(X 2 )(X 3 )(X 4 )Q(X 5 )VV, wherein: X 1 is S or G; X 2 is 5, F, Y, D, H or W; X 3 is N, D or S; X 4 is N, A, L, E or T; and X 5 is G, R, A or L (SEQ ID NO: 370).

In some embodiments, within CDR-H3: X 1 is R or L. Within CDR-L3: X 2 may be Y. Within CDR-L3: X 3 may be D; and X 4 may be T. In some preferred embodiments, within CDR-H3: X 1 is R or L (optionally R), within CDR-L3: X 2 is Y; and within CDR-L3: X 3 is D; and X 4 is T.

In alternative embodiments, within CDR-H3: X 1 is R or L (optionally R), within CDR-L3: X 2 is Y and within CDR-L3: X 3 is D; X 4 is N; and X 5 is A.

In some embodiments within CDR-L1: X 1 is S or H; X 2 is N or A; and X 3 is N, D or Y; within CDR-L2: X 1 is N or F; and X 2 is Q or V; and within CDR-L3: X 1 is S or G; X 2 is 5, Y, D or W; X 3 is D or S; X 4 is N, L or T; and X 5 is G, R, A or L. In some embodiments, within CDR-L1: X 1 is S; X 2 is N; and X 3 is N or Y; within CDR-L2: X 1 is N; and X 2 is Q or V; and within CDR-L3: X 1 is S or G; X 2 is 5, Y or W; X 3 is D; X 4 is N or T; and X 5 is G, R or A. In some embodiments, within CDR-L3: X 1 is S; X 2 is S or Y; X 3 is D; X 4 is N or T; and X 5 is G, R or A. In some embodiments, within CDR-L3: X 1 is S; X 2 is Y; X 3 is D; X 4 is N or T; and X 5 is G or A. In some preferred embodiments, within CDR-L3: X 1 is S; X 2 is Y; X 3 is D; X 4 is T; and X 5 is G.

In particularly preferred embodiments, the antibody or antigen-binding fragment has the CDRs of Ab42, e.g.: CDR-H1 comprising the amino acid sequence FTFRSYVMH (SEQ ID NO: 166); CDR-H2 comprising the amino acid sequence VISHEGSLKYYADSVKG (SEQ ID NO: 167); CDR-H3 comprising the amino acid sequence ARPRIAARRGGFGY (SEQ ID NO: 168); CDR-L1 comprising the amino acid sequence TRSSGNIDNNYVQ (SEQ ID NO: 169); CDR-L2 comprising the amino acid sequence EDNQRPS (SEQ ID NO: 170); and CDR-L3 comprising the amino acid sequence QSYDYDTQGVV (SEQ ID NO: 171).

In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region having an amino acid sequence that is at least 90% identical to SEQ ID NO: 318; and a light chain variable region having an amino acid sequence that is at least 90% identical to SEQ ID NO: 319.

In some embodiments, the antibody, or antigen-binding fragment, binds a human LTBP1-proTGFβ1 complex and/or a human LTBP3-TGFβ1 complex with a K D of <5 nM as measured by suitable in vitro binding assay, such as BLI. For example, the antibody, or antigen-binding fragment, may bind a human LTBP1-proTGFβ1 complex and a human LTBP3-TGFβ1 complex with a K D of <5 nM as measured by suitable in vitro binding assay, such as BLI. In some embodiments, the antibody, or antigen-binding fragment, binds the human LTBP1- and/or LTBP3-proTGFβ1 complex with a K D of <1 nM as measured by suitable in vitro binding assay, such as BLI.

In some embodiments, the antibody, or antigen-binding fragment thereof does not show detectable binding to a human GARP-proTGFβ1 complex, as measured by BLI, under the same assay conditions as used to measure binding to human LTBP1-proTGFβ1 complex and/or human LTBP3-TGFβ1. For example, the antibody, or antigen-binding fragment may not show detectable binding to a human GARP-proTGFβ1 complex, as measured by BLI, under the same assay conditions as used to measure binding to human LTBP1-proTGFβ1 complex and human LTBP3-TGFβ1 complex.

In some embodiments, the antibody, or antigen-binding fragment thereof, binds a human LTBP1-proTGFβ1 complex and/or a human LTBP3-TGFβ1 complex with a K D that is at least 50 times lower (e.g., at least 75 times lower, at least 100 times lower) than the K D when binding to a human GARP-proTGFβ1 complex under the same assay conditions. For example, the antibody, or antigen-binding fragment thereof, may bind a human LTBP1-proTGFβ1 complex and a human LTBP3-TGFβ1 complex with a K D that is at least 50 times lower (e.g., at least 75 times lower, at least 100 times lower) than the K D when binding to a human GARP-proTGFβ1 complex under the same assay conditions. In some embodiments, K D is as determined by BLI or SPR. In some embodiments, K D is as determined by SPR.

In some embodiments, the antibody, or antigen-binding fragment thereof does not show detectable binding to an LRRC33-proTGFβ1 latent complex, as measured by BLI, under the same assay conditions as used to measure binding to human LTBP1-proTGFβ1 complex and/or human LTBP3-TGFβ1. For example, the antibody, or antigen-binding fragment thereof may not show detectable binding to an LRRC33-proTGFβ1 latent complex, as measured by BLI, under the same assay conditions as used to measure binding to human LTBP1-proTGFβ1 complex and human LTBP3-TGFβ1.

In some embodiments, the antibody, or antigen-binding fragment thereof, binds a human LTBP1-proTGFβ1 complex and/or a human LTBP3-TGFβ1 complex with a K D that is at least 50 times lower (e.g., at least 75 times lower, at least 100 times lower) than the K D when binding to a human LRRC33-proTGFβ1 complex under the same assay conditions. For example, the antibody, or antigen-binding fragment thereof, may bind a human LTBP1-proTGFβ1 complex and a human LTBP3-TGFβ1 complex with a K D that is at least 50 times lower (e.g., at least 75 times lower, at least 100 times lower) than the K D when binding to a human LRRC33-proTGFβ1 complex under the same assay conditions. In some embodiments, K D is as determined by BLI or SPR. In some embodiments, K D is as determined by SPR.

In some embodiments, the antibody, or antigen-binding fragment thereof, is cross-reactive with mouse LTBP1-proTGFβ1. In some embodiments, the antibody, or antigen-binding fragment thereof, is cross-reactive with mouse LTBP3-proTGFβ1. In some embodiments, the antibody, or antigen-binding fragment thereof, binds a mouse LTBP1-proTGFβ1 complex with a K D of <10 nM as measured by BLI. In some embodiments, the antibody, or antigen-binding fragment thereof, binds a mouse LTBP3-proTGFβ1 complex with a K D of <10 nM as measured by BLI.

In further embodiments, the antibody which selectively binds a human LTBP1-TGFβ1 complex and/or a human LTBP3-TGFβ1 complex may not show meaningful binding (e.g., may not show a response of more than 0.1 units (nm)) on exposure to a human GARP-proTGFβ1 complex in a BLI assay (e.g., Octet) when the human GARP-proTGFβ1 complex is at a concentration of 200 nM.

In another aspect, the antibody, or antigen-binding fragment thereof, comprises the following six CDRs:

a) CDR-H1 comprising the amino acid sequence G(X 1 )I(X 2 )S(X 3 )SYYW(X 4 ), wherein, optionally: X 1 is S; X 2 is S, H or R; X 3 is S or G; and, X 4 is G, I, N or V (SEQ ID NO: 386); b) CDR-H2 comprising the amino acid sequence SISYS(X 1 )(X 2 )TYY, wherein, optionally: X 1 is G or A; and X 2 is S or T (SEQ ID NO: 398); c) CDR-H3 comprising the amino acid sequence A(X 1 )DPSYDS(X 2 )AGM(X 3 )V, wherein, optionally: X 1 is R, S or G; X 2 is A or I; and X 3 is D or Q (SEQ ID NO: 387); d) CDR-L1 comprising the amino acid sequence RAS(X 1 )(X 2 )IS(X 3 )YLN, wherein, optionally: X 1 is K or Q; X 2 is V or S; and X 3 is S or Y (SEQ ID NO: 389); e) CDR-L2 comprising the amino acid sequence (X 1 )AS(X 2 )(X 3 )QS, wherein, optionally: X 1 is Y, A or S; X 2 is S or N; and X 3 is L or R (SEQ ID NO: 390); f) CDR-L3 comprising the amino acid sequence QQ(X 1 )(X 2 )D(X 3 )P(X 4 )T, wherein, optionally: X 1 is S or G; X 2 is F or N; X 3 is W or F; and X 4 is F or L (SEQ ID NO: 391).

In some embodiments: within CDR-H1: X 1 is S; X 2 is S or R; X 3 is 5; and, X 4 is G; within CDR-H2: X 1 is G or A; and X 2 is S or T; within CDR-H3: X 1 is R, S or G; X 2 is A or I; and X 3 is D or Q; within CDR-L1: X 1 is K or Q; X 2 is V or S; and X 3 is S or Y; within CDR-L2: X 1 is Y, A or S; X 2 is S or N; and X 3 is L or R; and within CDR-L3: X 1 is S or G; X 2 is F or N; X 3 is W or F; and X 4 is F or L.

In some embodiments: CDR-H1 comprises the amino acid sequence GSIRSSSYYWG (SEQ ID NO: 292); CDR-H2 comprises the amino acid sequence SISYSATTYY (SEQ ID NO: 293); within CDR-H3: X 1 is S or G; X 2 is A or I; and X 3 is D or Q; within CDR-L1: X 1 is K or Q; X 2 is V or S; and X 3 is S or Y; within CDR-L2: X 1 is Y, A or S; X 2 is S or N; and X 3 is L or R; and within CDR-L3: X 1 is S or G; X 2 is F or N; X 3 is W or F; and X 4 is F or L.

In some embodiments: CDR-H1 comprises the amino acid sequence GSIRSSSYYWG (SEQ ID NO: 292); CDR-H2 comprises the amino acid sequence SISYSATTYY (SEQ ID NO: 293); CDR-H3 comprises the amino acid sequence AGDPSYDSIAGMQV (SEQ ID NO: 294); CDR-L1 comprises the amino acid sequence RASQSISSYLN (SEQ ID NO: 295); CDR-L2 comprises the amino acid sequence AASNLQS (SEQ ID NO: 296); and CDR-L3 comprises the amino acid sequence QQSFDWPLT (SEQ ID NO: 297).

In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region having an amino acid sequence that is at least 90% identical to SEQ ID NO: 360; and a light chain variable region having an amino acid sequence that is at least 90% identical to SEQ ID NO: 361.

In some embodiments, the antibody, or antigen-binding fragment, binds a human LTBP1-proTGFβ1 complex and/or a human LTBP3-TGFβ1 complex with a K D of <5 nM as measured by suitable in vitro binding assay, such as BLI. For example, the antibody, or antigen-binding fragment, may bind a human LTBP1-proTGFβ1 complex and a human LTBP3-TGFβ1 complex with a K D of <5 nM as measured by suitable in vitro binding assay, such as BLI. In some embodiments, the antibody, or antigen-binding fragment, binds the human LTBP1- and/or LTBP3-proTGFβ1 complex with a K D of <1 nM as measured by suitable in vitro binding assay, such as BLI.

In some embodiments, the antibody, or antigen-binding fragment thereof does not show detectable binding to a human GARP-proTGFβ1 complex, as measured by BLI, under the same assay conditions as used to measure binding to human LTBP1-proTGFβ1 complex and/or human LTBP3-TGFβ1. For example, the antibody, or antigen-binding fragment may not show detectable binding to a human GARP-proTGFβ1 complex, as measured by BLI, under the same assay conditions as used to measure binding to human LTBP1-proTGFβ1 complex and human LTBP3-TGFβ1 complex.

In some embodiments, the antibody, or antigen-binding fragment thereof, binds a human LTBP1-proTGFβ1 complex and/or a human LTBP3-TGFβ1 complex with a K D that is at least 50 times lower (e.g., at least 75 times lower, at least 100 times lower) than the K D when binding to a human GARP-proTGFβ1 complex under the same assay conditions. For example, the antibody, or antigen-binding fragment thereof, may bind a human LTBP1-proTGFβ1 complex and a human LTBP3-TGFβ1 complex with a K D that is at least 50 times lower (e.g., at least 75 times lower, at least 100 times lower) than the K D when binding to a human GARP-proTGFβ1 complex under the same assay conditions. In some embodiments, K D is as determined by BLI or SPR. In some embodiments, K D is as determined by SPR.

In some embodiments, the antibody, or antigen-binding fragment thereof does not show detectable binding to an LRRC33-proTGFβ1 latent complex, as measured by BLI, under the same assay conditions as used to measure binding to human LTBP1-proTGFβ1 complex and/or human LTBP3-TGFβ1. For example, the antibody, or antigen-binding fragment thereof may not show detectable binding to an LRRC33-proTGFβ1 latent complex, as measured by BLI, under the same assay conditions as used to measure binding to human LTBP1-proTGFβ1 complex and human LTBP3-TGFβ1.

In some embodiments, the antibody, or antigen-binding fragment thereof, binds a human LTBP1-proTGFβ1 complex and/or a human LTBP3-TGFβ1 complex with a K D that is at least 50 times lower (e.g., at least 75 times lower, at least 100 times lower) than the K D when binding to a human LRRC33-proTGFβ1 complex under the same assay conditions. For example, the antibody, or antigen-binding fragment thereof, may bind a human LTBP1-proTGFβ1 complex and a human LTBP3-TGFβ1 complex with a K D that is at least 50 times lower (e.g., at least 75 times lower, at least 100 times lower) than the K D when binding to a human LRRC33-proTGFβ1 complex under the same assay conditions. In some embodiments, K D is as determined by BLI or SPR. In some embodiments, K D is as determined by SPR.

In some embodiments, the antibody, or antigen-binding fragment thereof, is cross-reactive with mouse LTBP1-proTGFβ1. In some embodiments, the antibody, or antigen-binding fragment thereof, is cross-reactive with mouse LTBP3-proTGFβ1. In some embodiments, the antibody, or antigen-binding fragment thereof, binds a mouse LTBP1-proTGFβ1 complex with a K D of <10 nM as measured by BLI. In some embodiments, the antibody, or antigen-binding fragment thereof, binds a mouse LTBP3-proTGFβ1 complex with a K D of <10 nM as measured by BLI.

In further embodiments, the antibody which selectively binds a human LTBP1-TGFβ1 complex and/or a human LTBP3-TGFβ1 complex may not show meaningful binding (e.g., may not show a response of more than 0.1 units (nm)) on exposure to a human GARP-proTGFβ1 complex in a BLI assay (e.g., Octet) when the human GARP-proTGFβ1 complex is at a concentration of 200 nM.

In some embodiments, the “percent identity” of two amino acid sequences is determined using the algorithm of Karlin and Altschul Proc. Natl. Acad. Sci. USA 87:2264-68, 1990, modified as in Karlin and Altschul Proc. Natl. Acad. Sci. USA 90:5873-77, 1993. Such an algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. J. Mol. Biol. 215:403-10, 1990. BLAST protein searches can be performed with the XBLAST program, score=50, word length=3 to obtain amino acid sequences homologous to the protein molecules of interest. Where gaps exist between two sequences, Gapped BLAST can be utilized as described in Altschul et al., Nucleic Acids Res. 25(17):3389-3402, 1997. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

In any of the antibodies or antigen-binding fragments described herein, one or more conservative mutations can be introduced into the CDRs or framework sequences at positions where the residues are not likely to be involved in an antibody-antigen interaction. In some embodiments, such conservative mutation(s) can be introduced into the CDRs or framework sequences at position(s) where the residues are not likely to be involved in interacting with a LTBP1-TGFβ complex and/or a LTBP3-TGFβ complex, as determined based on the crystal structure. In some embodiments, the likely interface (e.g., residues involved in an antigen-antibody interaction) may be deduced from known structural information on another antigens sharing structural similarities.

As used herein, a “conservative amino acid substitution” refers to an amino acid substitution that does not alter the relative charge or size characteristics of the protein in which the amino acid substitution is made. Variants can be prepared according to methods for altering polypeptide sequence known to one of ordinary skill in the art such as are found in references which compile such methods, e.g., Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, or Current Protocols in Molecular Biology, F. M. Ausubel, et al., eds., John Wiley & Sons, Inc., New York. Conservative substitutions of amino acids include substitutions made amongst amino acids within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D.

In some embodiments, the antibodies provided herein comprise mutations that confer desirable properties to the antibodies. For example, to avoid potential complications due to Fab-arm exchange, which is known to occur with native IgG4 mAbs, the antibodies provided herein may comprise a stabilizing ‘Adair’ mutation (Angal et al., “A single amino acid substitution abolishes the heterogeneity of chimeric mouse/human (IgG4) antibody,” Mol Immunol 30, 105-108; 1993), where serine 228 (EU numbering; residue 241 Kabat numbering) is converted to proline resulting in an IgG1-like (CPPCP (SEQ ID NO: 45)) hinge sequence. Accordingly, any of the antibodies may include a stabilizing ‘Adair’ mutation or the amino acid sequence CPPCP (SEQ ID NO: 45). In one embodiment, an antibody described herein comprises a heavy chain immunoglobulin constant domain of a human IgG 4 having a backbone substitution of Ser to Pro, that produces an IgG 1 -like hinge and permits formation of inter-chain disulfide bonds.

Antibodies of this disclosure that selectively bind to a LTBP1-TGFβ complex and/or a LTBP3-TGFβ complex may optionally comprise antibody constant regions or parts thereof. For example, a V L domain may be attached at its C-terminal end to a light chain constant domain such as C κ or C λ . Similarly, a V H domain or portion thereof may be attached to all or part of a heavy chain such as IgA, IgD, IgE, IgG, and IgM, and any isotype subclass. Antibodies may include suitable constant regions (see, for example, Kabat et al., Sequences of Proteins of Immunological Interest, No. 91-3242, National Institutes of Health Publications, Bethesda, Md. (1991)). Therefore, antibodies within the scope of this may disclosure include V H and V L domains, or antigen-binding portions thereof, combined with any suitable constant region. In exemplary embodiments, the antibodies, or antigen-binding portions thereof, comprise a heavy chain immunoglobulin constant domain containing all or a portion of a human IgG 1 or a human IgG 4 constant domain. In some embodiments, the antibody, or antigen-binding portion thereof, comprises a light chain immunoglobulin constant domain containing all or a portion of a human Ig lambda constant domain or a human Ig kappa constant domain.

In some embodiments, antibodies that selectively bind to a LTBP1-TGFβ complex and/or a LTBP3-TGFβ complex may or may not include the framework region of the antibodies of SEQ ID NOs: 7 and 8. In some embodiments, antibodies that selectively bind to a LTBP1-TGFβ complex and/or a LTBP3-TGFβ complex are murine antibodies and include murine framework region sequences. In other embodiments, the antibodies are chimeric antibodies, or antigen-binding fragments thereof. In another embodiment, the antibodies are humanized antibodies, or antigen-binding fragments thereof. In another embodiment, the antibodies are fully human antibodies, or antigen-binding fragments thereof. In one embodiment, the antibody comprises a framework region comprising a human germline amino acid sequence.

The antibodies, and antigen-binding fragments thereof, described herein can have any configuration suitable for binding antigen. For example, in one embodiment, the antibody, or antigen-binding portion thereof, comprises four polypeptide chains, including two heavy chain variable regions and two light chain variable regions. In another embodiment, the antibody, or antigen-binding portion thereof, comprises one heavy chain variable region and one light chain variable region. In exemplary embodiments, the antibody, or antigen-binding portion thereof, is a Fab fragment, a F(ab′)2 fragment, a scFab fragment, an scFv, or a diabody.

In one embodiment, the antibody, or antigen-binding portion thereof, comprises a heavy chain immunoglobulin constant domain of a human IgG 1 constant domain or a human IgG 4 constant domain. In an exemplary embodiment, the heavy chain immunoglobulin constant domain is a human IgG 4 constant domain. In one embodiment, the antibody, or antigen-binding portion thereof, binds a conformational epitope. In one embodiment, the antibody, or antigen-binding portion thereof, binds a combinatorial epitope.

In one embodiment, the antibody, or antigen-binding portion thereof, comprises a heavy chain immunoglobulin constant domain of a human IgG 4 constant domain having a backbone substitution of Ser to Pro that produces an IgG 1 -like hinge and permits formation of inter-chain disulfide bonds. In one embodiment, the antibody, or antigen-binding portion thereof, further comprises a light chain immunoglobulin constant domain comprising a human Ig lambda constant domain, or a human Ig kappa constant domain.

In one embodiment, the antibody is an IgG having four polypeptide chains which are two heavy chains and two light chains. In exemplary embodiments, the antibody can be a humanized antibody, a human antibody, or a chimeric antibody. In one embodiment, the antibody comprises a framework having a human germline amino acid sequence.

In one embodiment, the invention provides an antibody, or antigen-binding portion thereof, that competes for binding with an antibody, or antigen-binding portion thereof, described herein. In one embodiment, the invention provides an antibody, or antigen-binding portion thereof, that binds to the same epitope as an antibody, or antigen-binding portion thereof, described herein. In one embodiment, the antibody, or antigen-binding fragment thereof, does not compete with antibody SR-Ab1 for binding to a human LTBP1-proTGFβ1 complex.

Binding Kinetics of Novel Antibodies

The novel antibodies and antigen-binding fragments thereof (e.g., Fabs) disclosed herein are characterized by enhanced binding properties. The antibodies and the fragments are capable of selectively binding to a LTBP1-TGFβ1 complex and/or a LTBP3-TGFβ1 complex (also known as large latent complexes (LLCs). Recombinantly produced, purified protein complexes may be used as antigens (e.g., antigen complexes) to screen, evaluate or confirm the ability of an antibody to bind the antigen complexes in suitable in vitro binding assays. Such assays are well known in the art and include but are not limited to: BLI-based assays (such as Octet®) and SPR-based assays (such as Biacore).

Previously, antibodies and fragments that exhibited high affinities (e.g., sub-nanomolar K D ) to the LLCs were identified. Here, advantageously, antibodies and fragments with particularly slow dissociation rates were specifically selected, aimed to achieve particularly durable inhibitory effects.

Accordingly, selection of suitable TGFβ inhibitors for carrying out the methods and therapeutic use in accordance with the present disclosure may include carrying out in vitro binding assays to measure binding kinetics. In preferred embodiments, the antibody or the antigen-binding fragment binds hLTBP1-pro TGFβ1 and/or hLTBP3-proTGFβ1 with high affinity and low dissociation rate k OFF , as described herein. Preferably, the antibody or the fragment further binds the murine LLC counterparts, namely, mLTBP1-proTGFβ1 and/or mLTBP3-proTGFβ1, with equivalent affinities as the human LLCs. In vitro binding kinetics may be readily determined by measuring interactions of test antibodies (such as antigen-binding fragments) and suitable antigen, such as LLCs and small latent complexes (SLCs). Suitable methods for in vitro binding assays to determine the parameters of binding kinetics include BLI-based assays such as Octet, and surface plasmon resonance-based assays, such as Biacore systems. An example of an Octet-based in vitro binding assay is provided in Example 9/Table 9. Several antibodies were shown in this experiment to have “OFF” rates (k OFF ) that are <5×10 −4 (1/s). These results are in stark contrast to the results shown for Ab10, for which binding to hLTBP1-pro TGFβ1 and/or hLTBP3-proTGFβ1 could not even be detected by the same Octet assay (Table 8). An example of SPR-based in vitro binding assay is provided in Example 9. Fab fragments of Ab42, Ab63 and Ab43, which are activation inhibitors of TGFβ1, were used in this experiment. As illustrated in this example, these antibodies have sub-nanomolar K D and “OFF” rates that are <5×10 −4 (1/s). Thus, for example, Ab42 is able to remain bound to the antigen for a much longer duration of time (e.g., greater t½) than an antibody with much higher “OFF” rate, which “falls off” (e.g., dissociates from) the antigen relatively quickly. Thus, the difference in the dissociation kinetics predominantly attributes to the notable difference in their overall affinities (K D ), which may result in enhanced potency. Therefore, characterization of binding kinetics provides useful information as to potential durability of effects and resulting in vivo potency.

