Linker-drug compounds and antibody-drug conjugates that bind to human oncology targets are disclosed. The linker-drug compounds and antibody-drug conjugates comprise a splicing modulator drug moiety. The disclosure further relates to methods and compositions for use in the treatment of neoplastic disorders by administering the antibody-drug conjugates provided herein. In an embodiment, the splicing modulator comprises a pladienolide or a pladienolide derivative.

Description

80 parts

›This is a continuation of application Ser. No…

This is a continuation of application Ser. No. 17/247,117, filed Nov. 30, 2020, which is a continuation of PCT/US2019/035015, filed May 31, 2019, which claims the benefit of U.S. Provisional No. 62/679,631, filed Jun. 1, 2018; U.S. Provisional No. 62/679,672, filed Jun. 1, 2018; and U.S. Provisional No. 62/779,324, filed Dec. 13, 2018, all of which are incorporated herein by reference.

The present disclosure relates to antibody-drug conjugates (ADCs) comprising a splicing modulator and an antibody or antigen binding fragment thereof that binds a human oncology antigen target. The disclosure further relates to methods and compositions useful in the treatment or diagnosis of cancers that express a target antigen and/or are amenable to treatment by disruption of RNA splicing, as well as methods of making those compositions.

The majority of protein-coding genes in the human genome are composed of multiple exons (coding regions) that are separated by introns (non-coding regions). Gene expression results in a single precursor messenger RNA (pre-mRNA). The intron sequences are subsequently removed from the pre-mRNA by a process called splicing, which results in the mature messenger RNA (mRNA). By including different combinations of exons, alternative splicing gives rise to mRNAs encoding distinct protein isoforms.

RNA splicing is catalyzed by the spliceosome, a dynamic multiprotein-RNA complex composed of five small nuclear RNAs (snRNAs U1, U2, U4, U5, and U6) and associated proteins. The spliceosome assembles on pre-mRNAs to establish a dynamic cascade of multiple RNA and protein interactions that catalyze excision of the introns and ligation of exons (Matera and Wang (2014) Nat Rev Mol Cell Biol. 15(2):108-21). Accumulating evidence has linked human diseases to dysregulation in RNA splicing that impact many genes (Scotti and Swanson (2016) Nat Rev Genet. 17(1):19-32).

The spliceosome is an important target in cancer biology. Several studies have now documented significant alterations in the splicing profile of cancer cells, as well as in the splicing factors themselves (Agrawal et al. (2018) Curr Opin Genet Dev. 48:67-74). Alternative splicing can lead to differential exon inclusion/exclusion, intron retention, or usage of cryptic splice sites (Seiler et al. (2018) Cell Rep. 23(1):282-296). Altogether, these events account for functional changes that may contribute to tumorigenesis or resistance to therapy (Siegfried and Kami (2018) Curr Opin Genet Dev. 48:16-21).

Certain natural products can bind the SF3b spliceosome complex. These small molecules modulate splicing by promoting intron retention and/or exon skipping (Teng et al. (2017) Nat Commun. 8:15522). A significant portion of the resulting transcripts contain premature stop codons triggering nonsense mediated mRNA decay (NMD). Furthermore, because canonical splicing is impaired, canonical transcripts are considerably reduced, which can negatively impact cell function and viability. For this reason, splicing modulators have become a promising class of drugs for the treatment of cancer (Puthenveetil et al. (2016) Bioconjugate Chem. 27:1880-8).

The proto-oncogene human epidermal growth factor receptor 2 (HER2) encodes a transmembrane tyrosine kinase receptor that belongs to the human epidermal growth factor receptor (EGFR) family (King et al. (1985) Science 229:974-6). Overexpression of HER2 enables constitutive activation of growth factor signaling pathways, such as the PI3K-AKT-mTOR pathway, and thereby serves as an oncogenic driver in several types of cancers, including approximately 20% of invasive breast carcinomas (Slamon et al. (1989) Science 244:707-12; Gajra and Chandarlapaty (2011) Expert Rev Anticancer Ther. 11:263-75). Given that HER2 amplification mediates the transformed phenotype, and because HER2 expression is largely restricted to malignant cells, HER2 is a promising antigen for targeting certain cancers and/or delivering novel cancer treatments (Parakh et al. (2017) Cancer Treat Rev. 59:1-21). Additional antigens for targeted delivery of cancer therapies include, but are not limited to, CD138 (also referred to as syndecan-1) and ephrin type-A receptor 2 (EPHA2).

CD138 is a cell surface heparan sulfate proteoglycan that is essential for maintaining cell morphology and interaction with the surrounding microenvironment (Akl et al. (2015) Oncotarget 6(30):28693-715; Szatmdri et al. (2015) Dis Markers 2015:796052). In general, the loss of CD138 expression in carcinoma cells reduces cell adhesion to the extracellular matrix and enhances cell motility and invasion (Teng et al. (2012) Matrix Biol. 31:3-16). Increased stromal CD138 expression also alters fibronectin production and extracellular matrix organization (Yang et al. (2011) Am J Pathol. 178:325-35). Additionally, increased expression of CD138 in stromal fibroblasts is associated with angiogenesis and cancer progression (Maeda et al. (2006) Oncogene 25:1408-12). CD138 expression increases during B cell development and its presence is a hallmark of plasma cells (Ribatti (2017) Immunol Lett. 188:64-7). CD138 expression is maintained in multiple myeloma, a malignancy of plasma cells. CD138 is therefore an attractive antigen for the targeted treatment of several cancers and other hematological malignancies (Sherbenou et al. (2015) Blood Rev. 29(2):81-91; Wijdenes et al. (1996) Br J Haematol. 94(2):318-23).

EPHA2 is a transmembrane glycoprotein that is abundantly overexpressed in several malignant cancer-derived cell lines and in advanced forms of cancer (Wykosky and Debinski (2008) Mol Cancer Ref. 6(12):1795-1806). For instance, EPHA2 is strongly overexpressed in approximately 61% of GBM patient tumors (Wykosky et al. (2008) Clin Cancer Res. 14:199-208), 76% of ovarian cancers (Thaker et al. (2004) Clin Cancer Res. 10:5145-50), and 85% of prostate adenocarcinomas (Zeng et al. (2003) Am J Pathol. 163:2271-6). The EPHA2 protein is highly overexpressed with regard to percentage of patient tumors and percentage of cells within a tumor, and is a plasma membrane-localized receptor that can internalize on ligand binding (Walker-Daniels et al. (2002) Mol Cancer Res. 1:79-87). Moreover, expression of EPHA2 is associated with poor prognosis, increased metastasis, and decreased survival. Thus, due to its expression pattern, localization, and functional importance in the outcome of cancer patients, EPHA2 is another attractive antigen for the targeted delivery of novel anti-cancer therapies.