Accordingly, the invention includes a method of selecting a TGFβ activation inhibitor for therapeutic use, wherein the method comprises selection of an antibody or antigen-binding fragment thereof that has a dissociation rate of <5×10 −4 ((1/s) as measured by SPR. In some embodiments, the antibody or the fragment binds antigen with an affinity of less than 1 nM (i.e., sub-nanomolar), e.g., less than 500 pM, 400 pM, 300 pM, 200 pM, 100 pM, or 50 pM.

The selection method may include determining or measuring dissociation half-time (also referred to as half binding time or t′) of test antibody by suitable means, such as BLI-based assays and SPR-based assays. Monovalent or multivalent (e.g., divalent) test antibodies may be used. For example, Fab fragments are suitable monovalent antibodies that can be used to determine dissociation half time. Similarly, full length immunoglobulins (e.g., IgGs) may be used to determine dissociation half time. In some embodiments, the method comprises selection of an antibody or antigen-binding fragment thereof that has t′ of 45 minutes or longer to human LTBP1-proTGFβ1 and/or human LTBP3-proTGFβ1. Preferably, the antibody or the fragment has t′ of 45 minutes or longer to each of human LTBP1-proTGFβ1 and human LTBP3-proTGFβ1. More preferably, the antibody or the fragment further has t′ of 45 minutes or longer to murine LTBP1-proTGFβ1 and/or murine LTBP3-proTGFβ1. Most preferably, the antibody or the fragment has t′ of 45 minutes or longer to each of murine LTBP1-proTGFβ1 and murine LTBP3-proTGFβ1. The method may further include selection of an antibody or an antigen-binding fragment that has t½ of 5 minutes or less to human GARP-proTGFβ1. Any of these antibodies with advantageous dissociation half time should preferably have a KD of less than 1 nM as measured by BLI (e.g., Octet) or SPR (e.g., Biacore). Preferably, SPR assays are used to determine dissociation half-time (t½).

Polypeptides

Some aspects of the disclosure relate to isolated polypeptides. For example, in one embodiment, the invention provides an isolated polypeptide comprising CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, or CDRL3, or combinations thereof, as provided in Table 5. In an exemplary embodiment, the isolated polypeptide can contain CDRH1, CDRH2, and CDRH3 as provided in Table 5. In other embodiments, the isolated polypeptide can contain CDRL1, CDRL2, and CDRL3 as provided in Table 5. In some embodiments, the polypeptide can contain up to 6, 5, 4, 3, 2, or 1 amino acid residue variations as compared to the corresponding CDR region in any one of CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, or CDRL3, or combinations thereof, as provided in Table 5. In one embodiment, the invention provides an isolated polypeptide comprising SEQ ID NO: 7. In another embodiment, the invention provides an isolated polypeptide comprising SEQ ID NO: 8. In another embodiment, the invention provides an isolated polypeptide comprising SEQ ID NO:7 and SEQ ID NO:8. In this embodiment, SEQ ID NO:7 and SEQ ID NO:8 can optionally be connected by a linker peptide. In some embodiments, the polypeptide is a heavy chain variable domain. In some embodiments, the polypeptide is at least 75% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to SEQ ID NO: 7. In some embodiments, the polypeptide is a light chain variable domain. In some embodiments, the polypeptide is at least 75% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to SEQ ID NO: 8.

In another embodiment, the invention provides an isolated polypeptide comprising a heavy chain variable region sequence set forth in Table 6. In one embodiment, the invention provides an isolated polypeptide comprising SEQ ID NO: 7, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, or SEQ ID NO: 106. In one embodiment, the invention provides an isolated polypeptide comprising SEQ ID NO: 8, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, or SEQ ID NO: 107. In one embodiment, the invention provides an isolated polypeptide comprising SEQ ID NO: 318. In one embodiment, the invention provides an isolated polypeptide comprising SEQ ID NO: 319. In one embodiment, the invention provides an isolated polypeptide comprising SEQ ID NO: 360. In one embodiment, the invention provides an isolated polypeptide comprising SEQ ID NO: 361.

In another embodiment, the invention provides an isolated polypeptide comprising a light chain variable region set forth in Table 6. In another embodiment, the invention provides an isolated polypeptide comprising a heavy chain variable region sequence set forth in Table 6 (e.g., SEQ ID NO: 7, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, or SEQ ID NO: 106) and a light chain variable region sequence set forth in Table 6 (e.g., SEQ ID NO: 8, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, or SEQ ID NO: 107). In this embodiment, the heavy chain and light chain sequences (e.g., SEQ ID NO:7 and SEQ ID NO:8) can optionally be connected by a linker peptide. In some embodiments, the polypeptide is a heavy chain variable domain. In some embodiments, the polypeptide is at least 75% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to SEQ ID NO: 7, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, or SEQ ID NO: 106. In some embodiments, the polypeptide is a light chain variable domain. In some embodiments, the polypeptide is at least 75% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to SEQ ID NO: 8, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, or SEQ ID NO: 107.

In another embodiment, the invention provides an isolated polypeptide comprising a heavy chain variable region sequence set forth in SEQ ID NO: 318 and a light chain variable region sequence set forth in SEQ ID NO: 319. In another embodiment, the invention provides an isolated polypeptide comprising a heavy chain variable region sequence set forth in SEQ ID NO: 360 and a light chain variable region sequence set forth in SEQ ID NO: 361. In this embodiment, the heavy chain and light chain sequences (e.g., SEQ ID NO: 318 and SEQ ID NO: 319) can be connected by a linker peptide. In some embodiments, the polypeptide is a heavy chain variable domain. In some embodiments, the polypeptide is at least 85% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to SEQ ID NO: 318 or SEQ ID NO: 360. In some embodiments, the polypeptide is a light chain variable domain. In some embodiments, the polypeptide is at least 85% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to SEQ ID NO: 319 or SEQ ID NO: 361.

Nucleic Acids

In some embodiments, antibodies, antigen-binding portions thereof, and/or compositions of the present disclosure may be encoded by nucleic acid molecules. Such nucleic acid molecules include, without limitation, DNA molecules, RNA molecules, polynucleotides, oligonucleotides, mRNA molecules, vectors, plasmids and the like. In some embodiments, the present disclosure may comprise cells programmed or generated to express nucleic acid molecules encoding compounds and/or compositions of the present disclosure.

In some embodiments, the invention provides a nucleic acid molecule that encodes the foregoing antibodies, or an antigen-binding portion thereof. For example, in one embodiment, the invention provides a nucleic acid molecule that encodes a polypeptide comprising CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, or CDRL3, or combinations thereof, as provided in Table 5. The nucleic acid molecule can, in some embodiments, encode a polypeptide comprising CDRH1, CDRH2, and CDRH3 as provided in Table 5. In some embodiments, the nucleic acid molecule can encode a polypeptide comprising CDRL1, CDRL2, and CDRL3 as provided in Table 5. In some embodiments, the nucleic acid molecule encodes a polypeptide that can contain up to 6, 5, 4, 3, 2, or 1 amino acid residue variations as compared to the corresponding CDR region in any one of CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, or CDRL3, or combinations thereof, as provided in Table 5. In an exemplary embodiment, the nucleic acid molecule encodes a polypeptide comprising a heavy chain variable domain having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 7, and/or a light chain variable domain having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 8. In one embodiment, the nucleic acid molecule encodes a polypeptide comprising a heavy chain variable domain having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to a heavy chain variable region sequence set forth in Table 6, and/or a light chain variable domain having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to a light chain variable region sequence set forth in Table 6. In an exemplary embodiment, the nucleic acid molecule encodes a polypeptide comprising a heavy chain variable domain having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 7, and/or a light chain variable domain having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 8. In some embodiments, the nucleic acid molecule encodes an antibody, or antigen-binding portion thereof, comprising a heavy chain variable domain amino acid sequence set forth in SEQ ID NO: 7, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, or SEQ ID NO: 106, and a light chain variable domain amino acid sequence set forth in SEQ ID NO: 8, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, or SEQ ID NO: 107. In some embodiments, the nucleic acid molecule encodes an antibody, or antigen-binding portion thereof, comprising a heavy chain variable domain amino acid sequence set forth in SEQ ID NO: 7, and a light chain variable domain amino acid sequence set forth in SEQ ID NO: 8.

In another embodiment, the invention provides nucleic acid molecule that encodes the foregoing antibodies, or an antigen-binding portion thereof. In an exemplary embodiment, the nucleic acid molecule encodes a polypeptide comprising a heavy chain variable domain having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 318, and/or a light chain variable domain having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 319. In another embodiment, the nucleic acid molecule encodes a polypeptide comprising a heavy chain variable domain having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 360, and/or a light chain variable domain having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence set forth in SEQ ID NO: 361.

In another embodiment, the invention provides a nucleic acid molecule that encodes a polypeptide comprising a heavy chain variable region sequence set forth in SEQ ID NO: 318 and a light chain variable region sequence set forth in SEQ ID NO: 319. In another embodiment, the invention provides a nucleic acid molecule that encodes an isolated polypeptide comprising a heavy chain variable region sequence set forth in SEQ ID NO: 360 and a light chain variable region sequence set forth in SEQ ID NO: 361. In this embodiment, the heavy chain and light chain sequences (e.g., SEQ ID NO: 318 and SEQ ID NO: 319) can be connected by a linker peptide. In some embodiments, the nucleic acid molecule encodes a polypeptide which is a heavy chain variable domain. In some embodiments, the nucleic acid molecule encodes a polypeptide which is at least 85% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to SEQ ID NO: 318 or SEQ ID NO: 360. In some embodiments, the nucleic acid molecule encodes a polypeptide which is a light chain variable domain. In some embodiments, the nucleic acid molecule encodes a polypeptide which is at least 85% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) identical to SEQ ID NO: 319 or SEQ ID NO: 361. In some embodiments, the nucleic acid molecule encodes an antibody, or antigen-binding portion thereof, comprising a heavy chain variable domain amino acid sequence set forth in SEQ ID NO: 318, and a light chain variable domain amino acid sequence set forth in SEQ ID NO: 319. In some embodiments, the nucleic acid molecule encodes an antibody, or antigen-binding portion thereof, comprising a heavy chain variable domain amino acid sequence set forth in SEQ ID NO: 360, and a light chain variable domain amino acid sequence set forth in SEQ ID NO: 361.

In some cases, nucleic acids of the disclosure include codon-optimized nucleic acids. Methods of generating codon-optimized nucleic acids are known in the art and may include, but are not limited to those described in U.S. Pat. Nos. 5,786,464 and 6,114,148, the contents of each of which are herein incorporated by reference in their entirety. Also provided herein are expression vectors comprising any of the aforementioned nucleus acid(s).

Production of Antibodies that Bind a LTBP1/3-TGFβ1 Complex

The art is familiar with various techniques and methods that may be used for obtaining antibodies, or antigen-binding fragments thereof, of the disclosure. For example, antibodies can be produced using recombinant DNA methods, hybridoma techniques, phage or yeast display technologies, transgenic animals (e.g., a XenoMouse®) or some combination thereof.

Immunization and Hybridomas

In some methods described herein, the specified antigen (e.g., an LTBP1-TGFβ1 complex and/or an LTBP3-TGFβ1 complex) can be used to immunize a non-human animal (“host”), e.g., a rodent, e.g., a mouse, hamster, or rat. In one embodiment, the non-human animal is a mouse. In another embodiment, the host may be a camelid.

After immunization, which may include single or multiple steps of antigen exposures (e.g., injections), splenocytes are harvested from the animal and the associated B cells are fused with immortalized myeloma cells to form hybridomas for antibody production. Hybridomas may be generated in accordance with known methods (see e.g., Kohler and Milstein (1975) Nature, 256: 495-499). Hybridomas formed in this manner are then screened using standard methods, such as enzyme-linked immunosorbent assay (ELISA), Bio-Layer Interferometry (BLI) technology (e.g., OCTET) and surface plasmon resonance (e.g., BIACORE) analysis, to identify one or more hybridomas that produce an antibody that specifically binds to a specified antigen. Any form of the specified antigen may be used as the immunogen, e.g., recombinant antigen, naturally occurring forms, any variants or fragments thereof, as well as antigenic peptide thereof (e.g., any of the epitopes described herein as a linear epitope or within a scaffold as a conformational epitope).

Screening Library/Libraries

In some embodiments, the method or process of making or identifying antibodies includes a step of screening protein expression libraries that express antibodies or fragments thereof (e.g., scFv), e.g., phage, yeast, or ribosome display libraries. For example, a library of human combinatorial antibodies or scFv fragments can synthesized on phages or yeast, the library is then screened with the antigen of interest or an antibody-binding portion thereof, and the phage or yeast that binds the antigen is isolated, from which one may obtain the antibodies or immunoreactive fragments (Vaughan et a/., 1996, PMID: 9630891; Sheets et a/., 1998, PMID: 9600934; Boder et a/., 1997, PMID: 9181578; Pepper et a/., 2008, PMID: 18336206).

Phage display is further described, for example, in Ladner et al., U.S. Pat. No. 5,223,409; Smith (1985) Science 228:1315-1317; Clackson et al. (1991) Nature, 352: 624-628; Marks et al. (1991) J. Mol. Biol., 222: 581-597; WO 92/18619; WO 91/17271; WO 92/20791; WO 92/15679; WO 93/01288; WO 92/01047; WO 92/09690; and WO 90/02809. Yeast display is further described, for example, in U.S. Pat. Nos. 7,700,302 and 8,877,688. In particular methods, the yeast display library expresses full-length antibodies (e.g., Adimab, LLC).

Kits for generating phage or yeast display libraries are commercially available. There also are other methods and reagents that can be used in generating and screening antibody display libraries (see U.S. Pat. No. 5,223,409; WO 92/18619, WO 91/17271, WO 92/20791, WO 92/15679, WO 93/01288, WO 92/01047, WO 92/09690; and Barbas et a/., 1991, PMID: 1896445). Such techniques advantageously allow for the screening of large numbers of candidate antibodies.

No matter how obtained, antibody producing cells (e.g., yeast colonies, hybridomas, etc.) may be selected, cloned and further screened for desirable characteristics including, for example, robust growth, high antibody production and desirable antibody characteristics such as high affinity for the antigen of interest. Methods of selecting, cloning and expanding colonies and/or hybridomas are well known to those of ordinary skill in the art. Once the desired antibodies are identified, the relevant genetic material may be isolated, manipulated, and expressed using common, art-recognized molecular biology and biochemical techniques.

Humanization

The antibodies or fragments of the present invention are preferably fully human antibodies or humanized antibodies. Thus, whatever the source, it will be appreciated that the method may comprise humanizing one or more antibodies or fragments thereof, wherein the human antibody sequences may be fabricated using art-known molecular engineering techniques and introduced into expression systems and host cells as described herein. Such non-natural recombinantly produced human antibodies (and subject compositions) are entirely compatible with the teachings of this disclosure and are expressly held to be within the scope of the instant invention. In certain select aspects, the LTBP1-TGFβ1 complex-binding and/or a LTBP3-TGFβ1 complex-binding antibodies of the invention will comprise a recombinantly produced human antibody.

Affinity Maturation

In some embodiments, antibodies produced by the methods described-above may be of moderate affinity (Ka of about 10 6 to 10 7 M-1). Accordingly, antibodies or fragments thereof may be subjected to a process of affinity maturation as part of optimization, if desired. The term “affinity maturation” shall have the meaning readily understood by the skilled artisan. Briefly, it refers to further modifying the amino acid sequence of candidate antibodies or fragments (often referred to as “parent”) to achieve improved binding profiles to the specific antigen. Typically, a parental antibody and an affinity-matured counterpart (sometimes referred to as “progeny” or “offspring”) retain the same epitope. Suitable in vitro binding assays may be carried out to screen for improved binders at appropriate step(s) during the affinity maturation process. Optionally, in some embodiments, functional assays (e.g., cell-based potency assays) may also be performed to confirm desired functionality.

Affinity maturation typically involves sequence diversification and/or mutagenesis, whilst the exact means of introducing or generating mutations is not limiting. In some embodiments, mutagenesis comprises introducing one or more changes (e.g., substitutions or deletions) in amino acid residues of one or more CDRs. Accordingly, in some embodiments the VR or CDR sequences described herein may comprise up to one, two, three, four, five, or six amino acid changes. In some embodiments, the VR or CDR sequences described herein may comprise up to one, two, three, four, five, or six amino acid substitutions. In some embodiments, the VR or CDR sequences described herein may comprise up to one, two, three, or four deletions. Additionally or alternatively, mutagenesis may comprise so-called oligo-walking of variable regions or CDRs.

For example, affinity maturation of antibodies can be accomplished by a number of methods including random mutagenesis (Gram H., et al. Proc. Natl. Acad. Sci. U.S.A. (1992) 89, 3576-3580, and Hawkins R. E., et al. J. Mol. Biol. (1992) 226, 889-896), random mutagenesis of CDR sequences, e.g., CDR walking (Yang W. P., et al., J. Mol. Biol. (1995) 254, 392-403), directed mutagenesis of residues (Ho M., et al., J. Biol. Chem. (2005) 280, 607-617 and Ho M., et al., Proc. Natl. Acad. Sci. U.S.A. (2006) 103, 9637-9642), and approaches that reproduce somatic hypermutation (SHM) in vitro (Bowers P. M., et al., Proc. Natl. Acad. Sci. U.S.A. (2011) 108, 20455-20460).

In one embodiment, antibodies may be affinity matured by conducting mutagenesis on the variable heavy or variable light chain regions. In another embodiment, antibodies may be affinity matured by conducting mutagenesis on any one of the variable heavy chain CDRs or variable light chain CDRs. In another embodiment, antibodies may be affinity matured by conducting mutagenesis on the variable heavy chain CDR3 (e.g., CDR-H3 mutagenesis). In another embodiment, antibodies may be affinity matured by conducting mutagenesis on the variable heavy chain CDR2 (e.g., CDR-H2 mutagenesis). In another embodiment, antibodies may be affinity matured by conducting mutagenesis on the variable heavy chain CDR1 (e.g., CDR-H1 mutagenesis). In another embodiment, antibodies may be affinity matured by conducting mutagenesis on the variable light chain CDR3 (e.g., CDR-L3 mutagenesis). In another embodiment, antibodies may be affinity matured by conducting mutagenesis on the variable light chain CDR2 (e.g., CDR-L2 mutagenesis). In another embodiment, antibodies may be affinity matured by conducting mutagenesis on the variable light chain CDR1 (e.g., CDR-L1 mutagenesis).

In some embodiments, an antibody may be affinity matured and/or optimized by separately conducting mutagenesis on some or all of the three light chain CDRs (i.e., CDR-L1, CDR-L2, and CDR-L3) to generate up to three different libraries of antibodies, each having unique mutations in one of the three CDRs. The new antibodies can then be screened for improved properties (e.g., antigen-binding). Then, if further affinity improvements are desired, unique CDRs from each of the libraries can be mixed and matched to generate a new library of antibodies and screened for improved properties (e.g., antigen-binding). In some embodiments, affinity maturation involves screening an antibody library comprising variants of one or more CDRs (“repertoire”), which may be combined with at least one CDR of a parent antibody. This process is sometimes called CDR shuffling or CDR diversification. In some embodiments, a variable heavy chain CDR3 (e.g., CDR-H3) may be used to screen a library that contains variable heavy chain CDR1 and CDR2 repertoires of variants (a.k.a., CDR-H1/H2 diversification). In some embodiments, variable light chain CDR3 (e.g., CDR-L3) may be used to screen a library that contains variable light chain CDR1 and CDR2 repertoires of variants (a.k.a., CDR-L1/L2 diversification).

In some embodiments, affinity maturation involves screening an antibody library comprising light chain variants (“repertoire”), which may be combined with a heavy chain of a parent antibody (light-chain shuffling). For example, in some embodiments, selected heavy chains are introduced into an antibody library comprising light chain variants thereby producing a new library of antibodies that can be screened for improved affinity. In some embodiments, repertoires of naturally occurring variable region variants may be obtained from unimmunized donors. Examples of heavy or light-chain shuffling are described in the following documents: Marks et al., (1992) Nature Biotech 10: 779-78; Schier et al., (1996) J. Mol. Biol. 255, 28-43; Park et al., (2000) BBRC. 275. 553-557; and Chames et al., (2002) J. Immunol 1110-1118. In some embodiments, the light chain library comprises lambda light chains variants. In some embodiments, the light chain library comprises kappa light chains variants. In some embodiments, the light chain library comprises both lambda and kappa light chains variants.

It should be appreciated that the various methods for affinity maturation may be combined in any order. For example, in one embodiment, a select antibody may undergo heavy chain CDR-H1/H2 diversification, followed by CDR-H3 mutagenesis. In another embodiment, a select antibody may undergo heavy chain CDR-H1/H2 diversification, followed by CDR-H3 mutagenesis, followed by light chain shuffling. In another embodiment, a select antibody may undergo heavy chain CDR-H1/H2 diversification, followed by light chain shuffling, followed by CDR-H3 mutagenesis. In another embodiment, a select antibody may undergo light chain shuffling, followed by heavy chain CDR-H1/H2 diversification, followed by CDR-H3 mutagenesis. In another embodiment, a select antibody may undergo light chain shuffling, followed by heavy chain CDR-H1/H2 diversification, followed by CDR-H3 mutagenesis, followed by CDR-L3 mutagenesis.

In some embodiment, a select antibody may undergo light chain shuffling, followed by heavy chain CDR-H1/H2 diversification, followed by CDR-H3 mutagenesis, followed by CDR-L1, CDR-L2, and/or CDRL3 mutagenesis. In some embodiments, a select antibody may undergo heavy chain CDR-H1/H2 diversification, followed by CDR-H3 mutagenesis, followed by CDR-L1, CDR-L2, and/or CDRL3 mutagenesis. In some embodiments, light chain CDR mutagenesis is separately conducted on some or all of the three light chain CDRs (CDR-L1, CDR-L2, and CDRL3) to produce up to three libraries of antibodies. In some embodiments, the light chain CDRs from each of the libraries are mixed and matched to generate a new library of antibodies having unique combinations of light chain CDR mutations.

In any of the methods for affinity maturation described above, the resulting new antibodies may be selected for binding to the target antigen (e.g., a LTBP1-TGFβ1 complex and/or a LTBP3-TGFβ1 complex) using known techniques (e.g., FACS). Binding specificity and affinity using FACS may be tested by varying antigen concentration and/or competition for unlabeled (cold) antigen. Binding affinity can be further assessed using other techniques known in the art, such as ELISA, BLI (e.g., OCTET), and SPR (e.g., BIACORE).

In addition to the affinity maturation process discussed above, further optimization may be performed to achieve desired product profiles. Thus, antibodies may be further subjected to a step of optimization and selected based on certain physicochemical properties that are advantageous. For therapeutic antibodies (biologics), physicochemical criteria for developability that may be evaluated include, but are not limited to: solubility, stability, immunogenicity, lack of self-association or aggregation, Fc functionality, internalization profiles, pH-sensitivity, glycosylation and manufacturability such as cell viability and/or gene expression. In some embodiments, the process of optimization involves mutagenesis of one or more amino acid sequences within the constant regions.

In one aspect, the invention provides a method for making a composition comprising an antibody, or antigen-binding fragment thereof, that specifically binds a human LTBP1-proTGFβ1 complex and/or a human LTBP3-proTGFβ1 complex, and does not bind a human GARP-proTGFβ1 complex; wherein the antibody, or antigen-binding fragment thereof, inhibits TGFβ1 but does not inhibit TGFβ2 or TGFβ3, the method comprising steps of i) providing at least one antigen comprising LTBP1-proTGFβ1 and/or LTBP3-proTGFβ31, ii) selecting a first pool of antibodies, or antigen-binding fragments thereof, that specifically bind the at least one antigen of step (i) so as to provide specific binders of LTBP1-proTGFβ1 and/or LTBP3-proTGFβ31; iii) selecting a second pool of antibodies, or antigen-binding fragments thereof, that inhibit activation of TGFβ1, so as to generate specific inhibitors of TGFβ1 activation; iv) formulating an antibody, or antigen-binding fragment thereof, that is present in the first pool of antibodies and the second pool of antibodies into a pharmaceutical composition, thereby making the composition comprising the antibody, or antigen-binding fragment thereof.

In one embodiment, the method further comprises a step of removing from the first pool of antibodies, or antigen-binding fragments thereof, any antibodies, or antigen-binding fragments thereof, that bind GARP-proTGFβ1, LRRC33-proTGFβ1, mature TGFβ1, GARP-proTGFβ2, LRRC33-proTGFβ2, mature TGFβ2, GARP-proTGFβ3, LRRC33-proTGFβ3, mature TGFβ3, or any combinations thereof. In one embodiment, the method further comprises a step of determining or confirming isoform-specificity of the antibodies, or antigen-binding fragments thereof, selected in steps (ii) and/or (iii). In one embodiment, the method further comprises a step of selecting for antibodies, or antigen-binding fragments thereof, that are cross-reactive to human and rodent antigens. In one embodiment, the method further comprises a step of generating a fully human or humanized antibody, or antigen-binding fragment thereof, of the antibody, or antigen-binding fragment thereof, that is present in the first pool of antibodies and the second pool of antibodies.

In one embodiment, the method further comprises a step of subjecting the antibody, or antigen-binding fragment thereof, that is present in the first pool of antibodies and/or the second pool of antibodies to affinity maturation and/or optimization, so as to provide an affinity matured and/or optimized antibody or fragment thereof. In one embodiment, the affinity maturation and/or optimization comprises a step of subjecting the antibody, or antigen-binding fragment thereof, to light chain shuffling as described herein. In one embodiment, the affinity maturation and/or optimization comprises the step of subjecting the antibody, or antigen-binding fragment thereof, to CDR H1/H2 diversification as described herein. In one embodiment, the affinity maturation and/or optimization comprises the step of subjecting the antibody, or antigen-binding fragment thereof, to CDR-H3 mutagenesis as described herein. In one embodiment, the affinity maturation and/or optimization comprises the step of subjecting antibody, or antigen-binding fragment thereof, to light chain CDR mutagenesis as described herein. In one embodiment, the affinity maturation and/or optimization comprises the step of subjecting the antibody, or antigen-binding fragment thereof, to light chain CDR L1/L2 diversification as described herein.

Further optimization steps may be carried out to provide physicochemical properties that are advantageous for therapeutic compositions. Such steps may include, but are not limited to, mutagenesis or engineering to provide improved solubility, lack of self-aggregation, stability, pH sensitivity, Fc function, and so on.

In one embodiment, the method further comprises a step of determining affinity of the antibodies, or antigen-binding fragments thereof, to human LTBP1-proTGFβ1 and/or human LTBP3-proTGFβ1. In some embodiments, the method further comprises a step of removing from the first and/or second pools of antibodies, or antigen-binding fragments thereof, any antibodies, or antigen-binding fragments thereof, that bind to human LTBP1-proTGFβ1 and/or human LTBP3-proTGFβ1 with a K D of >100 nM, as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI). In some embodiments, the method further comprises a step of removing from the first and/or second pools of antibodies, or antigen-binding fragments thereof, any antibodies, or antigen-binding fragments thereof, that bind to human LTBP1-proTGFβ1 and/or human LTBP3-proTGFβ1 with a K D of >50 nM, as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI). In some embodiments, the method further comprises a step of removing from the first and/or second pools of antibodies, or antigen-binding fragments thereof, any antibodies, or antigen-binding fragments thereof, that bind to human LTBP1-proTGFβ1 and/or human LTBP3-proTGFβ1 with a K D of >25 nM, as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI). In some embodiments, the method further comprises a step of removing from the first and/or second pools of antibodies, or antigen-binding fragments thereof, any antibodies, or antigen-binding fragments thereof, that bind to human LTBP1-proTGFβ1 and/or human LTBP3-proTGFβ1 with a K D of >10 nM, as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI).

In one embodiment, the method further comprises a step of determining affinity of the antibodies, or antigen-binding fragments thereof, from the first and/or second pools of antibodies to mouse LTBP1-proTGFβ1 and/or mouse LTBP3-proTGFβ1. In some embodiments, the method further comprises a step of removing from the first and/or second pools of antibodies, or antigen-binding fragments thereof, any antibodies, or antigen-binding fragments thereof, that bind to mouse LTBP1-proTGFβ1 and/or mouse LTBP3-proTGFβ1 with a K D of >100 nM, as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI). In some embodiments, the method further comprises a step of removing from the first and/or second pools of antibodies, or antigen-binding fragments thereof, any antibodies, or antigen-binding fragments thereof, that bind to mouse LTBP1-proTGFβ1 and/or mouse LTBP3-proTGFβ1 with a K D of >50 nM, as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI). In some embodiments, the method further comprises a step of removing from the first and/or second pools of antibodies, or antigen-binding fragments thereof, any antibodies, or antigen-binding fragments thereof, that bind to mouse LTBP1-proTGFβ1 and/or mouse LTBP3-proTGFβ1 with a K D of >25 nM, as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI). In some embodiments, the method further comprises a step of removing from the first and/or second pools of antibodies, or antigen-binding fragments thereof, any antibodies, or antigen-binding fragments thereof, that bind to mouse LTBP1-proTGFβ1 and/or mouse LTBP3-proTGFβ1 with a K D of >10 nM, as measured in a suitable in vitro binding assay such as Bio-Layer Interferometry (BLI).

In one embodiment, the method further comprises a step of removing from the first and/or second pool of antibodies, or antigen-binding fragments thereof, any antibodies, or antigen-binding fragments thereof, that do not bind mouse LTBP1-proTGFβ1 and/or mouse LTBP3-proTGFβ1.

In one embodiment, the method further comprises a step of determining the IC 50 of the antibodies, or antigen-binding fragments thereof, from the first and/or second pools of antibodies, or antigen-binding fragments thereof, as measured by a suitable functional in vitro cell-based assay such as a caga assay, as described herein. In some embodiments, the method comprises the step of removing antibodies, or antigen-binding fragments thereof, from the first and/or second pools of antibodies, or antigen-binding fragments thereof, that have an IC 50 of greater than 50 nM as measured by a caga assay as described herein. In some embodiments, the method comprises the step of removing antibodies, or antigen-binding fragments thereof, from the first and/or second pools of antibodies, or antigen-binding fragments thereof, that have an IC 50 of greater than 25 nM as measured by a caga assay as described herein. In some embodiments, the method comprises the step of removing antibodies, or antigen-binding fragments thereof, from the first and/or second pools of antibodies, or antigen-binding fragments thereof, that have an IC 50 of greater than 10 nM as measured by a caga assay as described herein. In some embodiments, the method comprises the step of removing antibodies, or antigen-binding fragments thereof, from the first and/or second pools of antibodies, or antigen-binding fragments thereof, that have an IC 50 of greater than 5 nM as measured by a caga assay as described herein.

In some embodiments, the method comprises the step of removing antibodies, or antigen-binding fragments thereof, from the first and/or second pools of antibodies, or antigen-binding fragments thereof, that have an IC 50 of greater than 100 nM as measured by an endogenous LTBP caga assay as described herein. In some embodiments, the method comprises the step of removing antibodies, or antigen-binding fragments thereof, from the first and/or second pools of antibodies, or antigen-binding fragments thereof, that have an IC 50 of greater than 50 nM as measured by an endogenous LTBP caga assay as described herein. In some embodiments, the method comprises the step of removing antibodies, or antigen-binding fragments thereof, from the first and/or second pools of antibodies, or antigen-binding fragments thereof, that have an IC 50 of greater than 25 nM as measured by an endogenous LTBP caga assay as described herein. In some embodiments, the method comprises the step of removing antibodies, or antigen-binding fragments thereof, from the first and/or second pools of antibodies, or antigen-binding fragments thereof, that have an IC 50 of greater than 10 nM as measured by an endogenous LTBP caga assay as described herein. In some embodiments, the method comprises the step of removing antibodies, or antigen-binding fragments thereof, from the first and/or second pools of antibodies, or antigen-binding fragments thereof, that have an IC 50 of greater than 5 nM as measured by an endogenous LTBP caga assay as described herein.

In some embodiments, the method comprises the step of removing antibodies, or antigen-binding fragments thereof, from the first and/or second pools of antibodies, or antigen-binding fragments thereof, that have an IC 50 of greater than 100 nM as measured by a human LTBP overexpression caga assay as described herein. In some embodiments, the method comprises the step of removing antibodies, or antigen-binding fragments thereof, from the first and/or second pools of antibodies, or antigen-binding fragments thereof, that have an IC 50 of greater than 50 nM as measured by a human LTBP overexpression caga assay as described herein. In some embodiments, the method comprises the step of removing antibodies, or antigen-binding fragments thereof, from first and/or second pools of antibodies, or antigen-binding fragments thereof, that have an IC 50 of greater than 25 nM as measured by a human LTBP overexpression caga assay as described herein. In some embodiments, the method comprises the step of removing antibodies, or antigen-binding fragments thereof, from the first and/or second pools of antibodies, or antigen-binding fragments thereof, that have an IC 50 of greater than 10 nM as measured by a human LTBP overexpression caga assay as described herein. In some embodiments, the method comprises the step of removing antibodies, or antigen-binding fragments thereof, from the first and/or second pools of antibodies, or antigen-binding fragments thereof, that have an IC 50 of greater than 5 nM as measured by a human LTBP overexpression caga assay as described herein.

In some embodiments, the method comprises the step of removing antibodies, or antigen-binding fragments thereof, from the first and/or second pools of antibodies, or antigen-binding fragments thereof, that have an IC 50 of greater than 100 nM as measured by a murine LTBP overexpression caga assay as described herein. In some embodiments, the method comprises the step of removing antibodies, or antigen-binding fragments thereof, from the first and/or second pools of antibodies, or antigen-binding fragments thereof, that have an IC 50 of greater than 50 nM as measured by a murine LTBP overexpression caga assay as described herein. In some embodiments, the method comprises the step of removing antibodies, or antigen-binding fragments thereof, from first and/or second pools of antibodies, or antigen-binding fragments thereof, that have an IC 50 of greater than 25 nM as measured by a murine LTBP overexpression caga assay as described herein. In some embodiments, the method comprises the step of removing antibodies, or antigen-binding fragments thereof, from first and/or second pools of antibodies, or antigen-binding fragments thereof, that have an IC 50 of greater than 10 nM as measured by a murine LTBP overexpression caga assay as described herein. In some embodiments, the method comprises the step of removing antibodies, or antigen-binding fragments thereof from first and/or second pools of antibodies, or antigen-binding fragments thereof, that have an IC 50 of greater than 5 nM as measured by a murine LTBP overexpression caga assay as described herein.

In another embodiment, a monoclonal antibody is obtained from the non-human animal, and then modified, e.g., made chimeric, using suitable recombinant DNA techniques. A variety of approaches for making chimeric antibodies have been described. See e.g., Morrison et al., Proc. Natl. Acad. Sci. U.S.A. 81:6851, 1985; Takeda et al., Nature 314:452, 1985, Cabilly et al., U.S. Pat. No. 4,816,567; Boss et al., U.S. Pat. No. 4,816,397; Tanaguchi et al., European Patent Publication EP171496; European Patent Publication 0173494, United Kingdom Patent GB 2177096B.

For additional antibody production techniques, see, e.g., Antibodies: A Laboratory Manual, eds. Harlow et al., Cold Spring Harbor Laboratory, 1988. The present disclosure is not necessarily limited to any particular source, method of production, or other special characteristics of an antibody.

Some aspects of the present disclosure relate to host cells transformed with a polynucleotide or vector. Host cells may be a prokaryotic or eukaryotic cell. The polynucleotide or vector which is present in the host cell may either be integrated into the genome of the host cell or it may be maintained extrachromosomally. The host cell can be any prokaryotic or eukaryotic cell, such as a bacterial, insect, fungal, plant, animal or human cell. In some embodiments, fungal cells are, for example, those of the genus Saccharomyces, in particular those of the species S. cerevisiae. The term “prokaryotic” includes all bacteria which can be transformed or transfected with a DNA or RNA molecules for the expression of an antibody or the corresponding immunoglobulin chains. Prokaryotic hosts may include gram negative as well as gram positive bacteria such as, for example, E. coli, S. typhimurium, Serratia marcescens and Bacillus subtilis . The term “eukaryotic” includes yeast, higher plants, insects and vertebrate cells, e.g., mammalian cells, such as NSO and CHO cells. Depending upon the host employed in a recombinant production procedure, the antibodies or immunoglobulin chains encoded by the polynucleotide may be glycosylated or may be non-glycosylated. Antibodies or the corresponding immunoglobulin chains may also include an initial methionine amino acid residue.

In some embodiments, once a vector has been incorporated into an appropriate host, the host may be maintained under conditions suitable for high level expression of the nucleotide sequences, and, as desired, the collection and purification of the immunoglobulin light chains, heavy chains, light/heavy chain dimers or intact antibodies, antigen-binding fragments or other immunoglobulin forms may follow; see, Beychok, Cells of Immunoglobulin Synthesis, Academic Press, N.Y., (1979). Thus, polynucleotides or vectors are introduced into the cells which in turn produce the antibody or antigen-binding fragments. Furthermore, transgenic animals, preferably mammals, comprising the aforementioned host cells may be used for the large scale production of the antibody or antibody fragments.

The transformed host cells can be grown in fermenters and cultured using any suitable techniques to achieve optimal cell growth. Once expressed, the whole antibodies, their dimers, individual light and heavy chains, other immunoglobulin forms, or antigen-binding fragments, can be purified according to standard procedures of the art, including ammonium sulfate precipitation, affinity columns, column chromatography, gel electrophoresis and the like; see, Scopes, “Protein Purification”, Springer Verlag, N.Y. (1982). The antibody or antigen-binding fragments can then be isolated from the growth medium, cellular lysates, or cellular membrane fractions. The isolation and purification of the, e.g., microbially expressed antibodies or antigen-binding fragments may be by any conventional means such as, for example, preparative chromatographic separations and immunological separations such as those involving the use of monoclonal or polyclonal antibodies directed, e.g., against the constant region of the antibody.

Aspects of the disclosure relate to a hybridoma, which provides an indefinitely prolonged source of monoclonal antibodies. As an alternative to obtaining immunoglobulins directly from the culture of hybridomas, immortalized hybridoma cells can be used as a source of rearranged heavy chain and light chain loci for subsequent expression and/or genetic manipulation. Rearranged antibody genes can be reverse transcribed from appropriate mRNAs to produce cDNA. In some embodiments, heavy chain constant region can be exchanged for that of a different isotype or eliminated altogether. The variable regions can be linked to encode single chain Fv regions. Multiple Fv regions can be linked to confer binding ability to more than one target or chimeric heavy and light chain combinations can be employed. Any appropriate method may be used for cloning of antibody variable regions and generation of recombinant antibodies.

In some embodiments, an appropriate nucleic acid that encodes variable regions of a heavy and/or light chain is obtained and inserted into an expression vectors which can be transfected into standard recombinant host cells. A variety of such host cells may be used. In some embodiments, mammalian host cells may be advantageous for efficient processing and production. Typical mammalian cell lines useful for this purpose include CHO cells, 293 cells, or NSO cells. The production of the antibody or antigen-binding fragment may be undertaken by culturing a modified recombinant host under culture conditions appropriate for the growth of the host cells and the expression of the coding sequences. The antibodies or antigen-binding fragments may be recovered by isolating them from the culture. The expression systems may be designed to include signal peptides so that the resulting antibodies are secreted into the medium; however, intracellular production is also possible.

The disclosure also includes a polynucleotide encoding at least a variable region of an immunoglobulin chain of the antibodies described herein. In some embodiments, the variable region encoded by the polynucleotide comprises at least one complementarity determining region (CDR) of the VH and/or VL of the variable region of the antibody produced by any one of the above described hybridomas.

Polynucleotides encoding antibody or antigen-binding fragments may be, e.g., DNA, cDNA, RNA or synthetically produced DNA or RNA or a recombinantly produced chimeric nucleic acid molecule comprising any of those polynucleotides either alone or in combination. In some embodiments, a polynucleotide is part of a vector. Such vectors may comprise further genes such as marker genes which allow for the β the vector in a suitable host cell and under suitable conditions.

In some embodiments, a polynucleotide is operatively linked to expression control sequences allowing expression in prokaryotic or eukaryotic cells. Expression of the polynucleotide comprises transcription of the polynucleotide into a translatable mRNA. Regulatory elements ensuring expression in eukaryotic cells, preferably mammalian cells, are well known to those skilled in the art. They may include regulatory sequences that facilitate initiation of transcription and optionally poly-A signals that facilitate termination of transcription and stabilization of the transcript. Additional regulatory elements may include transcriptional as well as translational enhancers, and/or naturally associated or heterologous promoter regions. Possible regulatory elements permitting expression in prokaryotic host cells include, e.g., the PL, Lac, Trp or Tac promoter in E. coli , and examples of regulatory elements permitting expression in eukaryotic host cells are the AOX1 or GAL 1 promoter in yeast or the CMV-promoter, SV40-promoter, RSV-promoter (Rous sarcoma virus), CMV-enhancer, SV40-enhancer or a globin intron in mammalian and other animal cells.

Beside elements which are responsible for the initiation of transcription such regulatory elements may also include transcription termination signals, such as the SV40-poly-A site or the tk-poly-A site, downstream of the polynucleotide. Furthermore, depending on the expression system employed, leader sequences capable of directing the polypeptide to a cellular compartment or secreting it into the medium may be added to the coding sequence of the polynucleotide and have been described previously. The leader sequence(s) is (are) assembled in appropriate phase with translation, initiation and termination sequences, and preferably, a leader sequence capable of directing secretion of translated protein, or a portion thereof, into, for example, the extracellular medium. Optionally, a heterologous polynucleotide sequence can be used that encode a fusion protein including a C- or N-terminal identification peptide imparting desired characteristics, e.g., stabilization or simplified purification of expressed recombinant product.

In some embodiments, polynucleotides encoding at least the variable domain of the light and/or heavy chain may encode the variable domains of both immunoglobulin chains or only one. Likewise, polynucleotides may be under the control of the same promoter or may be separately controlled for expression. Furthermore, some aspects relate to vectors, particularly plasmids, cosmids, viruses and bacteriophages used conventionally in genetic engineering that comprise a polynucleotide encoding a variable domain of an immunoglobulin chain of an antibody or antigen-binding fragment; optionally in combination with a polynucleotide that encodes the variable domain of the other immunoglobulin chain of the antibody.

In some embodiments, expression control sequences are provided as eukaryotic promoter systems in vectors capable of transforming or transfecting eukaryotic host cells, but control sequences for prokaryotic hosts may also be used. Expression vectors derived from viruses such as retroviruses, vaccinia virus, adeno-associated virus, herpes viruses, or bovine papilloma virus, may be used for delivery of the polynucleotides or vector into targeted cell population (e.g., to engineer a cell to express an antibody or antigen-binding fragment). A variety of appropriate methods can be used to construct recombinant viral vectors. In some embodiments, polynucleotides and vectors can be reconstituted into liposomes for delivery to target cells. The vectors containing the polynucleotides (e.g., the heavy and/or light variable domain(s) of the immunoglobulin chains encoding sequences and expression control sequences) can be transferred into the host cell by suitable methods, which vary depending on the type of cellular host.

Modifications

Antibodies, or antigen-binding portions thereof, of the disclosure may be modified with a detectable label or detectable moiety, including, but not limited to, an enzyme, prosthetic group, fluorescent material, luminescent material, bioluminescent material, radioactive material, positron emitting metal, nonradioactive paramagnetic metal ion, and affinity label for detection and/or isolation of a LTBP1-TGFβ1 complex or a LTBP3-TGFβ1 complex. The detectable substance or moiety may be coupled or conjugated either directly to the polypeptides of the disclosure or indirectly, through an intermediate (such as, for example, a linker (e.g., a cleavable linker)) using suitable techniques. Non-limiting examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, or acetylcholinesterase; non-limiting examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; non-limiting examples of suitable fluorescent materials include biotin, umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, or phycoerythrin; an example of a luminescent material includes luminol; non-limiting examples of bioluminescent materials include luciferase, luciferin, and aequorin; and examples of suitable radioactive material include a radioactive metal ion, e.g., alpha-emitters or other radioisotopes such as, for example, iodine ( 131 I, 125 I, 123 I, 121 I) carbon ( 14 C), sulfur ( 35 S), tritium ( 3 H), indium ( 115 mIn, 113 mIn, 112 In, 111 In), and technetium ( 99 Tc, 99 mTc), thallium ( 201 Ti), gallium ( 68 Ga, 67 Ga), palladium ( 103 Pd) molybdenum ( 99 Mo), xenon ( 133 Xe), fluorine ( 18 F), 153 Sm, Lu, 159 Gd, 149 Pm, 140 La, 175 Yb, 166Ho, 90 Y, 47 Sc, 86 R, 188 Re, 142 Pr, 105 Rh, 97 Ru, 68 Ge, 57 Co, 65 Zn, 85 Sr, 32 P, 153 Gd, 169 Yb, 51 Cr, 54 Mn, 75 Se, and tin ( 113 Sn, 117 Sn). The detectable substance may be coupled or conjugated either directly to the antibodies of the disclosure that bind selectively to a LTBP1-TGFβ1 complex and/or a LTBP3-TGFβ1 complex, or indirectly, through an intermediate (such as, for example, a linker) using suitable techniques. Any of the antibodies provided herein that are conjugated to a detectable substance may be used for any suitable diagnostic assays, such as those described herein.