›In various embodiments, the present disclosure provides, in…

In various embodiments, the present disclosure provides, in part, novel compounds with biological activity against neoplastic cells. The compounds may slow, inhibit, and/or reverse tumor growth in mammals, and may be useful for treating human cancer patients. In various embodiments, the disclosure provides novel antibody-drug conjugates employing the novel compounds or other functional splice inhibitor molecules.

The present disclosure more specifically relates, in various embodiments, to antibody-drug conjugate (ADC) compounds that are capable of binding and killing neoplastic cells. In various embodiments, the ADC compounds disclosed herein comprise a linker that attaches a splicing modulator to a full-length antibody or an antigen binding fragment. In various embodiments, the ADC compounds are also capable of internalizing into a target cell after binding.

In various embodiments, ADC compounds may be represented by Formula (I):

Ab-(L-D) p (I)

wherein Ab is an antibody or an antigen binding fragment thereof which targets a neoplastic cell or another oncology-related target;

D is a splicing modulator;

L is a linker which covalently attaches Ab to D; and

p is an integer from 1 to 15.

In various embodiments, ADC compounds may be represented by Formula (I):

Ab-(L-D) p (I)

wherein Ab is an antibody or an antigen binding fragment thereof which targets a neoplastic cell;

D is a splicing modulator of Formula (II):

or a pharmaceutically acceptable salt thereof, wherein:

R 1 is chosen from absent, hydrogen, C 1 -C 6 alkyl groups, C 1 -C 6 alkylalkoxy groups, C 1 -C 6 alkylamino groups, C 1 -C 6 alkylcarboxylic acid groups, C 1 -C 6 alkylhydroxy groups, C 3 -C 8 cycloalkyl groups, benzyl groups, C 3 -C 8 heterocyclyl groups, —O—C(═O)—(C 1 -C 6 alkyl) groups, and —CD 3 ;

R 3 is chosen from hydrogen, C 1 -C 6 alkyl groups, C 1 -C 6 alkylalkoxy groups, C 1 -C 6 alkylamino groups, C 1 -C 6 alkylcarboxylic acid groups, C 1 -C 6 alkylhydroxy groups, C 3 -C 8 cycloalkyl groups, benzyl groups, C 3 -C 8 heterocyclyl groups, and —O—C(═O)—(C 1 -C 6 alkyl) groups; and

R 4 , R 5 , and R 8 are each independently chosen from hydrogen, hydroxyl groups, —O—(C 1 -C 6 alkyl) groups, —O—C(═O)—(C 1 -C 6 alkyl) groups, and C 1 -C 6 alkyl groups;

R 6 and R 7 are each independently chosen from hydrogen, —O—R 17 , —O—C(═O)—R 17 , —O—C(═O)—NR 15 R 16 , C 1 -C 6 alkyl groups, and —NR 15 R 16 ;

R 15 and R 16 are each independently chosen from hydrogen, R 17 , —C(═O)—R 17 , and —C(═O)—O—R 17 ;

R 17 is chosen from hydrogen, C 1 -C 6 alkyl groups, C 3 -C 8 cycloalkyl groups, benzyl groups, and C 3 -C 8 heterocyclyl groups; and

Z is chosen from

—O—(C 1 -C 6 alkyl) groups, —NR 15 R 16 , C 3 -C 8 cycloalkyl groups, C 1 -C 6 alkylhydroxy groups, C 1 -C 6 alkylalkoxy groups, benzyl groups, and C 3 -C 8 heterocyclyl groups,

wherein at least one of R 6 and R 7 is hydrogen;

and wherein L is a linker which covalently attaches Ab to D; and

p is an integer from 1 to 15.

In some embodiments, the antibody or antigen binding fragment is capable of internalization into a target cell. In some embodiments, the linker covalently attaches to the splicing modulator of Formula (II) (“L-D”), and L-D has a structure of Formula (II-A):

or a pharmaceutically acceptable salt thereof,

wherein Z′ is chosen from

and

wherein all other variables are as defined for Formula (II).

In various other embodiments, ADC compounds may be represented by of Formula (I):

Ab-(L-D) p (1)

wherein Ab is an antibody or an antigen binding fragment thereof which targets a neoplastic cell;

D is a splicing modulator of Formula (IV):

or a pharmaceutically acceptable salt thereof, wherein:

R 1 is chosen from hydrogen, C 1 -C 6 alkyl groups, C 1 -C 6 alkylalkoxy groups, C 1 -C 6 alkylamino groups, C 1 -C 6 alkylcarboxylic acid groups, C 1 -C 6 alkylhydroxy groups, C 3 -C 8 cycloalkyl groups, benzyl groups, C 3 -C 8 heterocyclyl groups, —O—C(═O)—(C 1 -C 6 alkyl) groups, and —CD 3 ;

R 4 , R 5 , and R 8 are each independently chosen from hydrogen, hydroxyl groups, —O—(C 1 -C 6 alkyl) groups, —O—C(═O)—(C 1 -C 6 alkyl) groups, and C 1 -C 6 alkyl groups;

R 6 and R 7 are each independently chosen from hydrogen, —O—R 17 , —O—C(═O)—R 17 , —O—C(═O)—NR 15 R 16 , C 1 -C 6 alkyl groups, and —NR 15 R 16 ;

R 15 and R 16 are each independently chosen from hydrogen, R 17 , —C(═O)—R 17 , and —C(═O)—O—R 17 ; and

R 17 is chosen from hydrogen, C 1 -C 6 alkyl groups, C 3 -C 8 cycloalkyl groups, benzyl groups, and C 3 -C 8 heterocyclyl groups;

—O—(C 1 -C 6 alkyl) groups, —NR 15 R 16 , C 3 -C 8 cycloalkyl groups, C 1 -C 6 alkylhydroxy groups, C 1 -C 6 alkylalkoxy groups, benzyl groups, and C 3 -C 8 heterocyclyl groups,

wherein at least one of R 6 and R 7 is hydrogen;

and wherein L is a linker which covalently attaches Ab to D; and

p is an integer from 1 to 15.

In some embodiments, the antibody or antigen binding fragment is capable of internalization into a target cell. In some embodiments, the linker covalently attaches to the splicing modulator (“L-D”), and L-D has a structure of Formula (IV-A):

or a pharmaceutically acceptable salt thereof.