In addition, antibodies, or antigen-binding portions thereof, of the disclosure may also be modified with a drug to form, e.g., an antibody-drug conjugate. The drug may be coupled or conjugated either directly to the polypeptides of the disclosure, or indirectly, through an intermediate (such as, for example, a linker (e.g., a cleavable linker)) using suitable techniques.

Targeting Agents

In some embodiments methods of the present disclosure comprise the use of one or more targeting agents to target an antibody, or antigen-binding portion thereof, as disclosed herein, to a particular site in a subject for purposes of enriching or localizing such agent(s) to a niche of interest. In some embodiments, such targeting may achieve modulating mature TGFβ release from a LTBP1-TGFβ1 complex and/or a LTBP3-TGFβ1 complex. For example, LTBP1-TGFβ1 and LTBP3-TGFβ1 complexes are typically localized to extracellular matrix. Thus, in some embodiments, antibodies disclosed herein can be conjugated to extracellular matrix targeting agents for purposes of localizing the antibodies to sites where LTBP1-TGFβ1 and LTBP3-TGFβ1 complexes reside. In such embodiments, selective targeting of antibodies leads to selective modulation of LTBP1-TGFβ1 and/or LTBP3-TGFβ1 complexes. In some embodiments, selective targeting of antibodies leads to selective inhibition of LTBP1-TGFβ1 and/or LTBP3-TGFβ1 complexes (e.g., for purposes of treating fibrosis). In some embodiments, extracellular matrix targeting agents include heparin binding agents, matrix metalloproteinase binding agents, lysyl oxidase binding domains, fibrillin-binding agents, hyaluronic acid binding agents, and others.

In some embodiments, bispecific antibodies may be used having a first portion that selectively binds a LTBP1/3-TGFβ1 complex and a second portion that selectively binds a component of a target site, e.g., a component of the ECM (e.g., fibrillin).

In some embodiments, such a target agent may be coupled to another agent or therapeutics to carry or localize the complex to a niche of interest.

Safety/Toxicity Considerations

Histopathology, Toxicology

As mentioned above, known pan-inhibitors that antagonize all TGFβ isoforms, namely, TGFβ1, TGFβ2 and TGFβ3, have been documented to cause various toxicities across multiple mammalian species. Most notable known toxicities include cardiovascular toxicities (such as valvulopathy) and epithelial hyperplasia, skin lesions, inflammation and bleeding. More specifically, some of the observed toxicities associated with pan-TGFβ inhibitors (e.g., small molecule antagonists of the TGFβR and non-selective neutralizing antibodies) reported in the literature include the following.

Cardiovascular toxicities associated with TGFβ inhibition include, hyperplasia in aortic valve, right AV valve, and left AV valve; inflammation in aortic valve, left AV valve, and ascending aorta; hemorrhage in ascending aorta, aortic valve and left AV valve; connective tissue degeneration in ascending aorta (see for example, Strauber et al. (2014) “Nonclinical safety evaluation of a Transforming Growth Factor β receptor I kinase inhibitor in Fischer 344 rats and beagle dogs” J. Clin. Pract 4(3): 1000196).

In addition, neutralizing antibodies that bind all three TGFβ isoforms have been associated with certain epithelial toxicities, which are summarized in the table below.

Applicant of the present disclosure previously demonstrated the improved safety profiles of monoclonal antibodies that selectively block the activation step of TGFβ1 by targeting latent proTGFβ1 complex (see, for example, WO 2017/156500 and WO 2018/129329). In rat toxicology studies described therein, there were no observable test article-related toxicities when the animals were dosed with the inhibitors up to 100 mg/kg per week for 4 weeks.

Building upon the earlier recognition by the applicant of the present disclosure (see PCT/US2017/021972) that lack of isoform-specificity of conventional TGFβ antagonists may underlie the source of toxicities associated with TGFβ inhibition, the present inventors sought to further achieve context-selective TGFβ1 inhibition for treating various diseases that manifest TGFβ1 dysregulation, particularly fibrotic conditions, with enhanced safety/tolerability. The work presented herein therefore further provided, among high-affinity inhibitors, a subset of antibodies with particularly low dissociation rates (k OFF ) in order to improve durability.

Thus, in some embodiments, the novel antibody according to the present disclosure has the maximally tolerated dose (MTD) of >100 mg/kg when dosed weekly for at least 4 weeks (e.g., 4, 6, 8, 10, 12 weeks). In some embodiments, the novel antibody according to the present disclosure has the no observed adverse effect level (NOAEL) of up to 100 mg/kg when dosed weekly for at least 4 weeks in rats. In some embodiments, the antibody has a NOAEL of at least 100 mg/kg/week when dosed for 4 weeks or 12 weeks in mice. Suitable animal models to be used for conducting safety/toxicology studies for TGFβ inhibitors and TGFβ1 inhibitors include, but are not limited to: rats, dogs, cynos, and mice. In preferred embodiments, the minimum effective amount of the antibody based on a suitable preclinical efficacy study is below the NOAEL. More preferably, the minimum effective amount of the antibody is about one-third or less of the NOAEL. In particularly preferred embodiments, the minimum effective amount of the antibody is about one-sixth or less of the NOAEL. In some embodiments, the minimum effective amount of the antibody is about one-tenth or less of the NOAEL.

In some embodiments, the invention encompasses an isoform-selective antibody capable of inhibiting TGFβ1 signaling, which, when administered to a subject, does not cause cardiovascular or known epithelial toxicities at a dose effective to treat a TGFβ1-related indication. In some embodiments, the antibody has a minimum effective amount of about 3-10 mg/kg administered weekly, biweekly or monthly. Preferably, the antibody causes no to minimum toxicities at a dose that is at least six-times the minimum effective amount (e.g., a six-fold therapeutic window). More preferably, the antibody causes no to minimum toxicities at a dose that is at least ten-times the minimum effective amount (e.g., a ten-fold therapeutic window). Even more preferably, the antibody causes no to minimum toxicities at a dose that is at least fifteen-times the minimum effective amount (e.g., a fifteen-fold therapeutic window).

Thus, selection of an antibody or an antigen-binding fragment thereof for therapeutic use may include: selecting an antibody or antigen-binding fragment that meets the criteria of one or more of TGFβ inhibitors (such as monoclonal antibodies and antigen-binding fragments selected for example for having slow dissociation rates, e.g., k OFF of <5×10 (1/s)); carrying out an in vivo efficacy study in a suitable preclinical model to determine an effective amount of the antibody or the fragment; carrying out an in vivo safety/toxicology study in a suitable model to determine an amount of the antibody that is safe or toxic (e.g., MTD, NOAEL, or any art-recognized parameters for evaluating safety/toxicity); and, selecting the antibody or the fragment that provides at least a three-fold therapeutic window (preferably 6-fold, more preferably a 10-fold therapeutic window, even more preferably a 15-fold therapeutic window). The selected antibody or the fragment may be used in the manufacture of a pharmaceutical composition comprising the antibody or the fragment. Such pharmaceutical composition may be used in the treatment of a TGFβ1 indication in a subject as described herein. For example, the TGFβ1 indication may be a fibrotic disorder. Preferably, a TGFβ3 inhibitor to be selected for therapeutic use or large-scale manufacture, does not produce observable adverse effects in the treated animals after at least 4 week, e.g., 8 weeks, and 12 weeks, of sustained exposure. In some embodiments, certain toxicities observed in histopathological analyses are considered non-adverse.

Immune Safety Assessment

Cytokines play an important role in normal immune responses, but when the immune system is triggered to become hyperactive, the positive feedback loop of cytokine production can lead to a “cytokine storm” or hypercytokinemia, a situation in which excessive cytokine production causes an immune response that can damage organs, especially the lungs and kidneys, and even lead to death. Such condition is characterized by markedly elevated proinflammatory cytokines in the serum. Historically, a Phase 1 Trial of the anti-CD28 monoclonal antibody TGN1412 in healthy volunteers led to a life-threatening “cytokine storm” response resulted from an unexpected systemic and rapid induction of proinflammatory cytokines (Suntharalingam G et al. N Engl J Med. 2006 Sep. 7; 355(10):1018-28). This incident prompted heightened awareness of the potential danger associated with pharmacologic stimulation of T cells.

Whilst TGFβ-directed therapies do not target a specific T cell receptor or its ligand, it is contemplated that it is prudent to carry out immune safety assessment, including, for example, in vitro cytokine release assays, in vivo cytokine measurements from plasma samples of non-human primate treated with a TGFβ3 inhibitor, and platelet assays using human platelets.

In some embodiments, selection of a TGFβ3 inhibitor for therapeutic use and/or large-scale production thereof includes an assessment of the ability for the TGFβ3 inhibitor to trigger cytokine release from cytokine-producing cells. In such an assessment, one or more of the cytokines (e.g., inflammatory cytokines) IL-2, TNFα, IFNγ, IL-1β, CCL2 (MCP-1), and IL-6 may be assayed. In some embodiments, the cytokine-producing cells may include peripheral blood mononuclear cell (PBMC) constituents from heathy donors. Cytokine response after exposure to the TGFβ3 inhibitor (such as an antibody disclosed) herein may be compared to release after exposure to a control, e.g., an IgG isotype negative control, or any other suitable control depending on the TGFβ3 inhibitor being tested. Cytokine activation may be assessed in plate-bound (e.g., immobilized) and/or soluble assay formats. Levels of IFNγ, IL-2, IL-1β, TNFα, IL-6, and CCL2 (MCP-1) should not exceed 10-fold, e.g., 8-, 6-, 4-, or 2-fold the activation in the negative control. In some embodiments, a positive control may also be used to confirm cytokine activation in the sample, e.g., in the PBMCs. In some embodiments, these in vitro cytokine release results may be further confirmed in vivo, e.g., in an animal model such as a monkey toxicology study, e.g., a 4-week GLP or non-GLP repeat-dose monkey study.

In some embodiments, selection of an antibody or an antigen-binding fragment thereof for therapeutic use may include: identifying an antibody or antigen-binding fragment that meets the criteria of one or more of those described herein; carrying out an in vivo efficacy study in a suitable preclinical model to determine an effective amount of the antibody or the fragment; carrying out an in vivo safety/toxicology study in a suitable model to determine an amount of the antibody that is safe or toxic (e.g., MTD, NOAEL, or any art-recognized parameters for evaluating safety/toxicity); and, selecting the antibody or the fragment that provides at least a three-fold therapeutic window (preferably 6-fold, more preferably a 10-fold therapeutic window, even more preferably a 15-fold therapeutic window). In certain embodiments, the in vivo efficacy study is carried out in two or more suitable preclinical models that recapitulate human conditions. In some embodiments, such preclinical models comprise TGFβ1-positive fibrosis. In some embodiments, the preclinical models are selected from liver fibrosis model, kidney fibrosis model, lung fibrosis model, heart (cardiac) fibrosis model, skin fibrosis model.

Identification of an antibody or antigen-binding fragment thereof for therapeutic use may further include carrying out an immune safety assay, which may include, but is not limited to, measuring cytokine release and/or determining the impact of the antibody or antigen-binding fragment on platelet binding, activation, and/or aggregation. In certain embodiments, cytokine release may be measured in vitro using PBMCs or in vivo using a preclinical model such as non-human primates. In certain embodiments, the antibody or antigen-binding fragment thereof does not induce a greater than 10-fold release in IL-6, IFNγ, and/or TNFα levels as compared to levels in an IgG control sample in the immune safety assessment. In certain embodiments, assessment of platelet binding, activation, and aggregation may be carried out in vitro using PBMCs. In some embodiments, the antibody or antigen-binding fragment thereof does not induce a more than 10% increase in platelet binding, activation, and/or aggregation as compared to buffer or isotype control in the immune safety assessment.

The selected antibody or the fragment may be used in the manufacture of a pharmaceutical composition comprising the antibody or the fragment. Such pharmaceutical composition may be used in the treatment of a TGFβ indication in a subject as described herein. For example, the TGFβ indication may be a fibrotic disorder, such as organ fibrosis, e.g., liver fibrosis. Thus, the invention includes a method for manufacturing a pharmaceutical composition comprising a TGFβ inhibitor, wherein the method includes the step of selecting a TGFβ inhibitor which is tested for immune safety as assessed by immune safety assessment comprising a cytokine release assay and optionally further comprising a platelet assay. The TGFβ inhibitor selected by the method does not trigger unacceptable levels of cytokine release, as compared to control (such as IgG control). Similarly, the TGFβ inhibitor selected by the method does not cause unacceptable levels of platelet aggregation, platelet activation and/or platelet binding. Such TGFβ inhibitor is then manufactured at large-scale, for example 250 L or greater, e.g., 1000L, 2000L, 3000L, 4000L or greater, for commercial production of the pharmaceutical composition comprising the TGFβ inhibitor.

Pharmaceutical Compositions

The invention further provides pharmaceutical compositions used as a medicament suitable for administration in human and non-human subjects. One or more antibodies that selectively binds an LTBP1-TGFβ1 complex and/or an LTBP3-TGFβ1 complex can be formulated or admixed with a pharmaceutically acceptable carrier (excipient), including, for example, a buffer, to form a pharmaceutical composition. Such formulations may be used for the treatment of a disease or disorder that involves TGFβ signaling or dysregulation thereof. In some embodiments, such disease or disorder associated with TGFβ signaling involves one or more contexts, i.e., the TGFβ is associated with a particular type or types of presenting molecules. In some embodiments, such context occurs in a cell type-specific and/or tissue-specific manner. In some embodiments, for example, such context-dependent action of TGFβ signaling is mediated in part via GARP, LRRC33, LTBP1 and/or LTBP3.

In some embodiments, the antibody of the present invention binds selectively to a single context of TGFβ, such that the antibody binds TGFβ in a complex with LTBP presenting molecules, e.g., LTBP1 and/or LTBP3. Thus, such pharmaceutical compositions may be administered to patients for alleviating a TGFβ-related indication (e.g., fibrosis) associated with TGFβ1 activation/release from LTBP1 and/or LTBP3.

A pharmaceutically “acceptable” carrier (excipient) means that the carrier is compatible with the active ingredient of the composition (and preferably, capable of stabilizing the active ingredient) and not deleterious to the subject to be treated. Examples of pharmaceutically acceptable excipients (carriers), including buffers, would be apparent to the skilled artisan and have been described previously. See, e.g., Remington: The Science and Practice of Pharmacy 20th Ed. (2000) Lippincott Williams and Wilkins, Ed. K. E. Hoover. In one example, a pharmaceutical composition described herein contains more than one antibody that selectively binds a LTBP1-TGFβ1 complex and/or a LTBP3-TGFβ1 complex, where the antibodies recognize different epitopes/residues of the LTBP1-TGFβ1 complex and/or LTBP3-TGFβ1 complex.

The pharmaceutical compositions to be used in the present methods can comprise pharmaceutically acceptable carriers, excipients, or stabilizers in the form of lyophilized formulations or aqueous solutions (Remington: The Science and Practice of Pharmacy 20th Ed. (2000) Lippincott Williams and Wilkins, Ed. K. E. Hoover). Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations used, and may comprise buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrans; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG). Pharmaceutically acceptable excipients are further described herein.

The invention also includes pharmaceutical compositions that comprise an antibody or fragment thereof according to the present invention, and a pharmaceutically acceptable excipient.

Thus, the antibody or a molecule comprising an antigen-binding fragment of such antibody can be formulated into a pharmaceutical composition suitable for human administration.

The pharmaceutical formulation may include one or more excipients. In some embodiments, excipient(s) may be selected from the list provided in the following: https://www.accessdata.fda.gov/scripts/cderhig/index.Cfm?event=browseByLetter.page&Letter=A

The pharmaceutical composition is typically formulated to a final concentration of the active biologic (e,g., monoclonal antibody, engineered binding molecule comprising an antigen-binding fragment, etc.) to be between about 2 mg/mL and about 200 mg/mL. For example, the final concentration (wt/vol) of the formulations may range between about 2-200, 2-180, 2-160, 2-150, 2-120, 2-100, 2-80, 2-70, 2-60, 2-50, 2-40, 5-200, 5-180, 5-160, 5-150, 5-120, 5-100, 5-80, 5-70, 5-60, 5-50, 5-40, 10-200, 10-180, 10-160, 10-150, 10-120, 10-100, 10-80, 10-70, 10-60, 10-50, 10-40, 20-200, 20-180, 20-160, 20-150, 20-120, 20-100, 20-80, 20-70, 20-60, 20-50, 20-40, 30-200, 30-180, 30-160, 30-150, 30-120, 30-100, 30-80, 30-70, 30-60, 30-50, 30-40, 40-200, 40-180, 40-160, 40-150, 40-120, 40-100, 40-80, 40-70, 40-60, 40-50, 50-200, 50-180, 50-160, 50-150, 50-120, 50-100, 50-80, 50-70, 50-60, 60-200, 60-180, 60-160, 60-150, 60-120, 60-100, 60-80, 60-70, 70-200, 70-180, 70-160, 70-150, 70-120, 70-100, 70-80 mg/mL. In some embodiments, the final concentration of the biologic in the formulation is about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 mg/mL.

According to some embodiments, the TGFβ inhibitor is administered in an amount of about 3000 mg, 2400 mg, 1600 mg, 800 mg, 240 mg, 80 mg, or less.

The pharmaceutical compositions of the present invention are preferably formulated with suitable buffers. Suitable buffers include but are not limited to: phosphate buffer, citric buffer, and histidine buffer.

The final pH of the formulation is typically between pH 5.0 and 8.0. For example, the pH of the pharmaceutical composition may be about 5.0, 5.2, 5.5, 6.0, 6.2, 6.5, 6.8, 7.0, 7.2, 7.4, 7.5, 7.6, or 7.8.

The pharmaceutical composition of the present disclosure may comprise a surfactant, such as nonionic detergent, approved for the use in pharmaceutical formulations. Such surfactants include, for example, polysorbates, such as Polysorbate 20 (Tween-20), Polysorbate 80 (Tween-80) and NP-40.

The pharmaceutical composition of the present disclosure may comprise a stabilizer. For liquid-protein preparations, stability can be enhanced by selection of pH-buffering salts, and often amino acids can also be used. It is often interactions at the liquid/air interface or liquid/solid interface (with the packaging) that lead to aggregation following adsorption and unfolding of the protein. Suitable stabilizers include but are not limited to: sucrose, maltose, sorbitol, as well as certain amino acids such as histidine, glycine, methionine and arginine.

The pharmaceutical composition of the present disclosure may contain one or any combinations of the following excipients: Sodium Phosphate, Arginine, Sucrose, Sodium Chloride, Tromethamine, Mannitol, Benzyl Alcohol, Histidine, Sucrose, Polysorbate 80, Sodium Citrate, Glycine, Polysorbate 20, Trehalose, Poloxamer 188, Methionine, Trehalose, rhHyaluronidase, Sodium Succinate, Potassium Phosphate, Disodium Edetate, Sodium Chloride, Potassium Chloride, Maltose, Histidine Acetate, Sorbitol, Pentetic Acid, Human Serum Albumin, Pentetic Acid.

In some embodiments, the pharmaceutical composition of the present disclosure may contain a preservative.

The pharmaceutical composition of the present disclosure is typically presented as a liquid or a lyophilized form. Typically, the products can be presented in vial (e.g., glass vial). Products available in syringes, pens, or autoinjectors may be presented as pre-filled liquids in these container/closure systems.

In some examples, the pharmaceutical composition described herein comprises liposomes containing an antibody that selectively binds a LTBP1-TGFβ1 complex and/or a LTBP3-TGFβ1 complex, which can be prepared by any suitable method, such as described in Epstein et al., Proc. Natl. Acad. Sci . USA 82:3688 (1985); Hwang et al. Proc. Natl. Acad. Sci . USA 77:4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545. Liposomes with enhanced circulation time are disclosed in U.S. Pat. No. 5,013,556. Particularly useful liposomes can be generated by the reverse phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter.

In some embodiments, liposomes with targeting properties are selected to preferentially deliver or localize the pharmaceutical composition to certain tissues or cell types. For example, certain nanoparticle-based carriers with bone marrow-targeting properties may be employed, e.g., lipid-based nanoparticles or liposomes. See, for example, Sou (2012) “Advanced drug carriers targeting bone marrow”, ResearchGate publication 232725109.

The antibodies that selectively bind a LTBP1-TGFβ1 complex and/or a LTBP3-TGFβ1 complex may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions. Exemplary techniques have been described previously, see, e.g., Remington, The Science and Practice of Pharmacy 20th Ed. Mack Publishing (2000).

In other examples, the pharmaceutical composition described herein can be formulated in sustained-release format. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and 7 ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), sucrose acetate isobutyrate, and poly-D-(−)-3-hydroxybutyric acid.

The pharmaceutical compositions to be used for in vivo administration must be sterile. This is readily accomplished by, for example, filtration through sterile filtration membranes. Therapeutic antibody compositions are generally placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.

The pharmaceutical compositions described herein can be in unit dosage forms such as tablets, pills, capsules, powders, granules, solutions or suspensions, or suppositories, for oral, parenteral or rectal administration, or administration by inhalation or insufflation.

Suitable surface-active agents include, in particular, non-ionic agents, such as polyoxyethylenesorbitans (e.g., TWEEEN™ 20, 40, 60, 80 or 85) and other sorbitans (e.g., SPAN™ 20, 40, 60, 80 or 85). Compositions with a surface-active agent will conveniently comprise between 0.05 and 5% surface-active agent, and can be between 0.1 and 2.5%. It will be appreciated that other ingredients may be added, for example mannitol or other pharmaceutically acceptable vehicles, if necessary.

Suitable emulsions may be prepared using commercially available fat emulsions, such as INTRALIPID™, LIPSYN™, INFONUTROL™, LIPOFUNDIN™ and LIPIPHYSAN™. The active ingredient may be either dissolved in a pre-mixed emulsion composition or alternatively it may be dissolved in an oil (e.g., soybean oil, safflower oil, cottonseed oil, sesame oil, corn oil or almond oil) and an emulsion formed upon mixing with a phospholipid (e.g., egg phospholipids, soybean phospholipids or soybean lecithin) and water. It will be appreciated that other ingredients may be added, for example glycerol or glucose, to adjust the tonicity of the emulsion. Suitable emulsions will typically contain up to 20% oil, for example, between 5 and 20%.

The emulsion compositions can be those prepared by mixing an antibody that selectively binds a LTBP1-TGFβ1 complex and/or a LTBP3-TGFβ1 complex with Intralipid™ or the components thereof (soybean oil, egg phospholipids, glycerol and water).

Use of Inhibitors that Selectively Bind a LTBP1/3-TGFβ1 Complex

The inhibitors, e.g., antibodies and antigen-binding portions thereof, described herein that selectively bind a LTBP1/3-TGFβ1 complex can be used in a wide variety of applications in which modulation of TGFβ1 activity associated with LTBP1 or LTBP3 is desired.

In one embodiment, the invention provides a method of inhibiting TGFβ1 activation by exposing a LTBP1-TGFβ1 complex and/or a LTBP3-TGFβ1 complex to an inhibitor, e.g., antibody, or antigen-binding portion thereof, which selectively binds a LTBP1/3-TGFβ1 complex. The foregoing method can be performed in vitro, e.g., to inhibit TGFβ1 activation in cultured cells. The foregoing method can also be performed in vivo, e.g., in a subject in need of TGFβ1 inhibition, or in an animal model in which the effect of TGFβ1 inhibition is to be assessed.

Any inhibitor, e.g., antibody, or antigen-binding portion thereof, described herein which selectively binds a LTBP1-TGFβ1 complex and/or a LTBP3-TGFβ1 complex, and any pharmaceutical composition comprising such antibody, is suitable for use in the methods of the invention. For example, in one embodiment, the inhibitor, e.g., antibody, or antigen-binding portion thereof, selectively binds to a LTBP1-TGFβ1 complex and a LTBP3-TGFβ1 complex, but does not bind to one or more targets selected from LTBP1 alone, mature TGFβ1 alone, a GARP-TGFβ1 complex, a LRRC33-TGFβ1 complex, and combinations thereof. Exemplary inhibitor, e.g., antibodies, can inhibit the release of mature TGFβ1 from a LTBP1-proTGFβ1 complex and/or a LTBP3-proTGFβ1 complex, without inhibiting the release of mature TGFβ1 from a GARP-proTGFβ1 complex and/or a LRRC33-proTGFβ1 complex.

The antibody, or antigen-binding portion thereof, can, in some embodiments, bind a LTBP1-proTGFβ1 complex and/or a LTBP3-proTGFβ1 complex with a dissociation constant (K D ) of about 10 −8 M or less. In some embodiments, the antibody, or antigen-binding portion thereof has a K D value of about 10 −9 M or less. In some embodiments, the antibody, or antigen-binding portion thereof has a K D value of about 10 −10 M or less (e.g., about 10 −11 M or less). In some embodiments, the antibody, or antigen-binding portion thereof has a K D value of <10 nM, <5 nM<1 nM) towards a LTBP1-proTGFβ1 complex and/or a LTBP3-proTGFβ1 complex as measured in a suitable in vitro binding assay such as BLI (e.g., Octet). In one embodiment, the antibody, or antigen-binding portion thereof, comprises at least one (e.g., one, two, or three) heavy chain CDRs shown in Table 5, and/or at least one (e.g., one, two, three) light chain CDRs shown in Table 5. In an exemplary embodiment, the antibody, or antigen-binding portion thereof, comprises a heavy chain variable region comprising SEQ ID NO:7, and/or a light chain variable region comprising SEQ ID NO:8. Antibodies and antigen-binding portions thereof which bind the same epitope as the foregoing antibodies, and/or which compete for binding with the foregoing antibodies to LTBP1/3-proTGFβ1, are also useful in the methods described herein. Additional features of the antibodies, or antigen-binding portions thereof, that are suitable for practicing the methods of the invention are described herein.

In one embodiment, contacting a LTBP1-TGFβ1 complex and/or a LTBP3-TGFβ1 complex with the inhibitor, e.g., antibody, or antigen-binding portion thereof, inhibits the release of mature TGFβ1 from the LTBP1-TGFβ1 complex and/or the LTBP3-TGFβ1 complex. In one embodiment, said contacting does not inhibit the release of mature TGFβ1 from presenting molecules other than LTBP1 and LTBP3. For example, exposing a GARP-TGFβ1 complex or a LRRC33-TGFβ1 complex to a context-specific inhibitor, e.g., antibody, that selectively binds LTBP1/3-TGFβ1 but does not bind TGFβ1 in the context of GARP or LRRC33 will not inhibit the release of mature TGFβ1 from the GARP-TGFβ1 complex or the LRRC33-TGFβ1 complex.

LTBP1 and LTBP3 are generally deposited in the extracellular matrix. Accordingly, in one embodiment, complexes comprising LTBP1-TGFβ1 and/or LTBP3-TGFβ1 are associated with the extracellular matrix, e.g., bound to the extracellular matrix. In some embodiments, the LTBP1/3-TGFβ1 complexes are bound to extracellular matrix comprising fibrillin, and/or a protein containing an RGD motif.

The invention also provides a method of reducing TGFβ1 activation in a subject, by administering to the subject an inhibitor, e.g., antibody, or antigen-binding portion thereof, which selectively binds a LTBP1/3-TGFβ1 complex, as described herein. Any antibody, or antigen-binding portion thereof, described herein which selectively binds a LTBP1-TGFβ1 complex and/or a LTBP3-TGFβ1 complex, and any pharmaceutical composition comprising such antibody, is suitable for use in the methods of the invention.

Exemplary LTBP1/3 inhibitors, e.g., antibodies, bind a LTBP1/3-TGFβ1 complex, and inhibit TGFβ1 activation in a context-specific manner, by inhibiting release of TGFβ1 presented by LTBP1 and LTBP3, without inhibiting release of TGFβ1 presented by GARP and/or LRRC33. Such antibodies are useful for blocking a particular subset of TGFβ1 activity in vivo. In one embodiment, the context-specific antibodies provided herein can be used to inhibit TGFβ1 localized to the extracellular matrix. In another embodiment, the context-specific antibodies can inhibit TGFβ1 without modulating TGFβ1-associated immune activity or immune response, which is primarily mediated by TGFβ1 presented by GARP and LRRC33. In another embodiment, the context-specific antibodies can be used to inhibit TGFβ1 activity associated with the extracellular matrix (e.g., LTBP1-associated TGFβ1 activity and LTBP3-associated TGFβ1 activity) without modulating TGFβ1 activity associated with hematopoietic cells, e.g., hematopoietic cells that express GARP and/or LRRC33.

Clinical Applications

Applicant previously described so-called “context-independent” inhibitors of TGFβ1 (see, for example: PCT/US2017/021972 and PCT/US2018/012601) which may be useful for treating various diseases and disorders involving TGFβ1 dysregulation, including, but are not limited to, cancer and fibrosis. Unlike traditional TGFβ1 antagonists, these context-independent TGFβ1 inhibitors are capable of selectively targeting the TGFβ1 isoform. Within the multifaceted biological functions driven by the TGFβ1 isoform, however, the context-independent inhibitors do not discriminate tissue-specific (thus context-specific) proTGFβ1 complexes, such that such inhibitors are capable of binding and thereby inhibiting release or activation of mature growth factor from any of the presenting molecule-proTGFβ1 complexes.

Based at least in part on the recognition that it may be advantageous to provide even greater selectivity in targeting only a subset of TGFβ activities, context-selective inhibitors of the present disclosure have been generated. It is contemplated that by further narrowing particular biological contexts in which to inhibit TGFβ function, greater safety may be achieved in a subset of disease conditions or patient populations. Specifically, the inventors of the present invention have recognized that in certain conditions, systemic perturbation of immune regulation may be particularly undesirable. Because TGFβ plays an important role in mediating immune response and maintaining immune homeostasis, broad inhibition of TGFβ activities effectuated in a context-independent manner may lead to unwanted side effects without justifiable benefits. In these circumstances, it is envisaged that it is advantageous to specifically target and inhibit matrix-associated TGFβ function using a context-selective inhibitor, such as those encompassed herein, which does not inhibit the immune components of TGFβ1 function.

Accordingly, the context-specific antibodies can be used to inhibit LTBP1/3-associated TGFβ activity in applications in which TGFβ activation in the context of LTBP1 or LTBP3 is desirable, and in which TGFβ activation in the context of GARP and/or or LRRC33 is detrimental.

The disease may involve dysregulation or impairment of ECM components or function and comprises increased collagen deposition. In some embodiments, the dysregulation or impairment of ECM components or function may further comprise increased stiffness and/or ECM reorganization. In some embodiments, the dysregulation or impairment of ECM components or function includes increased myofibroblast cells within the disease site. In some embodiments, the dysregulation of the ECM includes increased stiffness of the matrix, which is implicated in the pathogenesis and/or disease progression of a variety of fibrotic conditions and tumors. In some embodiments, the dysregulation of the ECM involves fibronectin and/or fibrillin.

Rationale for the Development of Matrix-Targeted TGFβ Inhibitors that do not Inhibit GARP-Associated TGFβ

The invention includes context-specific inhibitors of LTBP1-associated and/or LTBP3-associated TGFβ. Such inhibitors therefore are capable of specifically targeting the ECM-associated latent TGFβ complexes (e.g., LTBP1-proTGFβ1 and/or LTBP3-proTGFβ1) thereby inhibiting the release of mature TGFβ growth factor from the latent complex at disease environments, e.g., fibrotic tissues. Such inhibitors show no significant binding activities towards a GARP-proTGFβ1 complex, thereby minimizing unwanted systemic immune modulations. Such antibodies may be advantageous for use in the treatment of conditions with ECM dysregulation, such as abnormal remodeling and/or stiffness of the ECM.

The context-selective antibodies provided herein may be used in the treatment of a condition where it is undesirable to stimulate the subject's immune response and/or in situations where the subject is expected to benefit from a long-term TGFβ inhibition therapy to manage a chronic condition, such as many types of fibrosis.

At least three bases for supporting potential benefits of a TGFβ inhibitor that does not target the GARP-proTGFβ1 complex expressed on regulatory T cells are discussed below.

First, GARP-expressing T regulatory cells are a component of the immune system that suppress or dampen immune responses of other cells. This notion may be referred to as “tolerance.” This is an important “self-check” built into the immune system to prevent excessive reactions that in some situations can result in life-threatening conditions, such as sepsis, cytokine release syndrome and cytokine storm. TGFβ inhibition therapies that exert Treg-inhibitory effects may, therefore, pose certain risk when the normal Treg function is impaired, particularly for a prolonged duration of time, e.g., therapeutic regimen involving treatment of six months or longer, and chronic treatment that is administered for an indefinite period of time. For this reason, patients in need of TGFβ inhibition therapies, particularly to avoid the risk of eliciting autoimmunity, may benefit from TGFβ1 inhibitors that do not directly perturb the normal Treg function. For example, patient populations in need of a long-term TGFβ inhibition therapy may include those with genetic or congenital conditions, such as DMD, CF and others. In addition, patient populations that suffer from conditions that include inflammation may benefit from a context-specific inhibitor that does not perturb the GARP/Treg function so as to minimize the risk of exacerbating the existing inflammatory conditions.

Second, increasing evidence points to a link between disproportionate Th17/Treg ratios and pathologies involving inflammation and/or fibrosis. It is generally accepted that the differentiation of the two cell types, Th17 and Treg, is negatively regulated with an inverse relationship. TGFβ1 appears to be a master gatekeeper of this process, such that, TGFβ1 exposure promotes naïve T cells to differentiate into Foxp3+Tregs, whereas TGFβ1 in combination with IL-6, promotes naïve T cells to differentiate into RORγt+Th17 cells instead. In addition, once differentiated, these cell populations negatively regulate each other.

Lines of evidence suggest that an imbalance in Th17/Treg ratios correlates with the pathogenesis and/or progression of fibrotic conditions involving chronic inflammation, or severity thereof.

For example, Shoukry et al. reported that Th17 cytokines drive liver fibrosis by regulating TGFβ signaling. The authors examined ex vivo the frequency of Th17 and Treg populations in liver biopsy samples and found that increased Th17/Treg ratio correlated with advanced fibrosis, as compared to moderate fibrosis or healthy tissue samples. Consistent with the observation, a strong bias towards Th17 cytokines, IL-22 in particular, was also detected in fibrotic livers. These data suggest that increased Th17/Treg ratios lead to an imbalance in pro-fibrotic Th17 cytokines, which correlate with severity of liver fibrosis.

Similar inverse correlations of Th17 and Treg populations are observed in other diseases.

For example, increased muscle expression of IL-17 has been reported in patients with Duchenne muscular dystrophy (DMD), which is a condition that manifests chronic inflammation. De Pasquale et al. (Neurology 78(17): 1309-14) found that DMD muscle biopsy samples contained higher levels of IL-17 (a Th17 marker) and lower levels of Foxp3 (a Treg marker) mRNA compared to control. Elevations in other proinflammatory cytokines, such as TNF-α and MCP-1, were also observed and were found to be associated with worse clinical outcome of patients. The authors concluded that the data point to a possible pathogenic role of IL-17.

Similarly, Jamshidian et al. (J Neuroimmunol 2013, 262(1-2): 106-12) reported biased Treg/Th17 balance away from regulatory toward inflammatory phenotype in patients with relapsed multiple sclerosis and its correlation with severity of clinical symptoms.

A role of regulatory T cells is also implicated in the pathogenesis of cystic fibrosis (CF). In particular, CF lungs affected by the disease are associated with exaggerated Th17 and Th2 cell responses, indicative of a classic inflammatory phenotype, but also with a deficiency in numbers or function (i.e., impairment) of Treg cells (McGuire (2015) Am J Respir Crit Care Med 191(8): 866-8).

Furthermore, Zhuang et al. (Scientific Reports (2017) 7: 40141) found imbalance of Th17/Treg cells in patients with acute anteir uveitis (anterior segment intraocular inflammation with the positive of human class I major histocompatibility complex), in which both a marked increase in Th17 cells and a marked decrease in Treg cells were seen.

Taken together, the inventors of the present disclosure recognized that what appears to be a common feature in these various diseases associated with elevated Th17/Treg rations is that the patient suffers from a fibrotic condition accompanied by an inflammatory component.

Thus, it is envisaged in the present disclosure that TGFβ inhibition therapy that spares the Treg/GARP-arm of the TGFβ function may be particularly advantageous for an effective treatment of diseases characterized by an elevated level of Th17/Treg ratios. In this way, the context-selective inhibitors of TGFβ according to the invention are aimed to avoid more systemic effects of TGFβ inhibition that may interfere with Treg function, which may lead to exacerbation of existing fibrotic/inflammatory conditions in patients. Thus, the matrix-targeted TGFβ inhibitors described herein are used in a method for treating a patient who has or at risk of developing a fibrotic disorder that comprises inflammation. In some embodiments, the patient has an elevated Th17-to-Treg cell ratio. In some embodiments, the elevated Th17/Treg ratio may be predominantly caused by an increased number of Th17 cells, while in other embodiments, the elevated Th17/Treg ratio may be predominantly caused by a decreased number of Treg cells in the patient (or a biological sample collected from the patient). Yet in further embodiments, the elevated Th17/Treg ratio may be caused by a combination of an increased number of Th17 cells and a decreased number of Treg cells. In some embodiments, elevated levels of IL-17 and/or IL-22 detected in patients (or measured in samples collected from the patients) are also indicative of fibrotic conditions accompanied by chronic inflammation. Such patients may be therefore selected as candidates for receiving a context-selective TGFβ1 inhibitor therapy disclosed herein.

The third line of reasoning for keeping the GARP-TGFβ1 axis intact in a TGFβ inhibition therapy relates to the benefit of maintaining normal Treg function. As mentioned, GARP is expressed on the cell surface of Tregs and are thought to play a role in TGFβ-mediated immunomodulation. Because Tregs are indispensable for immune homeostasis and the prevention of autoimmunity, unnecessary perturbation of which may put certain patient populations at higher risk of, for example, infections (reviewed, for example, by: Richert-Spuhler and Lund (2015) Prog Mol Biol Transl Sci.

The third line of reasoning for keeping the GARP-TGFβ1 axis intact in a TGFβ inhibition therapy is that regulatory T cells function as a “break” to modulate or dampen over-reactive immune response. The discovery of Foxp3 as the master regulator of Treg cell development and function was critical for the understanding of Treg cell biology. Inactivating mutations in Foxp3 result in the spontaneous development of severe autoimmunity with a scurfy phenotype in mice and IPEX syndrome (‘immune dysregulation, polyendocrinopathy, enteropathy, X-linked’) in humans (see Dominguez-Villear and Haler, Nature Immunology 19, 665-673, 2018). Thus, it raises the possibility that TGFb1 therapy that elicits inhibitory effects of the Treg/GARP arm of TGFb function, especially in a prolonged treatment, may cause or exacerbate autoimmune response.

Increasing evidence suggests that Tregs not only act to dampen over exuberant effector immune responses, they also have the ability to potentiate appropriate immune responses to pathogens, by participating in pathogen clearance and protection of the host from collateral damage. Such diverse function of Treg cells is particularly apparent in delicate tissues such as the lung, which is constantly exposed to an external environment from which a variety of pathogens and other foreign components (e.g., viral pathogens, bacterial pathogens, fungal pathogens, and allergens) may gain access to host cells.

For example, influenza virus infection elicits a strong proinflammatory cytokine response with abundance immune cell infiltration. In acute and/or severe infections, such response can cause serious sequelae in susceptible individuals. Tregs provide a mechanism for dampening viral infection-associated pathology by controlling the magnitude of immune response in the host. Indeed, pathogen-exposed Tregs retain protective effects in adoptive transfer. Moreover, such adoptive transfer of primed Tregs have been shown to ameliorate influenza virus-associated morbidity and to prolong survival in severe immunocompromised animal models.

Accordingly, the invention provides use of an ECM-targeted, context-selective TGFβ inhibitor (e.g., LTBP1-selective or LTBP1/3-selective inhibitors of TGFβ1 activation inhibitors) for the treatment of a disease that involves matrix-associated TGFβ dysregulation in a subject. The subject is suffering from or at risk of an infection. The infection can be viral infections (e.g., influenza virus, respiratory syncytial virus or RSV, human immunodeficiency virus or HIV, MARS, SARS, herpes simplex virus or HSV, hepatitis A virus or HAV, hepatitis B virus or HBV, hepatitis C virus or HCV, CMV, Dengue virus, lymphocytic choriomeningitis virus, and West Nile virus), bacterial infections (meningitis, Mycobacterium tuberculosis, Listeria monocytogenes, Citrobacter rodentium, Salmonella , and E. coli ), and/or fungal infections (e.g., Candida, Pneumocytis, Aspergillus, Cryptococcus , and Coccidioides ).

Typically, high-risk or at-risk populations (individuals that are considered particularly susceptible to severe infections or infection-triggered responses) include pediatric populations (infants, young children, e.g., human individuals under the age of 7); elderly populations (those who are 65 years or older); those with compromised immune system due to medical condition, health status, life styles such as smoking, and/or medications with immunosuppressive effects, etc.

For example, certain medications cause weakened immunity, such as chemotherapy, therapies that target hematopoietic cells such as CD33 therapy, steroids, immunosuppressants, and statins.

In some embodiments, high-risk or at-risk populations are those with existing medical conditions, such as those with chronic infections such as HIV, those with bone marrow transplantation, pre-diabetic individuals, diabetic individuals, those with autoimmune disorders such as RA, asthma and allergy.

Thus, matrix-targeted, context-selective TGFβ inhibitors encompassed herein may be particularly advantageous for treating patients who require a long-term or chronic TGFβ therapy since in these scenarios it is beneficial to avoid impairment of immune homeostasis and the normal immune function that provides the ability to respond effectively to possible infections caused by a variety of pathogens such as those listed above.

Accordingly, antibodies that selectively bind LTBP-TGFβ (e.g., LTBP1-TGFβ1 and LTBP3-TGFβ31), and that do not inhibit TGFβ in the context of the immune-associated TGFβ presenters GARP and LRRC33, are therapeutic candidates for the treatment of fibrotic indications such as organ fibrosis, and are aimed to avoid TGFβ-related global immune activation. In one embodiment, the context-specific antibodies can be used to inhibit LTBP1/3-associated TGFβ activity in applications in which TGFβ-mediated immune suppression is beneficial, e.g., in a subject who has received a transplant, who is a candidate for receiving a transplant, or who is expected to receive a transplant. In some embodiments, the subject has an advanced stage fibrosis and/or a bone marrow disease. In some embodiments, the subject has or is at risk of developing an autoimmune disorder.

The foregoing methods can be used to treat a subject having a condition for which inhibition or reduction in LTBP-associated TGFβ activity is beneficial. For example, the subject may have or be at risk for developing a disorder in which extracellular matrix-associated TGFβ activity has been implicated.

Integrin-mediated activation of latent TGFβ in the extracellular matrix is a key contributor to fibrosis. Without wishing to be bound by theory, it is presently understood that integrins, including αVβ6 and αVβ8, can trigger the release of TGFβ from presenting molecules including LTBP1 and LTBP3. Inhibiting release or activation of TGFβ in this context can reduce or eliminate fibrosis, and/or symptoms associated therewith.

As described, LTBP1 and LTBP3 are produced and are deposited extracellularly as components of the ECM, where they can “present” a proTGFβ complex (latent, inactive precursor of TGFβ1) within the ECM. Upon stimulation, the LTBP1/3-proTGFβ complex releases the TGFβ growth factor (the active, mature form of growth factor) which in turn is thought to be involved in the regulation of the local tissue microenvironment, such as ECM maintenance/remodeling and the process of fibrosis, possibly by responding to various cytokines, chemokines and growth factors, and by interacting with other ECM components, such as fibronectin, Fibrillin, collagen, elastin, and matrix metallopeptidases (MMPs).

In the normal wound healing process that occurs in response to an injury, for example, TGFβ is thought to facilitate granular tissue formation, angiogenesis, and collagen synthesis and production. TGFβ signaling is also implicated in abnormal tissue fibrogenesis (i.e., fibrosis), which results in formation of excess fibrous connective tissue in an organ or tissue in a reparative or reactive process characterized by the pathological accumulation of extracellular matrix (ECM) components, such as collagens. In these and other situations, the TGFβ axis may affect further aspects (in addition to fibrotic aspect), such as inflammation, recruitment and phenotypic switch of various cell types, which may be mediated by its interaction with one or more of the other presenting molecules, such as GARP/LRRC32 and LRRC33. In certain instances, it is advantageous to preferentially inhibit the LTBP1/3-context of TGFβ activation, without significantly inhibiting one or more of the other contexts of TGFβ1 activation, in situations where ECM-associated TGFβ that drives fibrosis is to be selectively inhibited.

Accordingly, in one embodiment, the invention provides a method of reducing TGFβ activation in a subject having, or at risk of developing, a fibrotic disorder by administering to the subject an antibody, or antigen-binding portion thereof, which selectively binds a LTBP1/3-TGFβ complex, as described herein. In another embodiment, the invention provides a method of treating a fibrotic disorder by administering to the subject an antibody, or antigen-binding portion thereof, which selectively binds a LTBP1/3-TGFβ complex, as described herein.

In one embodiment, the fibrotic disorder is an organ fibrosis, wherein optionally, the organ fibrosis is an advanced organ fibrosis. In a further embodiment, the organ fibrosis is selected from the group consisting of kidney fibrosis, liver fibrosis, lung fibrosis, cardiac fibrosis, pancreatic fibrosis, skin fibrosis, scleroderma, muscle fibrosis, uterine fibrosis and endometriosis. In another further embodiment, the fibrotic disorder comprising chronic inflammation is a muscular dystrophy, multiple sclerosis (MS), or Cystic Fibrosis (CF). In a further embodiment, the muscular dystrophy is Duchenne muscular dystrophy (DMD). In another further embodiment, the MS comprises perivascular fibrosis. In a further embodiment, the lung fibrosis is idiopathic pulmonary fibrosis (IPF). In another further embodiment, the subject has chronic kidney disease (CKD). In another embodiment, the subject has nonalcoholic steatohepatitis (NASH).

In exemplary embodiments, the fibrotic disorder is fibrosis, Alport syndrome, fibroids, desmoplasia, amyotrophic lateral sclerosis (ALS), or Duchenne muscular dystrophy (DMD).

In one embodiment, the subject has desmoplasia.