In various other embodiments, ADC compounds may be represented by Formula (I):

Ab-(L-D) p (I)

wherein Ab is an antibody or an antigen binding fragment thereof which targets a neoplastic cell;

D is a splicing modulator of Formula (VI):

›or a pharmaceutically acceptable salt thereof, wherein: R…

or a pharmaceutically acceptable salt thereof, wherein:

R 1 and R 9 are each independently chosen from hydrogen, C 1 -C 6 alkyl groups, C 1 -C 6 alkylalkoxy groups, C 1 -C 6 alkylamino groups, C 1 -C 6 alkylcarboxylic acid groups, C 1 -C 6 alkylhydroxy groups, C 3 -C 8 cycloalkyl groups, benzyl groups, C 3 -C 8 heterocyclyl groups, —O—C(═O)—(C 1 -C 6 alkyl) groups, and —CD 3 ;

R 4 , R 5 , and R 8 are each independently chosen from hydrogen, hydroxyl groups, —O—(C 1 -C 6 alkyl) groups, —O—C(═O)—(C 1 -C 6 alkyl) groups, and C 1 -C 6 alkyl groups;

R 6 and R 7 are each independently chosen from hydrogen, —O—R 17 , —O—C(═O)—R 17 , —O—C(═O)—NR 15 R 16 , C 1 -C 6 alkyl groups, —NR 15 R 16 , and a linker;

R 10 is chosen from hydrogen, C 1 -C 6 alkyl groups, —C(═O)—(C 1 -C 6 alkyl) groups, and —CD 3 ;

R 15 and R 16 are each independently chosen from hydrogen, R 17 , —C(═O)—R 17 , and —C(═O)—O—R 17 ;

R 17 is chosen from hydrogen, C 1 -C 6 alkyl groups, C 3 -C 8 cycloalkyl groups, benzyl groups, and C 3 -C 8 heterocyclyl groups; and

a is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

wherein R 1 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 15 , R 16 , and R 17 are each independently substituted with 0 to 3 groups independently chosen from halogens, hydroxyl groups, C 1 -C 6 alkyl groups, —O—(C 1 -C 6 alkyl) groups, —NR 15 R 16 , C 3 -C 8 cycloalkyl groups, C 1 -C 6 alkylhydroxy groups, C 1 -C 6 alkylalkoxy groups, benzyl groups, and C 3 -C 8 heterocyclyl groups;

wherein at least one of R 6 and R 7 is hydrogen; and

wherein R 1 and R 9 cannot both be absent;

and wherein L is a linker which covalently attaches Ab to D; and

p is an integer from 1 to 15.

In some embodiments, the antibody or antigen binding fragment is capable of internalization into a target cell. In some embodiments, the linker covalently attaches to the splicing modulator (“L-D”), and L-D has a structure of Formula (VI-A):

or a pharmaceutically acceptable salt thereof.

In various other embodiments, ADC compounds may be represented by Formula (I):

Ab-(L-D) p (I)

wherein Ab is an antibody or an antigen binding fragment thereof which targets a neoplastic cell;

D is a splicing modulator of Formula (VIII):

or a pharmaceutically acceptable salt thereof, wherein:

R 4 is chosen from hydrogen, hydroxyl groups, —O—(C 1 -C 6 alkyl) groups, —O—C(═O)—(C 1 -C 6 alkyl) groups, and C 1 -C 6 alkyl groups; and

R 10 is chosen from 3 to 10 membered carbocycles and 3 to 10 membered heterocycles, each of which is substituted with 0 to 3 R a , wherein each R a is independently chosen from halogens, C 1 -C 6 alkyl groups, —O—(C 1 -C 6 )alkyl groups, C 1 -C 6 alkylalkoxy groups, C 1 -C 6 alkylhydroxy groups, —S(═O) w -(4 to 7 membered heterocycles), 4 to 7 membered carbocycles, and 4 to 7 membered heterocycles;

R 15 and R 16 are each independently chosen from hydrogen, R 17 , —C(═O)—R 17 , and —C(═O)—O—R 17 ; and

R 17 is chosen from hydrogen, C 1 -C 6 alkyl groups, C 3 -C 8 cycloalkyl groups, benzyl groups, and C 3 -C 8 heterocyclyl groups;

wherein R 1 , R 3 , R 4 , R 10 , R 15 , R 16 , and R 17 are each independently substituted with 0 to 3 groups independently chosen from halogens, hydroxyl groups, C 1 -C 6 alkyl groups, —O—(C 1 -C 6 alkyl) groups, —NR 15 R 16 , C 3 -C 8 cycloalkyl groups, C 1 -C 6 alkylhydroxy groups, C 1 -C 6 alkylalkoxy groups, benzyl groups, and C 3 -C 8 heterocyclyl groups; and

wherein each R a is independently substituted with 0 to 3 groups independently chosen from halogens, hydroxyl groups, —NR 15 R 16 , C 1 -C 6 alkyl groups, —(C═O)—(C 1 -C 6 alkyl) groups,

—(C═O)—(C 1 -C 6 alkyl)-(C 3 -C 10 heterocyclyl groups), —S(═O) w —(C 3 -C 8 heterocyclyl) groups, and C 1 -C 6 alkylcarboxylic acid groups, each of which is substituted with 0, 1, or 2 groups independently chosen from halogens, hydroxyl groups, —NR 15 R 16 , and C 1 -C 3 alkyl groups; and

w is 0, 1, or 2;

and wherein L is a linker which covalently attaches Ab to D; and

p is an integer from 1 to 15.

In some embodiments, the antibody or antigen binding fragment is capable of internalization into a target cell. In some embodiments, the linker covalently attaches to the splicing modulator (“L-D”), and L-D has a structure of Formula (VIII-A):

or a pharmaceutically acceptable salt thereof.

In some embodiments, the splicing modulator comprises a modulator of the SF3b complex.

In some embodiments, the splicing modulator comprises a pladienolide or a pladienolide derivative. In some embodiments, the splicing modulator comprises pladienolide D or a pladienolide D derivative. In some embodiments, the pladienolide D or derivative comprises D2, D1, D4, D8, D10, D11 (E7107), D20, D21, D22, D12, or D25. In some embodiments, the pladienolide D or derivative comprises D2. In some embodiments, the pladienolide D or derivative comprises D1. In some embodiments, the pladienolide D or derivative comprises D4. In some embodiments, the pladienolide D or derivative comprises D12.

In some embodiments, the pladienolide D or derivative is a zwitterionic pladienolide D or derivative. In some embodiments, the zwitterionic pladienolide D or derivative comprises D22 or D25.

In some other embodiments, the splicing modulator comprises pladienolide B or a pladienolide B derivative. In some embodiments, the pladienolide B or derivative comprises D9, D18, D19, or D13.

›In some embodiments, the splicing modulator comprises an…

In some embodiments, the splicing modulator comprises an aryl pladienolide. In some embodiments, the aryl pladienolide comprises D15, D14, D16, D17, D26, or D33. In some embodiments, the aryl pladienolide comprises D15. In some embodiments, the aryl pladienolide is a zwitterionic aryl pladienolide. In some embodiments, the zwitterionic aryl pladienolide comprises D33.