In one embodiment, the subject has organ fibrosis, for example, kidney fibrosis (e.g., fibrosis associated with chronic kidney disease (CKD)), liver fibrosis (e.g., fibrosis associated with nonalcoholic steatohepatitis (NASH)), lung fibrosis (e.g., idiopathic pulmonary fibrosis (IPF)), cardiac fibrosis, and/or skin fibrosis (e.g., scleroderma). In some embodiments, the subject can have advanced organ fibrosis. For example, the subject may be in need of an organ transplant. In one embodiment, the subject may be in need of an organ transplant, and the compounds and compositions described herein are administered to prevent allograft fibrosis from developing in the subject following receipt of the transplant.

A recent study examined whether inhibiting integrin αVβ6 could prevent TGFβ-mediated allograft fibrosis after kidney transplantation (Lo et al., Am. J. Transplant. (2013), 13:3085-3093). Surprisingly, animals treated with an inhibitory anti-αVβ6 antibody experienced a significant decrease in rejection-free survival compared to placebo animals. The authors conclude that this result cautions against TGFβ inhibition in kidney transplantation, because the immunosuppressive properties of TGFβ help prevent allograft rejection. The inhibitors, e.g., antibodies, and antigen-binding portions thereof, described herein advantageously inhibit activation of TGFβ presented by LTBP1 or LTBP3 in the extracellular matrix, but do not inhibit activation of TGFβ presented by GARP or LRRC33 on immune cells. Accordingly, the context-specific LTBP1/3-TGFβ inhibitors, e.g., antibodies, described herein can prevent or reduce allograft fibrosis, without eliminating the immunosuppressive properties of TGFβ that are useful for preventing allograft rejection. Accordingly, in one aspect, the invention provides a method for treating a fibrotic disorder in a subject, comprising administering to the subject a therapeutically effective amount of an inhibitor of TGFβ signaling, wherein the inhibitor is a selective inhibitor of ECM-associated TGFβ; and, wherein the subject benefits from suppressed immunity. In one embodiment, the subject has a fibrotic condition and would benefit from an allograft transplant, or has received an allograft transplant.

Additional fibrotic conditions for which antibodies and/or compositions of the present disclosure may be used therapeutically include, but are not limited to, lung indications (e.g., idiopathic pulmonary fibrosis (IPF), chronic obstructive pulmonary disorder (COPD), allergic asthma, cystic fibrosis (CF), acute lung injury, eosinophilic esophagitis, pulmonary arterial hypertension and chemical gas-injury), kidney indications (e.g., diabetic glomerulosclerosis, focal segmental glomeruloclerosis (FSGS), chronic kidney disease, fibrosis associated with kidney transplantation and chronic rejection, IgA nephropathy, and hemolytic uremic syndrome), liver fibrosis (e.g., non-alcoholic steatohepatitis (NASH), chronic viral hepatitis, parasitemia, inborn errors of metabolism, toxin-mediated fibrosis, such as alcohol fibrosis, non-alcoholic steatohepatitis-hepatocellular carcinoma (NASH-HCC), primary biliary cirrhosis, and sclerosing cholangitis), cardiovascular fibrosis (e.g., cardiomyopathy, hypertrophic cardiomyopathy, atherosclerosis and restenosis,) systemic sclerosis, skin fibrosis (e.g., skin fibrosis in systemic sclerosis, diffuse cutaneous systemic sclerosis, scleroderma, pathological skin scarring, keloid, post-surgical scarring, scar revision surgery, radiation-induced scarring and chronic wounds), eye-related conditions such as subretinal fibrosis, uveitis syndrome, uveitis associated with idiopathic retroperitoneal fibrosis, extraocular muscle fibrosis, eye diseases associated with the major histocompatibility complex (MHC class I) or histocompatibility antigens, subretinal fibrosis in macular degeneration (e.g., age-related macular degeneration) and cancers or secondary fibrosis (e.g., myelofibrosis, head and neck cancer, M7 acute megakaryoblastic leukemia and mucositis). Other diseases, disorders or conditions related to fibrosis that may be treated using compounds and/or compositions of the present disclosure, include, but are not limited to Marfan's syndrome, stiff skin syndrome, scleroderma, rheumatoid arthritis, bone marrow fibrosis, Crohn's disease, ulcerative colitis, systemic lupus erythematosus, muscular dystrophy, (such as DMD), Dupuytren's contracture, Camurati-Engelmann disease, neural scarring, dementia, proliferative vitreoretinopathy, corneal injury, complications after glaucoma drainage surgery, and multiple sclerosis (MS). Many such fibrotic indications are also associated with inflammation of the affected tissue(s), indicating involvement of an immune component. Such inflammation may be accompanied by aberrant immune cell populations, such as increased numbers of Th17 cells, reduced numbers of Treg cells, and/or both. In each case, the affected patient may exhibit increased Th17/Treg cell ratios. In some embodiments, diseases to be treated with an antibody according to the present disclosure include metabolic disorders, such as metabolic liver disorders. Non-limiting examples of metabolic disorders include NASH, NAFLD, type 2 diabetes and obesity. In some embodiments, the disease to be treated with an antibody according to the present disclosure is aortic stenosis.

In another aspect, the invention provides a method of selecting an isoform-specific TGFβ1 inhibitor for the treatment of a fibrotic disorder in a subject, comprising: (a) determining whether the subject manifests clinical presentations including fibrosis and one or more of the following: (i) inflammation; (ii) immune suppression; (iii) proliferative dysregulation; (iv) need for an allograft transplant; (v) at risk of severe infection; (vi) in need of a long-term TGFβ1 inhibition therapy; and (vii) manifestation of an autoimmune conditions(s); and (b) selecting an isoform-specific, context-dependent TGFβ1 inhibitor or an isoform-specific, context-independent TGFβ1 inhibitor for treatment of the fibrotic disorder based on the clinical presentations determined in step (a).

In another aspect, the invention provides a method of treating a subject having a fibrotic disorder, comprising (a) selecting a treatment regimen comprising an isoform-specific TGFβ1 inhibitor for the subject, said selection comprising (i) determining whether the fibrotic disorder manifests clinical presentations including fibrosis and one or more of the following: inflammation, immune suppression, proliferative dysregulation, and need for an allograft transplant; and (ii) selecting a treatment regimen comprising an isoform-specific, context-dependent TGFβ1 inhibitor or an isoform-specific, context-independent TGFβ1 inhibitor, based on the clinical presentations determined in step (i); and (b) administering the selected treatment regimen to the subject.

In one embodiment of the foregoing aspects, the fibrotic disorder manifests clinical presentations comprising fibrosis, inflammation, immune suppression, and proliferative dysregulation. In an exemplary embodiment, the fibrotic disorder is myelofibrosis, and the selected isoform-specific TGFβ1 inhibitor is an isoform-specific, context-independent TGFβ1 inhibitor.

In another embodiment, the fibrotic disorder manifests clinical presentations comprising fibrosis, inflammation, and need for an allograft transplant. In one embodiment, the fibrotic disorder manifests clinical presentations comprising fibrosis and inflammation. In another embodiment, the fibrotic disorder is a degenerative disease.

In one embodiment, the fibrotic disorder manifests clinical presentations comprising immune suppression and proliferative dysregulation. In an exemplary embodiment, the fibrotic disorder is associated with a solid tumor, and the selected isoform-specific TGFβ1 inhibitor is an isoform-specific LTBP1/3-specific inhibitor and/or a GARP-selective inhibitor. In one embodiment, the solid tumor is a malignant tumor. In another embodiment, the tumor is a benign tumor. In one embodiment, the subject has desmoplasia, for example, pancreatic desmoplasia. In another embodiment, the subject has fibroids.

In another aspect, the invention provides a method of treating a subject having a fibrotic disorder with an isoform-specific, LTBP1/3-specific TGFβ1 inhibitor, comprising determining whether the fibrotic disorder manifests clinical presentations including fibrosis and the need for an allograft transplant; and administering an effective amount of an isoform-specific, LTBP1/3-specific TGFβ1 inhibitor to the subject if the fibrotic disorder manifests fibrosis and the need for an allograft transplant.

In another aspect, the invention provides a method of treating a subject having a fibrotic disorder with an isoform-specific, context-independent TGFβ1 inhibitor, comprising determining whether the fibrotic disorder manifests clinical presentations including fibrosis, immune suppression and/or proliferative dysregulation; and administering an effective amount of an isoform-specific, context-independent TGFβ1 inhibitor to the subject if the fibrotic disorder manifests fibrosis in conjunction with immune suppression and/or proliferative dysregulation.

The inhibitors, e.g., antibodies, described herein can be administered to a subject in an amount effective to treat or reduce symptoms of fibrosis. The effective amount of such an inhibitor is an amount effective to achieve both therapeutic efficacy and clinical safety in the subject. In one embodiment, an effective amount is an amount effective to reduce TGFβ1 activity in the extracellular matrix. In another embodiment, an effective amount is an amount effective to reduce fibrosis in a subject. In another embodiment, the effective amount does not inhibit TGFβ1-mediated immune suppression. In some embodiments, such an inhibitor, e.g., antibody, is a context-specific inhibitor that can block activation of TGFβ1 that is mediated by an LTBP-containing, ECM-associated TGFβ1. In some embodiments, the LTBP is LTBP1 and/or LTBP3. Assays useful for determining the efficacy of the inhibitors, e.g., antibodies, and/or compositions of the present disclosure for the alteration of fibrosis include, but are not limited to, histological assays for counting fibroblasts and basic immunohistochemical analyses known in the art.

Diseases Involving Proteases:

Activation of TGFβ from its latent complex may be triggered by integrin in a force-dependent manner, and/or by proteases. Evidence suggests that certain classes of proteases may be involved in the process, including but are not limited to Ser/Thr proteases such as thrombin, Kallikreins, chemotrypsin, elastases, plasmin, as well as zinc metalloproteases of ADAM family such as ADAM 10 and ADAM 17, as well as MMP family, such as MMP-2, MMP-9 and MMP-13. MMP-2 degrades the most abundant component of the basement membrane, Collagen IV, raising the possibility that it may play a role in ECM-associated TGFβ1 regulation. MMP-9 has been implicated to play a central role in tumor progression, angiogenesis, stromal remodeling and metastasis. Thus, protease-dependent activation of TGFβ1 in the ECM may be important for treating cancer.

Kallikreins (KLKs) are trypsin- or chymotrypsin-like serine proteases that include plasma Kallikreins and tissue Kallikreins. The ECM plays a role in tissue homeostasis acting as a structural and signaling scaffold and barrier to suppress malignant outgrowth. KLKs may play a role in degrading ECM proteins and other components which may facilitate tumor expansion and invasion. For example, KLK1 is highly upregulated in certain breast cancers and can activate pro-MMP-2 and pro-MMP-9. KLK2 activates latent TGFβ1, rendering prostate cancer adjacent to fibroblasts permissive to cancer growth. KLK3 has been widely studied as a diagnostic marker for prostate cancer (PSA). KLK3 may directly activate TGFβ1 by processing plasminogen into plasmin, which proteolytically cleaves LAP. KLK6 may be a potential marker for Alzheimer's disease.

Known activators of TGFβ1, such as plasmin, TSP-1 and αVβ6 integrin, all interact directly with LAP. It is postulated that proteolytic cleavage of LAP may destabilize the LAP-TGFβ interaction, thereby releasing active TGFβ1. It has been suggested that the region containing 54-LSKLRL-59 (SEQ ID NO: 388) is important for maintaining TGFβ1 latency. Thus, agents (e.g., antibodies) that stabilize the interaction, or block the proteolytic cleavage of LAP may prevent TGFβ activation.

Many of these proteases associated with pathological conditions (e.g., cancer) function through distinct mechanisms of action. Thus, targeted inhibition of particular proteases, or combinations of proteases, may provide therapeutic benefits for the treatment of conditions involving the protease-TGFβ axis. Accordingly, it is contemplated that inhibitors (e.g., TGFβ1 antibodies) that selectively inhibit protease-induced activation of TGFβ1 may be advantageous in the treatment of such diseases (e.g., cancer). Similarly, selective inhibition of TGFβ1 activation by one protease over another protease may also be preferred, depending on the condition being treated.

Plasmin is a serine protease produced as a precursor form called Plasminogen. Upon release, Plasmin enters circulation and therefore is detected in serum. Elevated levels of Plasmin appear to correlate with cancer progression, possibly through mechanisms involving disruption of the extracellular matrix (e.g., basement membrane and stromal barriers) which facilitates tumor cell motility, invasion, and metastasis. Plasmin may also affect adhesion, proliferation, apoptosis, cancer nutrition, oxygen supply, formation of blood vessels, and activation of VEGF (Didiasova et al., Int. J. Mol. Sci, 2014, 15, 21229-21252). In addition, Plasmin may promote the migration of macrophages into the tumor microenvironment (Philips et al., Cancer Res. 2011 Nov. 1; 71(21):6676-83 and Choong et al., Clin. Orthop. Relat. Res. 2003, 415S, S46-S58). Indeed, tumor-associated macrophages (TAMs) are well characterized drivers of tumorigenesis through their ability to promote tumor growth, invasion, metastasis, and angiogenesis.

Plasmin activities have been primarily tied to the disruption of the ECM. However, there is mounting evidence that Plasmin also regulate downstream MMP and TGF beta activation. Specifically, Plasmin has been suggested to cause activation of TGF beta through proteolytic cleavage of the Latency Associated Peptide (LAP), which is derived from the N-terminal region of the TGF beta gene product (Horiguchi et al., J Biochem. 2012 October; 152(4):321-9), resulting in the release of active growth factor. Since TGFβ1 may promote cancer progression, this raises the possibility that plasmin-induced activation of TGFb may at least in part mediate this process.

TGFβ1 has also been shown to regulate expression of uPA, which is a critical player in the conversion of Plasminogen into Plasmin (Santibanez, Juan F., ISRN Dermatology, 2013: 597927). uPA has independently been shown to promote cancer progression (e.g., adhesion, proliferation, and migration) by binding to its cell surface receptor (uPAR) and promoting conversion of Plasminogen into Plasmin. Moreover, studies have shown that expression of uPA and/or plasminogen activator inhibitor-1 (PAI-1) are predictors of poor prognosis in colorectal cancer (D. Q. Seetoo, et al., Journal of Surgical Oncology , vol. 82, no. 3, pp. 184-193, 2003), breast cancer (N. Harbeck et al., Clinical Breast Cancer , vol. 5, no. 5, pp. 348-352, 2004), and skin cancer (Santibanez, Juan F., ISRN Dermatology, 2013: 597927). Thus, without wishing to be bound by a particular theory, the interplay between Plasmin, TGFβ1, and uPA may create a positive feedback loop towards promoting cancer progression. Accordingly, inhibitors that selectively inhibit Plasmin-dependent TGFβ1 activation may be particularly suitable for the treatment of cancers reliant on the Plasmin/TGFβ1 signaling axis.

In one aspect of the invention, the isoform-specific inhibitors of TGFβ1 described herein include inhibitors that can inhibit protease-dependent activation of TGFβ1. In some embodiments, the inhibitors can inhibit protease-dependent TGFβ1 activation in an integrin-independent manner. In some embodiments, such inhibitors can inhibit TGFβ1 activation irrespective of the mode of activation, e.g., inhibit both integrin-dependent activation and protease-dependent activation of TGFβ1. In some embodiments, the protease is selected from the group consisting of: serine proteases, such as Kallikreins, Chemotrypsin, Trypsin, Elastases, Plasmin, as well as zinc metalloproteases (MMP family) such as MMP-2, MMP-9 and MMP-13.

In some embodiments, the inhibitors can inhibit Plasmin-induced activation of TGFβ1. In some embodiments, the inhibitors can inhibit Plasmin- and integrin-induced TGFβ1 activation. In some embodiments, the inhibitors are monoclonal antibodies that specifically bind TGFβ1. In some embodiments, the antibody is a monoclonal antibody that specifically binds proTGFβ1. In some embodiments, the antibody binds latent proTGFβ1 thereby inhibiting release of mature growth factor from the latent complex. In some embodiments, the high-affinity, LTBP-complex specific inhibitor of TGFβ1 activation suitable for use in the method of inhibiting Plasmin-dependent activation of TGFβ1. In some embodiments, the LTBP-complex specific inhibitor of TGFβ1 activation is selected from Ab31, Ab34, Ab37, Ab38, Ab39, Ab40, Ab41, Ab42, Ab43, Ab44, Ab45, Ab62, Ab63, and Ab64 (optionally Ab42 or Ab63) (i.e., an antibody or antigen-binding fragment having the heavy and light chain variable regions of the corresponding Ab, as provided herein) a variant/derivative or antigen-binding fragment thereof thereof, or an engineered molecule comprising an antigen-binding fragment thereof. In some preferred embodiments, the LTBP-complex specific inhibitor of TGFβ1 activation is Ab42, a variant/derivative or antigen-binding fragment thereof, or an engineered molecule comprising an antigen-binding fragment thereof. In preferred embodiments, the LTBP-complex specific inhibitor of TGFβ1 activation is Ab42 or an antigen-binding fragment thereof.

In some embodiments, the inhibitor (e.g., TGFβ1 antibody) inhibits cancer cell migration. In some embodiments, the inhibitor inhibits macrophage migration. In some embodiments, the inhibitor inhibits accumulation of TAMs.

In another aspect, provided herein is a method for treating cancer in a subject in need thereof, the method comprising administering to the subject an effective amount of an TGFβ1 inhibitor (e.g., TGFβ1 antibody), wherein the inhibitor inhibits protease-induced activation of TGFβ1 (e.g., Plasmin), thereby treating cancer in the subject.

In another aspect, provided herein is a method of reducing tumor growth in a subject in need thereof, the method comprising administering to the subject an effective amount of an TGFβ1 inhibitor (e.g., TGFβ1 antibody), wherein the inhibitor inhibits protease-induced activation of TGFβ1 (e.g., Plasmin), thereby reducing tumor growth in the subject.

Disease Involving ECM Dysregulation

The extracellular matrix is a cell-secreted network that surrounds cells and is primarily composed of proteoglycans and fibrous proteins, the most abundant of which is collagen. The novel antibodies disclosed herein may be used in the treatment of diseases associated with extracellular matrix dysregulation. The diseases associated with extracellular matrix dysregulation are typically myofibroblast-driven pathologies and include cancer, fibrosis, and cardiovascular disease (reviewed, for example, in: Lampi and Reinhart-King (2018) “Targeting extracellular matrix stiffness to attenuate disease: From molecular mechanisms to clinical trials” Sci Tarnsl Med 10(422): eaao0475). Progression of fibrotic conditions involves increased levels of matrix components deposited into the ECM and/or maintenance/remodeling of the ECM. TGFβ1 at least in part contributes to this process. This is supported, for example, by the observation that increased deposition of ECM components such as collagens can alter the mechanophysical properties of the ECM (e.g., the stiffness of the matrix/substrate) and this phenomenon is associated with TGFβ1 signaling. The inhibitors of TGFβ1, such as those described herein may be used to block this process to counter disease progression involving ECM alterations, such as fibrosis. The LTBP-arm of such inhibitors can directly block ECM-associated pro/latent TGFβ3 complexes which are presented by LTBP1 and/or LTBP3, thereby preventing activation/release of the growth factor from the complex in the disease niche. In some embodiments, the isoform-specific TGFβ1 inhibitors such as those described herein may normalize ECM stiffness to treat a disease that involves integrin-dependent signaling. In some embodiments, the integrin comprises an all chain, 131 chain, or both.

Thus, the antibody may be administered to a subject diagnosed with a disease with extracellular matrix dysregulation in an amount effective to treat the disease. Therapeutically effective amount of the antibody may be an amount sufficient to reduce expression of one or more markers of myofibroblasts, such as α-SMA. The amount may be an amount sufficient to reduce the stiffness of the extracellular matrix of an affected tissue (e.g., fibrotic tissues). The amount may be an amount sufficient to reduce TGFβ1 downstream effectors, such as phosphorylation of SMAD2 and/or SMAD3. In some embodiments, the isoform-selective activation inhibitor of TGFβ1 is selected from Ab31, Ab34, Ab37, Ab38, Ab39, Ab40, Ab41, Ab42, Ab43, Ab44, Ab45, Ab62, Ab63, and Ab64 (optionally Ab42 or Ab63) a variant/derivative or antigen-binding fragment thereof thereof, or an engineered molecule comprising an antigen-binding fragment thereof. In some preferred embodiments, the isoform-selective activation inhibitor of TGFβ1 is Ab42, a variant/derivative or antigen-binding fragment thereof, or an engineered molecule comprising an antigen-binding fragment thereof. In preferred embodiments, the TGFβ1-selective inhibitor is Ab42 or an antigen-binding fragment thereof.

Diseases Involving Epithelial-to-Mesenchymal Transition (EMT):

EMT (epithelial mesenchymal transition) is the process by which epithelial cells with tight junctions switch to mesenchymal properties (phenotypes) such as loose cell-cell contacts. The process is observed in a number of normal biological processes as well as pathological situations, including embryogenesis, wound healing, cancer metastasis and fibrosis (reviewed in, for example, Shiga et al. (2015) “Cancer-Associated Fibroblasts: Their Characteristics and Their Roles in Tumor Growth.” Cancers, 7: 2443-2458). Generally, it is believed that EMT signals are induced mainly by TGFβ. Many types of cancer, for example, appear to involve transdifferentiation of cells towards mesenchymal phenotype (such as CAFs) which correlate with poorer prognosis. Thus, LTBP-specific inhibitors of TGFβ1, such as those described herein, may be used to treat a disease that is initiated or driven by EMT. Indeed, data exemplified herein (e.g., FIGS. 12 and 13 ) show that such inhibitors have the ability to suppress expression of CAF markers in vivo, such as α-SMA, Col1 (Type I collagen), and FN (fibronectin).

Diseases Involving Matrix Stiffening and Remodeling

Progression of fibrotic conditions involves increased levels of matrix components deposited into the ECM and/or maintenance/remodeling of the ECM. TGFβ1 at least in part contributes to this process. This is supported, for example, by the observation that increased deposition of ECM components such as collagens can alter the mechanophysical properties of the ECM (e.g., the stiffness of the matrix/substrate) and this phenomenon is associated with TGFβ1 signaling. To confirm this notion, the present inventors have evaluated the role of matrix stiffness in affecting integrin-dependent activation of TGFβ in primary fibroblasts transfected with proTGFβ and LTBP1, and grown on silicon-based substrates with defined stiffness (e.g., 5 kPa, 15 kPa or 100 kPa). Matrices with greater stiffness enhance TGFβ1 activation, and this can be suppressed by antibodies, and antigen-binding portions thereof, which are capable of binding and thereby inhibiting TGFβ1 activation associated with LTBP1/3. These observations suggest that TGFβ1 influences ECM properties (such as stiffness), which in turn can further induce TGFβ1 activation, reflective of disease progression. Thus, antibodies, and antigen-binding portions thereof, that selectively bind complexes of LTBP1-TGFβ1 and/or LTBP3-TGFβ1, such as those described herein may be used to block this process to counter disease progression involving ECM alterations, such as fibrosis, tumor growth, invasion, metastasis and desmoplasia. Such inhibitors can directly block ECM-associated pro/latent TGFβ complexes which are presented by LTBP1 and/or LTBP3, thereby preventing activation/release of the growth factor from the complex in the disease niche.

Fibrosis:

In response to tissue injury or chronic insult due to physical damage/trauma, toxic substances, and/or infection, a natural reparative process begins which involves several cell types including fibroblasts, several different types of immune cells, and resident epithelial and endothelial cells. However, if left unchecked, this process can lead to excessive accumulation of extracellular matrix (ECM) and fibrosis, which in turn can lead to progressive loss of tissue function and organ failure (Caja et al., Int. J. Mol. Sci. 2018, 19, 1294).

Fibrosis can occur in several different organs, including lung, kidney, liver, heart, and skin. Independent of the organ, the fibrotic response is characterized by inflammation, altered epithelial-mesenchymal interactions, and proliferation of fibroblasts. One of the hallmarks of fibrosis is the differentiation of fibroblasts into myofibroblasts, which greatly contribute to the dysregulation of the ECM. However, myofibroblasts have also been proposed to come from other cellular sources (e.g., endothelial cells, epithelial cells, and mesenchymal stem cells (Kim, K K et al, Cold Spring Harb. Perspect. Biol., 2017; Okabe, H. Histol. Histophathol., 2016, 31, 141-148; and Li, C et al, Nat Commun., 2016, 7, 11455). Moreover, immune cells play an important role in the process by secreting cytokines and chemokines which promote differentiation of myofibroblasts, stimulate ECM deposition, and recruit additional immune cells to the damaged tissue (Caj a et al., Int. J. Mol. Sci. 2018, 19, 1294).

Similar to fibrotic tissue, activation of cancer-associated fibroblasts can occur in the tumor milieu, which produces excessive amounts of ECM. The ECM provides a scaffold for the infiltration of other cells (e.g., pro-tumorigenic immune cells) and a substrate for cell migration. In other cases, excessive ECM may act as a barrier against anti-tumorigenic immune cells.

TGFβ is recognized as the central orchestrator of the fibrotic response. TGFβ can promote myofibroblast differentiation, recruit immune cells, and affect epithelial and endothelial cell differentiation. Particularly, TGFβ upregulates the production of ECM and basement membrane proteins, such as fibronectin, collagen, laminin, osteopontin, tenascin, elastin, decorin. TGFβ-induced myofibroblast differentiation can lead to additional deposition of ECM proteins, secretion of matric metalloproteinase (MMPs), and myofibroblast proliferation (Fabregat et al, FEBS J. 2016, 283, 2219-2232; Meng et al, Nat. Rev. Nephrol. 2016, 12, 325-338; and Kulkarni et al., Am. J. Respir. Cell Mol. Biol., 2016, 54, 751-760). Additionally, TGFβ mediates phenotypic changes affecting contractile proteins and collagen I in vascular smooth muscle cells (VSCM), and can activate myofibroblasts and other stromal cells to enhance the synthesis of collagen cross-linking proteins, such as lysyl oxidase (LOX) family of matrix-remodeling enzymes (Busnadiego et al., Mol. Cell. Biol. 2013, 33, 2388-2401). Moreover, TGFβ has been shown to regulate both EMT and EndMT, which contributes to the differentiation of pro-fibrotic cell types, such as myofibroblasts and CAFs. Moreover, TGFβ has been shown to induce epithelial apoptosis, which can promote lung and liver fibrosis among other tissues (Barbas-Filho et al., J. Clin. Pathol. 2001, 54, 132-138; and Wang et al., Dev. Dyn. 2017, 247, 492-508).

Whether innate or recruited, macrophages are thought to play an important role in responding to tissue damage and repair. However, upon certain signals they can become pro-fibrotic. TGFβ, among other cytokines, has also been shown to activate M2 macrophages, which are pro-inflammatory. Upon activation, these macrophages secrete their own cytokines, including TGFβ, ECM components, angiogenic factors, and chemotactic factors. M2 macrophages have been shown to be essential for TGFβ-driven lung fibrosis (Murray et al., Int. J. Biochem. Cell Biol. 2011, 43, 154-162).

In light of increasing evidence pointing to the importance of M2-type macrophages for diseases progression in many types of fibrosis, a question remained as to whether context-selective inhibition of LTBP1/3-associated TGFβ1 alone (that is, without addressing the macrophage-associated, LRRC33-arm of TGFβ1 activity) might be sufficient to produce a potent anti-fibrotic effect in vivo. Surprisingly, however, data presented herein suggest that selectively targeting the matrix-associated TGFβ1 (e.g., LTBP1/3-proTGFβ31) appears to be just as effective—if not better—in achieving anti-fibrotic effects in multiple preclinical models, as targeting all four known LLCs (e.g., LTBP1/3-proTGFβ1, GARP-proTGFβ1 and LRRC33-proTGFβ1) with the use of a so-called context-independent inhibitor of TGFβ1 (see, for example, FIGS. 19 and 20 ) and do so without triggering T cell stimulation mediated via GARP-proTGFβ1 inhibition. Moreover, previously disclosed LTBP1/3 complex-selective antibodies lacked robust species cross-reactivity that would be advantageous for both preclinical (e.g., rodent) and clinical (e.g., human) use. It was not clear whether the rare epitopes being sought which would confer both isoform-selectivity and context-selectivity would also enable favorable species cross-reactivity profiles. Advantageously, novel antibodies disclosed herein possess all of these criteria.

According to the invention, isoform-specific TGFβ1 such as those described herein are used in the treatment of fibrosis (e.g., fibrotic indications, fibrotic conditions) in a subject. Suitable inhibitors to carry out the present invention include antibodies and/or compositions according to the present disclosure which may be useful for altering or ameliorating fibrosis. More specifically, such antibodies and/or compositions are selective antagonists of TGFβ1 that are capable of targeting TGFβ1 presented by various types of presenting molecules. TGFβ1 is recognized as the central orchestrator of the fibrotic response. Antibodies targeting TGFβ decrease fibrosis in numerous preclinical models. Such antibodies and/or antibody-based compounds include LY2382770 (Eli Lilly, Indianapolis, IN). Also included are those described in U.S. Pat. Nos. 6,492,497, 7,151,169, 7,723,486 and U.S. Appl. Publ. No. 2011/0008364, the contents of each of which are herein incorporated by reference in their entirety. Prior art TGFβ antagonists include, for example, agents that target and block integrin-dependent activation of TGFβ.

However, evidence suggests that such prior art agents may not mediate isoform-specific inhibition and may cause unwanted effects by inadvertently blocking normal function of TGFβ2 and/or TGFβ3. Indeed, data presented herein support this notion. Normal (undiseased) lung tissues contain relatively low but measurable levels of TGFβ2 and TGFβ3, but notably less TGFβ1. In comparison, in certain disease conditions such as fibrosis, TGFβ1 becomes preferentially upregulated relative to the other isoforms. Preferably, TGFβ antagonists for use in the treatment of such conditions exert their inhibitory activities only towards the disease-induced or disease-associated isoform, while preserving the function of the other isoforms that are normally expressed to mediate tonic signaling in the tissue. Prior art inhibitors (LY2109761, a small molecule TGFβ receptor antagonist, and a monoclonal antibody that targets αVβ6 integrin) both are shown to inhibit TGFβ downstream tonic signaling in non-diseased rat BAL, raising the possibility that these inhibitors may cause unwanted side effects. Alternatively or additionally, agents that target and block integrin-dependent activation of TGFβ may be capable of blocking only a subset of integrins responsible for disease-associated TGFβ1 activation, among numerous integrin types that are expressed by various cell types and play a role in the pathogenesis. Furthermore, even where such antagonists may selectively block integrin-mediated activation of the TGFβ1 isoform, it may be ineffective in blocking TGFβ1 activation triggered by other modes, such as protease-dependent activation. Accordingly, the isoform-specific inhibitors of TGFβ1 such as those described herein are aimed to prevent the activation step of TGFβ1 regardless of the particular mode of activation, while maintaining isoform selectivity.

It is further contemplated that isoform-specific TGFβ1 inhibitors that preferentially inhibit matrix-associated over cell-associated antigen complexes (i.e., display context-bias) may offer a therapeutic advantage in certain clinical situations (e.g., the LTBP-specific inhibitors described herein). For example, TGFβ1 context-independent inhibitors (which target all four antigen complexes), may increase immune activation through the targeting of cell-associated TGFβ1 (e.g., GARP-TGFβ1 which is expressed on regulatory T cells) Immune activation may be disadvantageous for certain patients, e.g., patients with autoimmune disease or who are at risk of sepsis. Accordingly, context-bias antibodies may be useful for treating diseases associate with matrix-associated TGFβ1 complexes (e.g., fibrosis), while minimizing immune activation.

Previously, it was contemplated that isoform-specific TGFβ3 inhibitors might offer an added therapeutic benefit in particular disease states. For example, certain fibrotic diseases to be treated with a TGFβ1 inhibitor may also be TGFβ3-positive (i.e., TGFβ31+/TGFβ3+fibrotic tissue) characterized in that the disease tissue (e.g., fibrotic tissue) expresses both the isoforms. Accordingly, the invention includes the use of isoform-selective TGFβ1 inhibitor in conjunction with an isoform-selective TGFβ3 inhibitor in the treatment of such conditions.

Fibrotic indications for which antibodies and/or compositions of the present disclosure may be used therapeutically include, but are not limited to lung indications (e.g., idiopathic pulmonary fibrosis (IPF), chronic obstructive pulmonary disorder (COPD), allergic asthma, acute lung injury, eosinophilic esophagitis, pulmonary arterial hypertension and chemical gas-injury), kidney indications (e.g., diabetic glomerulosclerosis, focal segmental glomeruloclerosis (FSGS), chronic kidney disease (CKD), fibrosis associated with kidney transplantation and chronic rejection, IgA nephropathy, and hemolytic uremic syndrome), liver fibrosis (e.g., non-alcoholic steatohepatitis (NASH), chronic viral hepatitis, parasitemia, inborn errors of metabolism, toxin-mediated fibrosis, such as alcohol fibrosis, non-alcoholic steatohepatitis-hepatocellular carcinoma (NASH-HCC), primary biliary cirrhosis, and sclerosing cholangitis), cardiovascular fibrosis (e.g., cardiomyopathy, hypertrophic cardiomyopathy, atherosclerosis and restenosis,) systemic sclerosis, skin fibrosis (e.g., skin fibrosis in systemic sclerosis, diffuse cutaneous systemic sclerosis, scleroderma, pathological skin scarring, keloid, post-surgical scarring, scar revision surgery, radiation-induced scarring and chronic wounds), eye-related conditions such as subretinal fibrosis, uveitis syndrome, uveitis associated with idiopathic retroperitoneal fibrosis, extraocular muscle fibrosis, eye diseases associated with the major histocompatibility complex (MHC class I) or histocompatibility antigens, subretinal fibrosis in macular degeneration (e.g., age-related macular degeneration), and cancers or secondary fibrosis (e.g., myelofibrosis, head and neck cancer, M7 acute megakaryoblastic leukemia and mucositis). Other diseases, disorders or conditions related to fibrosis (including degenerative disorders) that may be treated using compounds and/or compositions of the present disclosure, include, but are not limited to adenomyosis, endometriosis, Marfan's syndrome, stiff skin syndrome, scleroderma, rheumatoid arthritis, bone marrow fibrosis, Crohn's disease, ulcerative colitis, systemic lupus erythematosus, muscular dystrophy (such as DMD), Parkinson's disease, ALS, Dupuytren's contracture, Camurati-Engelmann disease, neural scarring, dementia, proliferative vitreoretinopathy, corneal injury, complications after glaucoma drainage surgery, and multiple sclerosis (MS). Many such fibrotic indications are also associated with inflammation of the affected tissue(s), indicating involvement of an immune component. Such inflammation may be accompanied by aberrant immune cell populations, such as increased numbers of Th17 cells, reduced numbers of Treg cells, and/or both. In each case, the affected patient may exhibit increased Th17/Treg cell ratios.

In some embodiments, fibrotic indications that may be treated with the compositions and/or methods described herein include organ fibrosis, such as fibrosis of the lung (e.g., IPF), fibrosis of the kidney (e.g., fibrosis associated with CKD), fibrosis of the liver, fibrosis of the heart or cardiac tissues, fibrosis of the skin (e.g., scleroderma), fibrosis of the uterus (e.g., endometrium, myometrium), and fibrosis of the bone marrow. In some embodiments, such therapy may reduce or delay the need for organ transplantation in patients. In some embodiments, such therapy may prolong the survival of the patients.

To treat IPF, patients who may benefit from the treatment include those with familial IPF and those with sporadic IPF. Administration of a therapeutically effective amount of an isoform-specific inhibitor of TGFβ1 may reduce myofibroblast accumulation in the lung tissues, reduce collagen deposits, reduce IPF symptoms, improve or maintain lung function, and prolong survival. In some embodiments, the inhibitor blocks activation of ECM-associated TGFβ1 (e.g., pro/latent TGFβ1 presented by LTBP1/3) within the fibrotic environment of IPF.

Nonalcoholic fatty liver disease (NAFLD) includes a spectrum of histological changes that begin with simple fatty infiltration of the liver, also known as simple or isolated steatosis or nonalcoholic fatty liver (NAFL), which may gradually, sometimes over decades, progress to the development of chronic inflammation (steatohepatitis or NASH), fibrosis, and ultimately cirrhosis. Only a subgroup of patients with NAFL will progress to NASH and subsequent cirrhosis. Currently, there are no clear criteria to identify this group of patients. NAFLD is the most common cause of chronic liver disease in North America. Currently, there are no approved drugs for the treatment of NASH. Given the high prevalence of NASH, the associated morbidity, the growing burden of end-stage liver disease, and limited availability of livers for organ transplantation, identifications of therapies that will slow the progress of, halt, or reverse NASH and NAFLD will address an unmet medical need.

There is a consensus that TGFβ is a central player in liver fibrosis (reviewed in, for example, Dewidar et al., Cells 2019, 8, 1419, the contents of which are incorporated herein by reference). The isoform-specific TGFβ1 inhibitors such as those provided herein (i.e., isoform-specific inhibitors of TGFβ1 that are selective for LTBP1/3-TGFβ1 complexes, or “matrix-targeted” inhibitors) may be used to treat fibrotic conditions of the liver, such as nonalcoholic fatty liver (NAFL) and fibrosis associated with fatty liver (e.g., NASH). The fatty liver may or may not be inflamed. Inflammation of the liver due to fatty liver (i.e., steatohepatitis) may develop into scarring (fibrosis), which then often progresses to cirrhosis (scarring that distorts the structure of the liver and impairs its function). The inhibitor may therefore be used to treat such conditions. In some embodiments, the inhibitor blocks activation of ECM-associated TGFβ1 (e.g., pro/latent TGFβ1 presented by LTBP1/3) within the fibrotic environment of the liver. Administration of the inhibitor in a subject with such conditions may reduce one or more symptoms, prevent or retard progression of the disease, reduce or stabilize fat accumulations in the liver, reduce disease-associated biomarkers (such as serum collagen fragments), reduce liver scarring, reduce liver stiffness, and/or otherwise produce clinically meaningful outcome in a patient population treated with the inhibitor, as compared to a control population not treated with the inhibitor. In some embodiments, an effective amount of the inhibitor may achieve both reduced liver fat and reduced fibrosis (e.g., scarring) in NASH patients. In some embodiment, an effective amount of the inhibitor may achieve improvement in fibrosis by at least one stage with no worsening steatohepatitis in NASH patients. In some embodiments, an effective amount of the inhibitor may reduce the rate of occurrence of liver failure and/or liver cancer in NASH patients. In some embodiments, an effective amount of the inhibitor may normalize, as compared to control, the levels of multiple inflammatory or fibrotic serum biomarkers as assessed following the start of the therapy, at, for example, 12-36 weeks. In some embodiments in NASH patients, the isoform-specific TGFβ1 inhibitors may be administered in patients who receive one or more additional therapies, including, but are not limited to myostatin inhibitors, which may generally enhance metabolic regulation in patients with clinical manifestation of metabolic syndrome, including NASH.

In some embodiments, in NASH or NAFLD patients, the isoform-specific, matrix-targeted, TGFβ1 inhibitors may be administered in patients who receive an Acetyl CoA Carboxylase inhibitor (ACCi) (e.g., firsocostat (aka GS-0976) or PF-05221304). Other therapeutics which may be useful in combination with the improved isoform-specific TGFβ1 inhibitors described herein, include, but are not limited to: GLP-1 receptor agonists or analgues (e.g., semaglutide), farnesoid X receptor (FXR) agonists (e.g., GS-9674; aka Cilofexor), ASK1 inhibitors (e.g., selonsertib); obeticholic acid, PPAR agonists (e.g., GFT505; aka elafibranor); nitazoxanide, ketohexokinase (KHK) inhibitors (e.g., PF-06835919); myostatin inhibitors and/or Diacylglycerol 0-Acyltransferase 2 (DGAT2) inhibitors (e.g., PF-06865571). In some embodiments, any one or more of the above-mentioned therapeutics can be used in combination with an isoform specific TGFβ1 inhibitor of the present disclosure, for example, an isoform-specific TGFβ1 inhibitor in combination with a FXR agonist, an ACC inhibitor, and/or a GLP-1 analogue. In some embodiments, TGFβ inhibitors may be used in combination with a myostatin inhibitor in the treatment of a metabolic liver disease in a subject, such as NASH and NAFLD, and liver fibrosis associated therewith. The subject may also suffer from type 2 diabetes and/or obesity. The TGFβ inhibitors used are preferably TGFβ31-selective inhibitors, more preferably context-selective TGFβ1-selective inhibitors that target LTBP1/2-associated TGFβ1, such as those disclosed herein. The myostatin inhibitor is preferably a myostatin-selective inhibitor, such as SRK-015 (e.g., see WO2017/218592A1) and trevogrumab, or any variant thereof, or an antibody according to WO 2016/098357.

In some embodiments, treatment with the isoform specific TGFβ1 inhibitors alone or in combination with one or more additional therapeutics reduces hepatic fat as measured by MRI-PDFF. In some embodiments, the reduction of hepatic fat is at least ≥20%, e.g., ≥20%, ≥25%, ≥30%, ≥35%, ≥40%, ≥45%, or ≥50%. In some embodiments, treatment with the isoform specific TGFβ1 inhibitors alone or in combination with one or more additional therapeutics reduces serum ALT and/or GGT by at least 20%, e.g., ≥20%, ≥25%, ≥30%, ≥35%, ≥40%, ≥45%, or ≥50%. In some embodiments, treatment with the isoform specific TGFβ1 inhibitors alone or in combination with one or more additional therapeutics reduces bile acid synthesis.

In some embodiments, either as monotherapy or in conjunction with one or more additional therapy (e.g., combination therapy), the TGFβ1 inhibitors of the present disclosure may be effective to treat NASH. “Effective treatment” may refer to improvements in hepatic steatosis, liver stiffness, liver biochemistry and serum fibrosis markers. In some embodiments, a 12-week treatment may result in significant decline of at least 30 percent in hepatic fat measured by magnetic resonance imaging-proton density fat fraction (MRI-PDFF) from baseline to 12 weeks in at least 50% percent of patients. Improvements in liver biochemistry tests including serum ALT of median relative reduction of at least 25% and GGT of at least 25% along with markers of reduced bile acid synthesis, may be achieved at 12 weeks.

In some embodiments, the NASH patients may have advanced liver fibrosis (stage F3/F4). In some embodiments, such patients have stage F3 advanced liver fibrosis. In some embodiments, such patients have stage F4 liver fibrosis characterized by cirrhosis. In some embodiments, the NASH patients develop or at risk of developing hepatocellular carcinoma and/or esophageal varices.

Fibrosis staging in non-alcoholic fatty liver disease according to the classification derived by the Nonalcoholic Steatohepatitis Clinical Research Network Pathology Committee is provided below:

Therapeutic benefits may by also evaluated by burden of disease and patient-reported outcomes. NASH also has an impact on quality of life for those living with the condition, measured through patient-reported outcomes (PROs). PROs may be assessed using tools such as the Chronic Liver Disease Questionnaire (CLDQ-NASH) prior to treatment (e.g., baseline), particularly those related to physical health-related scores,

Treatment with a TGFβ1 inhibitor (such as LTBP1/3 complex-selective inhibitors described herein) either alone (e.g., monotherapy) or in combination with another therapy, may be effective to improve the PROs as compared to the baseline, or as compared to those of population norms. In some embodiments, diabetes mellitus may be associated with impairment in PROs including physical functioning, bodily pain, general health and vitality. Treatment with a TGFβ1 inhibitor (such as LTBP1/3 complex-selective inhibitors described herein) either alone (e.g., monotherapy) or in combination with another therapy, may be effective to improve physical health-related scores (such as PROs) among subjects with diabetes (e.g., type 2 diabetes) and/or obesity.

Published studies in the literature suggest that regulatory T cells (Tregs) may play a role in the progression of liver disease into later-stage fibrosis with greater severity. For example, Zhang et al. reported that persistence of liver cirrhosis is maintained by intrahepatic regulatory T cells that inhibit the process of fibrosis resolution (Transl Res. 2016; 169: 67-79.e1-2). Kobayashi et al. suggested that Tregs are involved in the progression of liver fibrosis into hepatocellular carcinoma (HCC), a process referred to as hepatocarcinogenesis (Clin Cancer Res. 2007; 13(3): 902-911).

Accordingly, it is contemplated that careful selection of suitable TGFβ inhibitor tailored to the disease type and stage of the disease progression should be considered to maximize therapeutic benefit to a particular patient or patient population.

In some embodiments, a TGFβ1-selective, context-selective inhibitor that targets matrix-associated TGFβ1 (e.g., LTBP1-proTGFβ1 and/or LTBP3-proTGFβ31), such as those disclosed herein, is selected for use in the treatment of an early-stage liver disease such as nonalchoholic fatty liver (“NAFL”) and noncirrhotic liver fibrosis associated with NASH. The noncirrhotic liver fibrosis includes liver fibrosis of stages 1-3. The TGFβ1-selective, context-selective inhibitor is administered to the subject in an amount effective to treat the disease, e.g., slow the progress of, halt, or reverse NAFL and noncirrhotic NASH. Preferably, the effective amount is sufficient to prevent progression to cirrhosis and cirrhosis complications, reduce the need for liver transplantation, and/or improve survival. In some embodiments, efficacy may be shown by, for example, reduction of inflammatory changes, improvement in fibrosis, or both. In some embodiments, the subject has a metabolic condition, such as obesity, type 2 diabetes. The subject with noncirrhotic NASH may include those with a NASH activity score (NAS) greater than or equal to 4 with at least 1 point each in inflammation and ballooning along with a NASH Clinical Research Network (CRN) fibrosis score greater than stage 1 fibrosis but less than stage 4 fibrosis. In some embodiments, the treatment achieves resolution of steatohepatitis on overall histopathological reading and no worsening of liver fibrosis on NASH CRN fibrosis score. Resolution of steatohepatitis is defined as absent fatty liver disease or isolated or simple steatosis without steatohepatitis and a NAS score of 0-1 for inflammation, 0 for ballooning, and any value for steatosis; or, improvement in liver fibrosis greater than or equal to one stage (NASH CRN fibrosis score) and no worsening of steatohepatitis (defined as no increase in NAS for ballooning, inflammation or steatosis); or, both resolution of steatohepatitis and improvement in fibrosis as defined above.