In some embodiments, the splicing modulator comprises D1:

In some embodiments, the splicing modulator comprises D2:

In some embodiments, the splicing modulator comprises D3:

In some embodiments, the splicing modulator comprises D4:

In some embodiments, the splicing modulator comprises D4′:

In some embodiments, the splicing modulator comprises D5:

In some embodiments, the splicing modulator comprises D6:

In some embodiments, the splicing modulator comprises D7:

In some embodiments, the splicing modulator comprises D8:

In some embodiments, the splicing modulator comprises D9:

In some embodiments, the splicing modulator comprises D10:

In some embodiments, the splicing modulator comprises D11:

In some embodiments, the splicing modulator comprises D12:

In some embodiments, the splicing modulator comprises D13:

In some embodiments, the splicing modulator comprises D14:

In some embodiments, the splicing modulator comprises D15:

In some embodiments, the splicing modulator comprises D16:

In some embodiments, the splicing modulator comprises D17:

In some embodiments, the splicing modulator comprises D18:

In some embodiments, the splicing modulator comprises D19:

In some embodiments, the splicing modulator comprises D20:

In some embodiments, the splicing modulator comprises D21:

In some embodiments, the splicing modulator comprises D22:

In some embodiments, the splicing modulator comprises D23:

In some embodiments, the splicing modulator comprises D24:

In some embodiments, the splicing modulator comprises D25:

In some embodiments, the splicing modulator comprises D26:

In some embodiments, the splicing modulator comprises D27:

In some embodiments, the splicing modulator comprises D28:

In some embodiments, the splicing modulator comprises D29:

In some embodiments, the splicing modulator comprises D30:

In some embodiments, the splicing modulator comprises D31:

In some embodiments, the splicing modulator comprises D32:

In some embodiments, the splicing modulator comprises D33:

In some embodiments, the splicing modulator comprises D34:

In some embodiments, the splicing modulator comprises D35:

In some embodiments, the splicing modulator comprises one of the drug moieties listed in Table 7. In some embodiments, the splicing modulator comprises D1, D2, D3, D4, D4′, D5, D6, D7, D8, D9, D10, D11, D12, D13, D14, D15, D16, D17, D18, D19, D20, D21, D22, D23, D24, D25, D26, D27, D28, D29, D30, D31, D32, D33, D34, and/or D35.

In some embodiments, a splicing modulator is disclosed, as well as its use as a therapeutic alone or as part of an ADC. In some embodiments, the splicing modulator comprises D4, D4′, D12, D15, D8, D9, D10, D13, D18, D19, D20, D21, D22, D25, or D33.

In some embodiments, the splicing modulator comprises D4 and the linker comprises MC-Val-Cit-pABC. In some embodiments, the splicing modulator comprises D4 and the linker comprises MC-β-glucuronide. In some embodiments, the splicing modulator comprises D12 and the linker comprises MC-Val-Cit-pABC. In some embodiments, the splicing modulator comprises D12 and the linker comprises MC-s-glucuronide. In some embodiments, the splicing modulator comprises D15 and the linker comprises MC-Val-Ala-pAB.

In various embodiments, the linker used in an ADC disclosed herein is stable outside a cell, such that the ADC remains intact when present in extracellular conditions, but is capable of being cleaved upon internalization into a cell, e.g., a tumor or cancer cell. In some embodiments, the splicing modulator is cleaved from the antibody or antigen binding fragment when the ADC enters a cell that expresses an antigen targeted by the antibody or antigen binding fragment of the ADC. In some embodiments, the linker is a cleavable linker.

In some embodiments, the linker comprises a cleavable peptide moiety. In some embodiments, the cleavable peptide moiety is cleavable by an enzyme. In some embodiments, the cleavable peptide moiety or linker comprises an amino acid unit. In some embodiments, the amino acid unit comprises valine-citrulline (“Val-Cit” or “VC”).

In some other embodiments, the amino acid unit comprises valine-alanine (“Val-Ala” or “VA”). In some other embodiments, the amino acid unit comprises glutamic acid-valine-citrulline (“Glu-Val-Cit” or “EVC”). In some other embodiments, the amino acid unit comprises alanine-alanine-asparagine (“Ala-Ala-Asn” or “AAN”).

In some embodiments, the linker comprises a cleavable glucuronide moiety. In some embodiments, the cleavable glucuronide moiety is cleavable by an enzyme. In some embodiments, the cleavable glucuronide moiety is cleavable by a glucuronidase. In some embodiments, the cleavable glucuronide moiety is cleavable by β-glucuronidase.

In some embodiments, the linker comprises at least one spacer unit. In some embodiments, the spacer unit or linker comprises a polyethylene glycol (PEG) moiety. In some embodiments, the PEG moiety comprises -(PEG) m - and m is an integer from 1 to 10. In some embodiments, m is 2. In some other embodiments, the spacer unit or linker comprises an alkyl moiety. In some embodiments, the alkyl moiety comprises —(CH 2 ) n — and n is an integer from 1 to 10. In some embodiments, n is 2. In some embodiments, n is 5. In some embodiments, n is 6.

In some embodiments, the spacer unit attaches to the antibody or antigen binding fragment via a maleimide (Mal) moiety (“Mal-spacer unit”). In some embodiments, the Mal-spacer unit is reactive with a cysteine residue on the antibody or antigen binding fragment. In some embodiments, the Mal-spacer unit is joined to the antibody or antigen binding fragment via a cysteine residue on the antibody or antigen binding fragment.

In some embodiments, the linker comprises the Mal-spacer unit and a cleavable peptide moiety. In some embodiments, the cleavable peptide moiety comprises an amino acid unit. In some embodiments, the cleavable peptide moiety or amino acid unit comprises Val-Cit. In some embodiments, the cleavable peptide moiety or amino acid unit comprises Val-Ala. In some embodiments, the cleavable peptide moiety or amino acid unit comprises Glu-Val-Cit. In some embodiments, the cleavable peptide moiety or amino acid unit comprises Ala-Ala-Asn. In some embodiments, the Mal-spacer unit comprises an alkyl moiety. In some embodiments, the Mal-spacer unit comprises a PEG moiety. In some embodiments, the Mal-spacer unit comprises a maleimidocaproyl (MC).

›In some embodiments, the Mal-spacer unit attaches the…

In some embodiments, the Mal-spacer unit attaches the antibody or antigen binding fragment to the cleavable moiety in the linker. In some embodiments, the cleavable moiety in the linker comprises a cleavable peptide moiety. In some embodiments, the cleavable peptide moiety comprises an amino acid unit. In some embodiments, the cleavable peptide moiety or amino acid unit comprises Val-Cit, Val-Ala, Glu-Val-Cit, or Ala-Ala-Asn. In some embodiments, the linker comprises MC-Val-Cit. In some embodiments, the linker comprises MC-Val-Ala. In some embodiments, the linker comprises MC-Glu-Val-Cit. In some embodiments, the linker comprises MC-Ala-Ala-Asn. In some embodiments, the Mal-spacer unit comprises an alkyl moiety. In some embodiments, the Mal-spacer unit comprises a PEG moiety. In some embodiments, the Mal-spacer unit comprises a maleimidocaproyl (MC).

In some embodiments, the cleavable moiety in the linker is directly joined to the splicing modulator, or a spacer unit attaches the cleavable moiety in the linker to the splicing modulator. In some embodiments, cleavage of the conjugate releases the splicing modulator from the antibody or antigen binding fragment and linker. In some embodiments, the spacer unit attaching the cleavable moiety in the linker to the splicing modulator is self-immolative.