In some embodiments, a TGFβ1-selective, context-selective inhibitor that targets matrix-associated TGFβ1 (e.g., LTBP1-proTGFβ1 and/or LTBP3-proTGFβ31), such as those disclosed herein, is selected for use in the treatment of NASH with compensated cirrhosis. NASH-associated compensated cirrhosis is characterized by significant scar formation that is evident by histopathology, with hepatocytes clustered in nodules surrounded by dense extracellular matrix. The TGFβ1-selective, context-selective inhibitor is administered to the subject in an amount effective to halt or slow progression of fibrosis, prevent clinical decompensation, reduce the need for liver transplantation, and/or improve survival.

NASH with decompensated cirrhosis may be characterized by one or more of the following criteria: portal hypertension (evidence of portal hypertension may include low platelet counts, esophageal varices, ascites, history of hepatic encephalopathy, splenomegaly); elevated bilirubin; or elevated international normalized ratio or prolonged prothrombin time. Thus, patients having NASH with compensated cirrhosis may be those not meeting one or more of the aforementioned decompensated cirrhosis criteria.

In some embodiments, a TGFβ1-selective, context-selective inhibitor that targets matrix-associated TGFβ1 (e.g., LTBP1-proTGFβ1 and/or LTBP3-proTGFβ31), such as those disclosed herein, is selected for use in the treatment of NASH with decompensated cirrhosis or HCC associated with liver fibrosis. In some embodiments, upon progression of the disease into a late-stage or end-stage liver disease characterized by manifestation of cirrhosis or HCC, the TGFβ1-selective, context-selective inhibitor is replaced with a TGFβ1-selective, context-independent inhibitor capable of targeting both matrix-associated and immune cell-associated TGFβ1, e.g., LTBP1-proTGFβ1, LTBP3-proTGFβ1, GARP-proTGFβ1 and LRRC33-proTGFβ1 (see, for example, WO 2020/014473) in an amount effective to treat liver cirrhosis or HCC. In some embodiments, the TGFβ1-selective, context-selective inhibitor and/or the TGFβ1-selective, context-independent inhibitor may be used as monotherapy or in conjunction with one or more additional therapy.

In some embodiments, an effective amount of the inhibitor may normalize, as compared to control, the levels of multiple inflammatory or fibrotic serum biomarkers as assessed following the start of the therapy, at, for example, 12-36 weeks. In some embodiments, inflammatory or fibrotic biomarkers may be used to assess severity of NAFLD (by measure levels of hepatic steatosis), select patients for treatment, and/or monitor disease progression or treatment response. For example, blood biomarkers and panels may include, but are not limited to:

i) the Fatty liver index (BMI, waist circumference, serum triglycerides, and gamma-glutamyltransferase (GGT); ii) the Hepatic steatosis index (serum aspartate aminotransferase (AST):alanine aminotransferase (ALT) ratio, BMI, gender, and presence of diabetes mellitus); i) the NAFLD liver fat score (serum ALT, HDL cholesterol, triglicerides, haemoglobin A 1c and leukocyte count); ii) the SteatoTest (BioPredictive) (serum levels of total bilirubin, GGT, α2-macroglobin, haptoglobin, ALT, apolipoprotein AI, total cholesterol, triglycerides, glucose (adjusted for age and gender) and BMI); and iii) the NAFLD ridge score (serum levels of ALT, HDL cholesterol, triglycerides, haemoglobin A 1c , leukocyte count, and comorbidity data (and the presence of hypertension)).

In some embodiments, imaging biomarkers can be used to assess levels of hepatic steatosis. For example, imaging biomarkers may include but are not limited to: ultrasonography, controlled attenuation parameter (CAP), MRI-estimated proton density fat fraction (MRI-PDFF), and magnetic resonance spectroscopy (MRS).

Liver biopsies are the current standard for diagnosis NASH, however, variability among pathologists limits the effectiveness of such diagnostic method. Accordingly, use of the Fatty Liver Inhibition of Progression (FLIP) algorithm (comprising histological steatosis, activity and fibrosis scores) may be used to improve the consistency of NASH diagnosis by biopsy. Moreover, many noninvasive biomarkers may also be useful for diagnosing and monitoring disease. Accordingly, in some embodiments, inflammatory or fibrotic biomarkers may be used to assess severity of NASH, select patients for treatment, and/or monitor disease progression or treatment response. Blood biomarkers may include:

i) apoptosis markers, such as CK18 fragments, total cytokeratin and sFAS; ii) inflammatory markers, such as CRP, TNF, IL-8, and CXCL10; iii) lipid oxidation products, such as 11-HETE, 9-HODE, 13-HODE, 12-oxo-ODE, LA-13-HODE (oxNASHscore), and 11,12-diHETrE; iv) lysosomal enzymes, such as cathepsin D; and v) combination panels, such as NASHTest (BioPredictive) and NASH Diagnostics Panel (comprising, presence of diabetes mellitus, sex, BMI, and serum levels of triglyceride, CK18 fragments, and total CK18).

In some embodiments, biomarkers and related panels may be useful in diagnosis levels of fibrosis and/or cirrhosis, select patients for treatment, and/or monitor disease progression or treatment response. For example, noninvasive tests of liver fibrosis and cirrhosis include, but are not limited to: AST:ALT ratio, AST:platelet ratio index, fibrosis-4 index (age, AST, ALT, and platelet count), NAFLD fibrosis score (age, BMI, impaired fasting glucose and/or diabetes, AST ALT, platelet count, and albumin), BARD score (AST, ALT, BMI, and diabetes).

Specific fibrosis markers and panels may also be useful, and include, but are not limited to: hyaluronic acid; PIIPNP; Pro-C3; TIMP1; Laminin; enhanced liver fibrosis (ELF) panel (PIINP, hyaluronic acid, TIMP1); FibroTest (GGT, total bilirubin, atm, apolipoprotein AI, and haptoglobin); and FibroMeter NAFLD (body weight, prothrombin index, ALT, AST, ferritin, and fasting glucose). Imaging biomarkers for liver fibrosis may include, but are not limited to: FibroScan (TE), point shear wave elastography (pSWE) (aka acoustic radiation force impulse (ARFI)), 2D-3D SWE, magnetic resonance elastography (MRE), and multiparameteric MRI.

Any RGFb-related disease with an inflammatory or auto inflammatory element may benefit from the novel inhibitors of the present disclosure which spare the regulatory T cell function. Particularly in liver diseases, e.g., metabolic liver conditions, it may be advantageous to select a TGFb inhibitor that selectivity target the matrix-associated TGFb1 signaling.

In one embodiment, the methods and compositions for use as described herein are useful for treating a subject having primary biliary cholangitis (PBC). In one embodiment, the subject having PBC has been nonresponsive to UDCA (ursodeoxycholic acid) treatment. In one embodiment, the subject has Barcelona, Paris-I, Toronto, Rotterdam, or Paris-II insufficient response to UCDA. In one embodiment, the subject having PBC has ALP≥2×ULN (upper limit of normal), and bilirubin >1×ULN despite an at least 1 year therapy with UDCA at the standard recommended dose (10-15 mg/kg b.w./day).

In another embodiment, the methods and compositions for use as described herein are useful for treating a subject having primary sclerosing cholangitis (PSC). In one embodiment, the subject having PSC has an elevated ALP, an abnormal cholangiography, endoscopic retrograde cholangiopancreatography, or percutaneous transhepatic cholangiography. In one embodiment, the subject having PSC has a model for end-stage liver disease (MELD) score of at least 14.

In another embodiment, the methods and compositions for use as described herein are useful for treating a subject having NASH. In one embodiment, a subject has fibrosis stage 2 NASH, fibrosis stage 3 NASH, or fibrosis stage 4 NASH. In one embodiment, a subject has a NAS (NAFLD activity score) of at least 4, or of at least 5. In one embodiment, a subject with NASH has a NAS>4 and fibrosis stage 2 or stage 3. In one embodiment, a subject with NASH has a NAS>5 and fibrosis stage 2 or stage 3. In one embodiment, a subject with NASH has a model for end-stage liver disease (MELD) score of at least 14.

The isoform-specific TGFβ1 inhibitors such as those provided herein may be used to treat fibrotic conditions of the kidney, e.g., diseases characterized by extracellular matrix accumulation (IgA nephropathy, focal and segmental glomerulosclerosis, crescentic glomerulonephritis, lupus nephritis and diabetic nephropathy) in which significantly increased expression of TGFβ in glomeruli and the tubulointerstitium has been observed. While glomerular and tubulointerstitial deposition of two matrix components induced by TGFβ, fibronectin EDA+ and PAI-1, was significantly elevated in all diseases with matrix accumulation, correlation analysis has revealed a close relationship primarily with the TGFβ1 isoform. Accordingly, the isoform-specific TGFβ1 inhibitors are useful as therapeutic for a spectrum of human glomerular disorders, in which TGFβ is associated with pathological accumulation of extracellular matrix.

In some embodiments, the fibrotic condition of the kidney is associated with chronic kidney disease (CKD). CKD is caused primarily by high blood pressure or diabetes and claims more than one million lives each year. CKD patients require lifetime medical care that ranges from strict diets and medications to dialysis and transplants. In some embodiments, the TGFβ1 inhibitor therapy described herein may reduce or delay the need for dialysis and/or transplantation. In some embodiments, such therapy may reduce the need (e.g., dosage, frequency) for other treatments. In some embodiments, the isoform-specific TGFβ1 inhibitors may be administered in patients who receive one or more additional therapies, including, but are not limited to myostatin inhibitors, which may generally enhance metabolic regulation in patients with CKD.

Fibrotic conditions that may be treated with the TGFβ1 inhibitor of the present disclosure include conditions involving fibrosis and/or chronic inflammation. Such conditions may be neuromuscular disorders, including but are not limited to Duchenne muscular dystrophy (DMD), and other genetic disorders such as multiple sclerosis (MS) and cystic fibrosis (CF). Through the inhibition of both the ECM- and immune cell-associated TGFβ1 arms, the TGFβ1 inhibitor such as those described herein is thought to suppress fibrotic progression and restore M1/M2 macrophage polarization.

The organ fibrosis which may be treated with the methods provided herein includes cardiac (e.g., cardiovascular) fibrosis. In some embodiments, the cardiac fibrosis is associated with heart failure, e.g., chronic heart failure (CHF). In some embodiments, the heart failure may be associated with myocardial diseases and/or metabolic diseases. In some embodiments, the isoform-specific, TGFβ1 inhibitors may be administered in patients who receive one or more additional therapies, including, but are not limited to myostatin inhibitors in patients with cardiac dysfunction that involves heart fibrosis and metabolic disorder.

In some embodiments, fibrotic conditions that may be treated with the compositions and/or methods described herein include desmoplasia. Desmoplasia may occur around a neoplasm, causing dense fibrosis around the tumor (e.g., desmoplastic stroma), or scar tissue within the abdomen after abdominal surgery. In some embodiments, desmoplasia is associated with malignant tumor. Due to its dense formation surrounding the malignancy, conventional anti-cancer therapeutics (e.g., chemotherapy) may not effectively penetrate to reach cancerous cells for clinical effects. Isoform-specific, inhibitors of TGFβ1 such as those described herein may be used to disrupt the desmoplasia, such that the fibrotic formation can be loosened to aid effects of anti-cancer therapy. In some embodiments, the isoform-specific inhibitors of TGFβ1 can be used as monotherapy (more below).

In some embodiments, a patient has a fibrotic solid tumor (e.g., desmoplasia) and is or has been excluded from a surgical candidate pool, such that the fibrotic solid tumor is considered to be non-resectable or non-operable (e.g., risk of surgical intervention outweighs potential benefit thereof). Such patient may be a candidate for receiving a TGFβ1 inhibition therapy of the present disclosure. The TGFβ1 inhibition therapy of the present invention administered to such patients may render the tumor become resectable or operable so that the patient may become a candidate for surgical resection.

To treat patients with fibrotic conditions, TGFβ1 isoform-specific inhibitors are administered to a subject in an amount effective to treat the fibrosis. The effective amount of such an antibody is an amount effective to achieve both therapeutic efficacy and clinical safety in the subject. In some embodiments, the inhibitor is an antibody that can block activation of an LTBP-mediated TGFβ1 localized (e.g., tethered) in the ECM. In some embodiments, the LTBP is LTBP1 and/or LTBP3. In some embodiments, a LTBP-specific inhibitor of TGFβ1 can be combined with an inhibitor of LRRC33-proTGFβ.

Assays useful in determining the efficacy of the antibodies and/or compositions of the present disclosure for the alteration of fibrosis include, but are not limited to, histological assays for counting fibroblasts and basic immunohistochemical analyses known in the art.

In some embodiments, circulating LAP fragment(s) may be used as a serum marker of fibrogenesis. See for example, U.S. Pat. No. 8,198,412, the contents of which are incorporated herein by reference.

There are many animal models that have been developed to study fibrosis. For example, certain high fat diets in mice has been shown to mimic both the histopathology and pathogenesis of human NAFLD. Moreover, some genetic models also display features of human metabolic syndrome and NAFLD, such as db/db and ob/ob mouse models. There are also animal models for the study of NASH, which mainly consist of various diet-induced models, including, but not limited to, methionine and choline-deficient diet (MCD), high-cholesterol diet (HCD), choline-deficient high fat diet (CDHFD), choline-deficient L-amino acid-deficient diet, choline-deficient L-amino acid-deficient diet+carbon tetrachloride, high-fat diet +streptozotocin, high fat +high cholesterol diet (HFHC), high-fructose diet (HFD), and high-fructose high fat diet (HFHF). Genetic mouse models for the study of NASH include, but are not limited to foz/foz mice, Hepatocyte-specific PTEN-deficient mice, Db/db mice+diethylnitrosamine (DEN), and db/db mice+MCD. The details of all of these models, including the pluses and minus of each, are outlined in Jennie Ka Ching Lau et al., J Pathol 2017; 241: 36-44; the contents of which are incorporated herein by reference.

Other models useful for testing the efficacy of TGFβ inhibitors in fibrosis include the carbon tetrachloride (CCL 4 )-induced liver fibrosis model and adenine-induced kidney fibrosis model. Another model useful for testing the efficacy of isoform-specific TGFβ inhibitors in liver fibrosis include the bile duct ligation (BDL) model (see, e.g., Tag et al., J Vis Exp. 2015; (96): 52438). A useful genetic model of kidney fibrosis includes Alport model (discussed elsewhere herein).

In any of such preclinical models, efficacy in fibrosis (e.g., pro-fibrotic or anti-fibrotic effects in vivo) may be assessed by any suitable methods, such as: percent of picosirius red (PSR)-positive area in tissue sections; contents (quantification) of hydroxyproline in tissue; and immunohistochemical detection and quantification of collA staining on tissue sections. In some embodiments, histopathological appraisal may be carried out, in which fibrosis scoring system can be employed. Pharmachological effects of test articles (e.g., TGFβ inhibitors) may be examined by suitable pharmacodynamics (PD) measures, such as measuring downstream signal transduction events. In case of the TGFβ pathway, for example, suitable PD measure includes relative phosphorylation of SMAD2/3.

Muscle Conditions Associated with Fibrosis

Accumulating evidence indicates that TGFβ plays an important role in muscle homeostasis, repair, and regeneration. Agents, such as monoclonal antibodies described herein, that selectively modulate LTBP-associated TGFβ signaling may be effective for treating damaged muscle fibers, such as in chronic/genetic muscular dystrophies, congenital fibrosis of ocular/extraocular muscles, and acute muscle injuries, without the toxicities associated with more broadly-acting TGFβ inhibitors.

Accordingly, the present invention provides methods for treating damaged muscle fibers using an agent that preferentially modulates a subset, but not all, of TGFβ effects in vivo. Such agents can selectively modulate TGFβ1 signaling (“isoform-specific modulation”) in a particular context, i.e., when presented by LTBP1 or LTBP3.

In skeletal muscle, TGFβ plays a variety of roles including inhibition of proliferation and differentiation, induction of atrophy, and development of fibrosis. TGFβ reduces satellite cell proliferation and prevents differentiation (via inhibition of MyoD and myogenin) (Allen, R. E. and L. K. J Cell Physiol, 1987. 133(3): p. 567-72; Brennan, T. J., et al., Proc Natl Acad Sci USA, 1991. 88(9): p. 3822-6; Massague, J., et al., Proc Natl Acad Sci USA, 1986. 83(21): p. 8206-10; Olson, E. N., et al., J Cell Biol, 1986. 103(5): p. 1799-805). The isoform of TGFβ (i.e., TGFβ31, 2, or 3) is not specified in these early papers, but is presumed to be TGFβ1. TGFβ also contributes to muscle fibrosis; direct injection of recombinant TGFβ1 results in skeletal muscle fibrosis, and pan-TGFβ inhibition decreases fibrosis in acute and chronically injured muscle (Li, Y., et al., Am J Pathol, 2004. 164(3): p. 1007-19; Mendias, C. L., et al., Muscle Nerve, 2012. 45(1): p. 55-9; Nelson, C. A., et al., Am J Pathol, 2011. 178(6): p. 2611-21). TGFβ1 is expressed by myofibers, macrophages, regulatory T cells, fibroblasts, and fibrocytes within the skeletal muscle (Li, Y., et al., Am J Pathol, 2004. 164(3): p. 1007-19; Lemos, D. R., et al., Nat Med, 2015. 21(7): p. 786-94; Villalta, S. A., et al., Sci Transl Med, 2014. 6(258): p. 258ra142; Wang, X., et al., J Immunol, 2016. 197(12): p. 4750-4761); and expression is increased upon injury and in disease (Li, Y., et al., Am J Pathol, 2004. 164(3): p. 1007-19; Nelson, C. A., et al., Am J Pathol, 2011. 178(6): p. 2611-21; Bernasconi, P., et al., J Clin Invest, 1995. 96(2): p. 1137-44; Ishitobi, M., et al., Neuroreport, 2000. 11(18): p. 4033-5). TGFβ2 and TGFβ3 are also upregulated (at the mRNA level) in mdx muscle (a mouse model of Duchenne muscular dystrophy), although to a lesser extent than TGFβ1 (Nelson, C. A., et al., Am J Pathol, 2011. 178(6): p. 2611-21; Zhou, L., et al., Neuromuscul Disord, 2006. 16(1): p. 32-8). Pessina, et al., recently used lineage tracing experiments to show that cells of multiple origins within dystrophic muscle adopt a fibrogenic fate via a TGFβ-dependent pathway (Pessina, P., et al., Stem Cell Reports, 2015. 4(6): p. 1046-60).

TGFβ1 has been implicated in human muscular dystrophies. Duchenne muscular dystrophy (DMD) is a severe, progressive, and ultimately fatal disease caused by the absence of dystrophin (Bushby, K., et al., Lancet Neurol, 2010. 9(1): p. 77-93). Lack of dystrophin results in increased susceptibility to contraction-induced injury, leading to continual muscle degeneration (Petrof, B. J., et al., Proc Natl Acad Sci USA, 1993. 90(8): p. 3710-4; Dellorusso, C., et al., J Muscle Res Cell Motil, 2001. 22(5): p. 467-75; Pratt, S. J., et al., Cell Mol Life Sci, 2015. 72(1): p. 153-64). Repeated rounds of repair contribute to chronic inflammation, fibrosis, exhaustion of the satellite cell pool, eventual loss of mobility and death (Bushby, K., et al., Lancet Neurol, 2010. 9(1): p. 77-93; McDonald, C. M., et al., Muscle Nerve, 2013. 48(3): p. 343-56). Expression of TGFβ1 is significantly increased in patients with DMD and correlates with the extent of fibrosis observed in these patients (Bernasconi, P., et al., J Clin Invest, 1995. 96(2): p. 1137-44; Chen, Y. W., et al., Neurology, 2005. 65(6): p. 826-34). Excessive ECM deposition has detrimental effects on the contractile properties of the muscle and can limit access to nutrition as the myofibers are isolated from their blood supply (Klingler, W., et al., Acta Myol, 2012. 31(3): p. 184-95). Recently, additional data has further implicated TGFβ1 in muscular dystrophies. Variants in LTBP4 have been found to modify disease severity in mouse and human. In mouse, a variant of LTBP4 is protective in mice lacking dystrophin or γ-sarcoglycan (Coley, W. D., et al., Hum Mol Genet, 2016. 25(1): p. 130-45; Heydemann, A., et al., J Clin Invest, 2009. 119(12): p. 3703-12). In humans, two groups independently identified a variant of LTBP4 as protective in DMD, delaying loss of ambulation by several years (Flanigan, K. M., et al., Ann Neurol, 2013. 73(4): p. 481-8; van den Bergen, J. C., et al., J Neurol Neurosurg Psychiatry, 2015. 86(10): p. 1060-5).

Although the nature of the genetic variants in mouse and human differs, in both species the protective variant results in decreased TGFβ signaling (Heydemann, A., et al., J Clin Invest, 2009. 119(12): p. 3703-12); Ceco, E., et al., Sci Transl Med, 2014. 6(259): p. 259ra144). Many of the functions of TGFβ1 in skeletal muscle biology have been inferred from experiments in which purified active growth factor is injected into animals or added to cells in culture (Massague, J., et al., Proc Natl Acad Sci USA, 1986. 83(21): p. 8206-10; Li, Y., et al., Am J Pathol, 2004. 164(3): p. 1007-19; Mendias, C. L., et al., Muscle Nerve, 2012. 45(1): p. 55-9). Given the importance of cellular context for specific functions of TGFβ1 (see, for example, Hinck et al., Cold Spring Harb. Perspect. Biol, 2016. 8(12)) it is possible that some of the effects observed in these experiments do not reflect the endogenous role(s) of the cytokine in vivo. For example, treatment of human dermal fibroblasts with recombinant TGFβ1, myostatin, or GDF11 results in nearly identical changes in gene expression in these cells, although in vivo the roles of these proteins are quite different (Tanner, J. W., Khalil, A., Hill, J., Franti, M., MacDonnell, S. M., Growth Differentiation Factor 11 Potentiates Myofibroblast Activation, in Fibrosis: From Basic Mechanisms to Targeted therapies. 2016: Keystone, CO).

Multiple investigators have used inhibitors of TGFβ to clarify the role of the growth factor in vivo. Treatment of mdx mice with the pan-TGFβ neutralizing antibody 1D11 clearly results in reduced fibrosis (by histology and hydroxyproline content), reduced muscle damage (reduced serum creatine kinase and greater myofiber density), and improved muscle function (by plethysmography, force generation of isolated EDL muscles, and increased forelimb grip strength) (Nelson, C. A., et al., Am J Pathol, 2011. 178(6): p. 2611-21; Andreetta, F., et al., J Neuroimmunol, 2006. 175(1-2): p. 77-86; Gumucio, J. P., et al., J Appl Physiol (1985), 2013. 115(4): p. 539-45). In addition, myofiber-specific expression of a dominant negative TGFβ type II receptor protects against muscle damage after cardiotoxin injury and in 6-sarcoglycan−/− mice (Accornero, F., et al., Hum Mol Genet, 2014. 23(25): p. 6903-15). The proteoglycan decorin, which is abundant in skeletal muscle and inhibits TGFβ activity, decreases muscle fibrosis in mdx mice and following laceration injury (Li, Y., et al., Mol Ther, 2007. 15(9): p. 1616-22; Gosselin, L. E., et al., Muscle Nerve, 2004. 30(5): p. 645-53). Other molecules with TGFβ inhibitory activity, such as suramin (an anti-neoplastic agent) and losartan (an angiotensin receptor blocker) have been effective in improving muscle pathology and reducing fibrosis in mouse models of injury, Marfan's syndrome, and muscular dystro