In some embodiments, the spacer unit attaching the cleavable moiety in the linker to the splicing modulator comprises a p-aminobenzyloxycarbonyl (pABC). In some embodiments, the pABC attaches the cleavable moiety in the linker to the splicing modulator. In some embodiments, the cleavable moiety in the linker comprises a cleavable peptide moiety. In some embodiments, the cleavable peptide moiety comprises an amino acid unit. In some embodiments, the cleavable peptide moiety or amino acid unit comprises Val-Cit, Val-Ala, Glu-Val-Cit, or Ala-Ala-Asn. In some embodiments, the linker comprises Val-Cit-pABC. In some other embodiments, the linker comprises Val-Ala-pABC. In some embodiments, the linker comprises Glu-Val-Cit-pABC. In some embodiments, the linker comprises Ala-Ala-Asn-pABC.

In some embodiments, the spacer unit attaching the cleavable moiety in the linker to the splicing modulator comprises a p-aminobenzyl (pAB). In some embodiments, the pAB attaches the cleavable moiety in the linker to the splicing modulator. In some embodiments, the cleavable moiety in the linker comprises a cleavable peptide moiety. In some embodiments, the cleavable peptide moiety comprises an amino acid unit. In some embodiments, the cleavable peptide moiety or amino acid unit comprises Val-Cit, Val-Ala, Glu-Val-Cit, or Ala-Ala-Asn. In some embodiments, the linker comprises Val-Cit-pAB. In some other embodiments, the linker comprises Val-Ala-pAB. In some other embodiments, the linker comprises Glu-Val-Cit-pAB. In some other embodiments, the linker comprises Ala-Ala-Asn-pAB.

In various embodiments, the linker is a non-cleavable linker. In some embodiments, the splicing modulator of the ADC is released by degradation of the antibody or antigen binding fragment. In some embodiments, the linker remains covalently associated with at least one amino acid of the antibody and drug upon internalization by and degradation within the target cell.

In some embodiments, the linker is a non-cleavable linker comprising at least one spacer unit. In some embodiments, the spacer unit or linker comprises a polyethylene glycol (PEG) moiety. In some embodiments, the PEG moiety comprises -(PEG), and m is an integer from 1 to 10. In some embodiments, m is 2. In some other embodiments, the spacer unit or linker comprises an alkyl moiety. In some embodiments, the alkyl moiety comprises —(CH 2 ) n — or —(CH 2 ) n —O—(CH 2 ) n and n is an integer from 1 to 10. In some embodiments, n is 2. In some embodiments, n is 5. In some embodiments, n is 6.

In some embodiments, the spacer unit in a non-cleavable linker attaches to the antibody or antigen binding fragment via a maleimide (Mal) moiety (“Mal-spacer unit”). In some embodiments, the Mal-spacer unit is reactive with a cysteine residue on the antibody or antigen binding fragment. In some embodiments, the Mal-spacer unit is joined to the antibody or antigen binding fragment via a cysteine residue on the antibody or antigen binding fragment. In some embodiments, the Mal-spacer unit comprises an alkyl moiety. In some embodiments, the Mal-spacer unit comprises a PEG moiety. In some embodiments, the linker or Mal-spacer unit comprises a maleimidocaproyl (MC). In some embodiments, the linker or Mal-spacer unit comprises a maleimidocaproyl (MC) and at least one additional spacer unit. In some embodiments, the linker or Mal-spacer unit comprises MC-(PEG) 2 . In some embodiments, the linker or Mal-spacer unit comprises MC-(PEG) 2 and at least one additional spacer unit. In some embodiments, the linker or Mal-spacer unit comprises Mal-Hex. In some embodiments, the linker or Mal-spacer unit comprises Mal-Hex and at least one additional spacer unit. In some embodiments, the linker or Mal-spacer unit comprises Mal-Et. In some embodiments, the linker or Mal-spacer unit comprises Mal-Et and at least one additional spacer unit. In some embodiments, the linker or Mal-spacer unit comprises Mal-Et-O-Et. In some embodiments, the linker or Mal-spacer unit comprises Mal-Et-O-Et and at least one additional spacer unit. In some embodiments, the Mal-spacer unit attaches the antibody or antigen binding fragment to the splicing modulator.

In various embodiments, ADC compounds may be represented by Formula (I):

Ab-(L-D) p (I)

wherein Ab is an antibody or an antigen binding fragment thereof which targets a neoplastic cell or another oncology-related target such as a cancer antigen (e.g., any of the antibody or binding domain sequences disclosed herein); D is any small molecule suitable for treating a cancer (e.g., a splicing modulator, e.g., any of the splicing modulators disclosed herein); L is a linker which covalently attaches Ab to D (e.g., any of the linkers disclosed herein); and p is an integer from 1 to 15.

›In some embodiments, Ab is selected from any…

In some embodiments, Ab is selected from any of the antibody or binding domain sequences disclosed herein. In some embodiments, Ab is an antibody or binding domain sequence which targets HER2 and/or a HER2-expressing neoplastic cell. In some embodiments, Ab is an antibody or binding domain sequence which targets CD138 and/or a CD138-expressing neoplastic cell. In some embodiments, Ab is an antibody or binding domain sequence which targets EPHA2 and/or an EPHA2-expressing neoplastic cell. In some embodiments, Ab is an antibody or binding domain sequence which targets MSLN and/or a MSLN-expressing neoplastic cell. In some embodiments, Ab is an antibody or binding domain sequence which targets FOLH1 and/or a FOLH1-expressing neoplastic cell. In some embodiments, Ab is an antibody or binding domain sequence which targets CDH6 and/or a CDH6-expressing neoplastic cell. In some embodiments, Ab is an antibody or binding domain sequence which targets CEACAM5 and/or a CEACAM5-expressing neoplastic cell. In some embodiments, Ab is an antibody or binding domain sequence which targets CFC1B and/or a CFC1B-expressing neoplastic cell. In some embodiments, Ab is an antibody or binding domain sequence which targets ENPP3 and/or an ENPP3-expressing neoplastic cell. In some embodiments, Ab is an antibody or binding domain sequence which targets FOLR1 and/or a FOLR1-expressing neoplastic cell. In some embodiments, Ab is an antibody or binding domain sequence which targets HAVCR1 and/or a HAVCR1-expressing neoplastic cell. In some embodiments, Ab is an antibody or binding domain sequence which targets KIT and/or a KIT-expressing neoplastic cell. In some embodiments, Ab is an antibody or binding domain sequence which targets MET and/or a MET-expressing neoplastic cell. In some embodiments, Ab is an antibody or binding domain sequence which targets MUC16 and/or a MUC16-expressing neoplastic cell. In some embodiments, Ab is an antibody or binding domain sequence which targets SLC39A6 and/or a SLC39A6-expressing neoplastic cell. In some embodiments, Ab is an antibody or binding domain sequence which targets SLC44A4 and/or a SLC44A4-expressing neoplastic cell. In some embodiments, Ab is an antibody or binding domain sequence which targets STEAP1 and/or a STEAP1-expressing neoplastic cell. In some embodiments, Ab is an antibody or binding domain sequence which targets another cancer antigen.

In some embodiments, D is a splicing modulator. In some embodiments, D is selected from any of the splicing modulators disclosed herein. In some embodiments, D is a splicing modulator selected from D2, D1, D4, D8, D10, D11 (E7107), D20, D21, D22, D12, D25, D9, D18, D19, D13, D15, D14, D16, D17, D26, and D33, or any derivative thereof. In some embodiments, D is a splicing modulator selected from D4, D12, D15, D8, D9, D10, D13, D18, D19, D20, D21, D22, D25, and D33, or any derivative thereof. In some embodiments, D is a splicing modulator comprising D2 or any derivative thereof. In some embodiments, D is a splicing modulator comprising D1 or any derivative thereof.

In some embodiments, L is selected from any of the linkers disclosed herein, or any combination of linker components disclosed herein. In some embodiments, L is a linker comprising MC-Val-Cit-pABC, Mal-(PEG) 2 -CO, MC-Val-Ala-pAB, MC-Val-Ala-pABC, MC-Val-Cit-pAB, Mal-Hex, Mal-Et, or Mal-Et-O-Et. In some embodiments, the linker may also comprise one or more additional spacer units. In some embodiments, L is an ADL1, ADL2, ADL5, ADL6, ADL7, ADL10, ADL12, ADL13, ADL14, ADL15, ADL21, ADL22, or ADL23 linker. In some embodiments, L is an ADL1, ADL2, ADL5, ADL6, ADL7, ADL12, ADL13, ADL14, ADL21, ADL23, or ADL15 linker. In some embodiments, L is an ADL12, ADL14, or ADL15 linker. In some embodiments, the ADL1, ADL2, ADL5, ADL6, ADL7, ADL12, ADL13, ADL14, ADL21, ADL23, or ADL15 linker may also comprise one or more additional spacer units. In some embodiments, L is an ADL1 linker and may optionally comprise one or more additional spacer units. In some embodiments, L is an ADL2 linker and may optionally comprise one or more additional spacer units. In some embodiments, L is an ADL5 linker and may optionally comprise one or more additional spacer units. In some embodiments, L is an ADL6 linker and may optionally comprise one or more additional spacer units. In some embodiments, L is an ADL7 linker and may optionally comprise one or more additional spacer units. In some embodiments, L is an ADL12 linker and may optionally comprise one or more additional spacer units. In some embodiments, L is an ADL14 linker and may optionally comprise one or more additional spacer units. In some embodiments, L is an ADL15 linker and may optionally comprise one or more additional spacer units. In various embodiments of the ADCs described herein, p is from 1 to 10. In various embodiments, p is from 2 to 8. In various embodiments, p is from 4 to 8. In some embodiments, p is 4. In some embodiments, p is 8.

In some embodiments, L-D of Formula (I) is ADL1-D1. In some embodiments, L-D of Formula (I) is ADL6-D1. In some embodiments, L-D of Formula (I) is ADL5-D2. In some embodiments, L-D of Formula (I) is ADL1-D18. In some embodiments, L-D of Formula (I) is ADL5-D19. In some embodiments, L-D of Formula (I) is ADL14-D1. In some embodiments, L-D of Formula (I) is ADL12-D1. In some embodiments, L-D of Formula (I) is ADL15-D1. In some embodiments, L-D of Formula (I) is ADL12-D20. In some embodiments, L-D of Formula (I) is ADL10-D1. In some embodiments, L-D of Formula (I) is ADL12-D2. In some embodiments, L-D of Formula (I) is ADL15-D2. In some embodiments, L-D of Formula (I) is ADL12-D21. In some embodiments, L-D of Formula (I) is ADL6-D9. In some embodiments, L-D of Formula (I) is ADL1-D4. In some embodiments, L-D of Formula (I) is ADL1-D3. In some embodiments, L-D of Formula (I) is ADL1-D12. In some embodiments, L-D of Formula (I) is ADL1-D7. In some embodiments, L-D of Formula (I) is ADL1-D6. In some embodiments, L-D of Formula (I) is ADL1-D5. In some embodiments, L-D of Formula (I) is ADL22-D4. In some embodiments, L-D of Formula (I) is ADL5-D10. In some embodiments, L-D of Formula (I) is ADL5-D11. In some embodiments, L-D of Formula (I) is ADL1-D13. In some embodiments, L-D of Formula (I) is ADL1-D8. In some embodiments, L-D of Formula (I) is ADL1-D22. In some embodiments, L-D of Formula (I) is ADL5-D25. In some embodiments, L-D of Formula (I) is ADL12-D22. In some embodiments, L-D of Formula (I) is ADL5-D15. In some embodiments, L-D of Formula (I) is ADL1-D14. In some embodiments, L-D of Formula (I) is ADL5-D26. In some embodiments, L-D of Formula (I) is ADL1-D16. In some embodiments, L-D of Formula (I) is ADL5-D17. In some embodiments, L-D of Formula (I) is ADL1-D33. In some embodiments, L-D of Formula (I) is ADL1-D28. In some embodiments, L-D of Formula (I) is ADL1-D31. In some embodiments, L-D of Formula (I) is ADL1-D29. In some embodiments, L-D of Formula (I) is ADL1-D35. In some embodiments, L-D of Formula (I) is ADL5-D32. In some embodiments, L-D of Formula (I) is ADL5-D27. In some embodiments, L-D of Formula (I) is ADL12-D35. In some embodiments, L-D of Formula (I) is ADL12-D28. In some embodiments, L-D of Formula (I) is ADL1-D23. In some embodiments, L-D of Formula (I) is ADL1-D24.

›In some embodiments, a pool of ADCs is…

In some embodiments, a pool of ADCs is provided whereby random conjugation occurs, and the average p in the pool is between about 2 and about 8. In some embodiments, a pool of ADCs is provided whereby random conjugation occurs, and the average p in the pool is between about 4 and about 8. In some embodiments, a pool of ADCs is provided whereby random conjugation occurs, and the average p in the pool is about 4. In some embodiments, a pool of ADCs is provided whereby random conjugation occurs, and the average p in the pool is about 8. Compositions (e.g., pharmaceutical compositions) comprising multiple copies of any of the described ADCs, wherein the average drug loading (average p) of the ADCs in the composition is from about 3.5 to about 5.5 (e.g., about 4), or from about 7 to about 9 (e.g., about 8) are provided herein.

In some embodiments, the antibody or antigen binding fragment (Ab) of the ADC targets a neoplastic cell derived from a hematological malignancy or a solid tumor. In some embodiments, the antibody or antigen binding fragment targets a neoplastic cell derived from a hematological malignancy. In some embodiments, the hematological malignancy is selected from a B-cell malignancy, a leukemia (e.g., acute myeloid leukemia), a lymphoma, and a myeloma (e.g., multiple myeloma). In some embodiments, the hematological malignancy is selected from acute myeloid leukemia and multiple myeloma. In some embodiments, the antibody or antigen binding fragment targets a neoplastic cell derived from a solid tumor. In some embodiments, the solid tumor is selected from breast cancer (e.g., HER2-positive breast cancer), gastric cancer (e.g., gastric adenocarcinoma), prostate cancer, ovarian cancer, lung cancer (e.g., lung adenocarcinoma), uterine cancer (e.g., uterine serous endometrial carcinoma), salivary duct carcinoma, melanoma, colon cancer, cervical cancer, pancreatic cancer, kidney cancer, colorectal cancer, and esophageal cancer. In some embodiments, the solid tumor is selected from HER2-positive breast cancer, gastric adenocarcinoma, prostate cancer, and osteosarcoma.

In various embodiments, the antibody or antigen binding fragment (Ab) of the ADC is an anti-HER2 antibody or an antigen binding fragment thereof. In some embodiments, the antibody or antigen binding fragment binds to HER2 and targets HER2-expressing neoplastic cells (i.e., the ADC targets HER2-expressing neoplastic cells). In some embodiments, the antibody or antigen binding fragment of the ADC is an internalizing anti-HER2 antibody or internalizing antigen binding fragment thereof.

In some embodiments, the anti-HER2 antibody or antigen binding fragment comprises three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) comprising amino acid sequences of SEQ ID NO:1 (HCDR1), SEQ ID NO:2 (HCDR2), and SEQ ID NO:3 (HCDR3); and three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) comprising amino acid sequences of SEQ ID NO:4 (LCDR1), SEQ ID NO:5 (LCDR2), and SEQ ID NO:6 (LCDR3). In some embodiments, the anti-HER2 antibody or antigen binding fragment is an internalizing antibody or internalizing antigen binding fragment. In some embodiments, the anti-HER2 antibody or antigen binding fragment comprises human framework sequences. In some embodiments, the anti-HER2 antibody or antigen binding fragment comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO:19, and a light chain variable region comprising an amino acid sequence of SEQ ID NO:20. In some embodiments, the anti-HER2 antibody or antigen binding fragment comprises a human IgG heavy chain constant region. In some embodiments, the anti-HER2 antibody or antigen binding fragment comprises a human IgG1 heavy chain constant region. In some embodiments, the anti-HER2 antibody or antigen binding fragment comprises a human Ig kappa or lambda light chain constant region. In some embodiments, the anti-HER2 antibody or antigen binding competes for binding and/or binds the same epitope as an antibody comprising a heavy chain variable domain of SEQ ID NO:19 and a light chain variable domain of SEQ ID NO:20.

In various embodiments, the antibody or antigen binding fragment (Ab) of the ADC is an anti-CD138 antibody or an antigen binding fragment thereof. In some embodiments, the antibody or antigen binding fragment binds to CD138 and targets CD138-expressing neoplastic cells (i.e., the ADC targets CD138-expressing neoplastic cells). In some embodiments, the antibody or antigen binding fragment of the ADC is an internalizing anti-CD138 antibody or internalizing antigen binding fragment thereof.

In some embodiments, the anti-CD138 antibody or antigen binding fragment comprises three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) comprising amino acid sequences of SEQ ID NO:7 (HCDR1), SEQ ID NO:8 (HCDR2), and SEQ ID NO:9 (HCDR3); and three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) comprising amino acid sequences of SEQ ID NO:10 (LCDR1), SEQ ID NO:11 (LCDR2), and SEQ ID NO:12 (LCDR3). In some embodiments, the anti-CD138 antibody or antigen binding fragment is an internalizing antibody or internalizing antigen binding fragment. In some embodiments, the anti-CD138 antibody or antigen binding fragment comprises human framework sequences. In some embodiments, the anti-CD138 antibody or antigen binding fragment comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO:21, and a light chain variable region comprising an amino acid sequence of SEQ ID NO:22. In some embodiments, the anti-CD138 antibody or antigen binding fragment comprises a murine IgG2a heavy chain constant region. In some embodiments, the anti-CD138 antibody or antigen binding fragment comprises a murine Ig kappa light chain constant region. In some embodiments, the anti-CD138 antibody or antigen binding fragment comprises a human IgG heavy chain constant region. In some embodiments, the anti-CD138 antibody or antigen binding fragment comprises a human IgG2a heavy chain constant region. In some embodiments, the anti-CD138 antibody or antigen binding fragment comprises a human Ig kappa or lambda light chain constant region. In some embodiments, the anti-CD138 antibody or antigen binding competes for binding and/or binds the same epitope as an antibody comprising a heavy chain variable domain of SEQ ID NO:21 and a light chain variable domain of SEQ ID NO:22.

›In various embodiments, the antibody or antigen binding…

In various embodiments, the antibody or antigen binding fragment (Ab) of the ADC is an anti-EPHA2 antibody or an antigen binding fragment thereof. In some embodiments, the antibody or antigen binding fragment binds to EPHA2 and targets EPHA2-expressing neoplastic cells (i.e., the ADC targets EPHA2-expressing neoplastic cells). In some embodiments, the antibody or antigen binding fragment of the ADC is an internalizing anti-EPHA2 antibody or internalizing antigen binding fragment thereof.

In some embodiments, the anti-EPHA2 antibody or antigen binding fragment comprises three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) comprising amino acid sequences of SEQ ID NO:13 (HCDR1), SEQ ID NO:14 (HCDR2), and SEQ ID NO:15 (HCDR3); and three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) comprising amino acid sequences of SEQ ID NO:16 (LCDR1), SEQ ID NO:17 (LCDR2), and SEQ ID NO:18 (LCDR3). In some embodiments, the anti-EPHA2 antibody or antigen binding fragment is an internalizing antibody or internalizing antigen binding fragment. In some embodiments, the anti-EPHA2 antibody or antigen binding fragment comprises human framework sequences. In some embodiments, the anti-EPHA2 antibody or antigen binding fragment comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO:23, and a light chain variable region comprising an amino acid sequence of SEQ ID NO:24. In some embodiments, the anti-EPHA2 antibody or antigen binding fragment comprises a human IgG heavy chain constant region. In some embodiments, the anti-EPHA2 antibody or antigen binding fragment comprises a human IgG1 heavy chain constant region. In some embodiments, the anti-EPHA2 antibody or antigen binding fragment comprises a human Ig kappa or lambda light chain constant region. In some embodiments, the anti-EPHA2 antibody or antigen binding competes for binding and/or binds the same epitope as an antibody comprising a heavy chain variable domain of SEQ ID NO:23 and a light chain variable domain of SEQ ID NO:24.

Also provided herein, in various embodiments, are compounds comprising a linker-drug defined by the generic formula: L-D, wherein L=a linker moiety, and D=a drug moiety (e.g., a splicing modulator drug moiety). In various embodiments, the linker-drug (L-D) compounds disclosed herein may attach to an antibody or antigen binding fragment and/or are suitable for use in the ADCs disclosed herein, e.g., in ADCs of Formula (I).

In various embodiments, the linker-drug (L-D) compounds disclosed herein comprise a linker-drug structure according to Formula (III). In various embodiments, the present disclosure provides a linker-drug (L-D) compound of Formula (III):

or a pharmaceutically acceptable salt thereof, wherein:

R 2 is absent or a linker;

R 4 , R 5 , and R 8 are each independently chosen from hydrogen, hydroxyl groups, —O—(C 1 -C 6 alkyl) groups, —O—C(═O)—(C 1 -C 6 alkyl) groups, and C 1 -C 6 alkyl groups;

R 6 and R 7 are each independently chosen from hydrogen, —O—R 17 , —O—C(═O)—R 17 , —O—C(═O)—NR 15 R 16 , C 1 -C 6 alkyl groups, —NR 15 R 16 , and a linker;

R 15 and R 16 are each independently chosen from hydrogen, R 17 , —C(═O)—R 17 , and —C(═O)—O—R 17 ;

R 17 is chosen from hydrogen, C 1 -C 6 alkyl groups, C 3 -C 8 cycloalkyl groups, benzyl groups, and C 3 -C 8 heterocycyl groups; and

Z″ is chosen from

wherein R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 15 , R 16 , and R 17 are each independently substituted with 0 to 3 groups independently chosen from halogens, hydroxyl groups, C 1 -C 6 alkyl groups, —O—(C 1 -C 6 alkyl) groups, —NR 15 R 16 , C 3 -C 8 cycloalkyl groups, C 1 -C 6 alkylhydroxy groups, C 1 -C 6 alkylalkoxy groups, benzyl groups, and C 3 -C 8 heterocyclyl groups;

wherein at least one of R 6 and R 7 is hydrogen; and

wherein if R 2 is a linker, then neither R 6 or R 7 is a linker, and if R 6 or R 7 is a linker, then R 2 is absent.

In various other embodiments, the linker-drug (L-D) compounds disclosed herein comprise a linker-drug structure according to Formula (V). In various embodiments, the present disclosure provides a linker-drug (L-D) compound of Formula (V):

or a pharmaceutically acceptable salt thereof, wherein:

R 2 is absent or a linker;

R 4 , R 5 , and R 8 are each independently chosen from hydrogen, hydroxyl groups, —O—(C 1 -C 6 alkyl) groups, —O—C(═O)—(C 1 -C 6 alkyl) groups, and C 1 -C 6 alkyl groups;

R 6 and R 7 are each independently chosen from hydrogen, —O—R 17 , —O—C(═O)—R 17 , —O—C(═O)—NR 15 R 16 , C 1 -C 6 alkyl groups, —NR 15 R 16 , and a linker;

R 15 and R 16 are each independently chosen from hydrogen, R 17 , —C(═O)—R 17 , and —C(═O)—O—R 17 ; and

R 17 is chosen from hydrogen, C 1 -C 6 alkyl groups, C 3 -C 8 cycloalkyl groups, benzyl groups, and C 3 -C 8 heterocyclyl groups;

›wherein R 1 , R 2 , R…

wherein at least one of R 6 and R 7 is hydrogen; and

wherein if R 2 is a linker, then neither R 6 or R 7 is a linker, and if R 6 or R 7 is a linker, then R 2 is absent.

In various other embodiments, the linker-drug (L-D) compounds disclosed herein comprise a linker-drug structure according to Formula (VII). In various embodiments, the present disclosure provides a linker-drug (L-D) compound of Formula (VII):

or a pharmaceutically acceptable salt thereof, wherein:

R 1 and R 9 are each independently chosen from absent, hydrogen, C 1 -C 6 alkyl groups, C 1 -C 6 alkylalkoxy groups, C 1 -C 6 alkylamino groups, C 1 -C 6 alkylcarboxylic acid groups, C 1 -C 6 alkylhydroxy groups, C 3 -C 8 cycloalkyl groups, benzyl groups, C 3 -C 8 heterocyclyl groups, —O—C(═O)—(C 1 -C 6 alkyl) groups, and —CD 3 ;

R 2 is absent or a linker;

R 3 is chosen from hydrogen, C 1 -C 6 alkyl groups, C 1 -C 6 alkylalkoxy groups, C 1 -C 6 alkylamino groups, C 1 -C 6 alkylcarboxylic acid groups, C 1 -C 6 alkylhydroxy groups, C 3 -C 8 cycloalkyl groups, benzyl groups, C 1 -C 6 heterocyclyl groups, and —O—C(═O)—(C 1 -C 6 alkyl) groups;

R 4 , R 5 , and R 8 are each independently chosen from hydrogen, hydroxyl groups, —O—(C 1 -C 6 alkyl) groups, —O—C(═O)—(C 1 -C 6 alkyl) groups, and C 1 -C 6 alkyl groups;

R 6 and R 7 are each independently chosen from hydrogen, —O—R 17 , —O—C(═O)—R 17 , —O—C(═O)—NR 15 R 16 , C 1 -C 6 alkyl groups, —NR 15 R 16 , and a linker;

R 10 is chosen from hydrogen, C 1 -C 6 alkyl groups, —C(═O)—(C 1 -C 6 alkyl) groups, and —CD 3 ;

R 15 and R 16 are each independently chosen from hydrogen, R 17 , —C(═O)—R 17 , and —C(═O)—O—R 17 ;

R 17 is chosen from hydrogen, C 1 -C 6 alkyl groups, C 3 -C 8 cycloalkyl groups, benzyl groups, and C 3 -C 8 heterocyclyl groups; and

a is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

wherein R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 15 , R 16 , and R 17 are each independently substituted with 0 to 3 groups independently chosen from halogens, hydroxyl groups, C 1 -C 6 alkyl groups, —O—(C 1 -C 6 alkyl) groups, —NR 15 R 16 , C 3 -C 8 cycloalkyl groups, C 1 -C 6 alkylhydroxy groups, C 1 -C 6 alkylalkoxy groups, benzyl groups, and C 3 -C 8 heterocyclyl groups;

wherein at least one of R 6 and R 7 is hydrogen;

wherein if R 2 is a linker, then neither R 6 or R 7 is a linker, and if R 6 or R 7 is a linker, then R 2 is absent; and

wherein R 1 and R 9 cannot both be absent.

In various other embodiments, the linker-drug (L-D) compounds disclosed herein comprise a linker-drug structure according to Formula (IX). In various embodiments, the present disclosure provides a linker-drug (L-D) compound of Formula (IX):

or a pharmaceutically acceptable salt thereof, wherein:

R 2 is a linker;

R 4 is chosen from hydrogen, hydroxyl groups, —O—(C 1 -C 6 alkyl) groups, —O—C(═O)—(C 1 -C 6 alkyl) groups, and C 1 -C 6 alkyl groups;

R 15 and R 16 are each independently chosen from hydrogen, R 17 , —C(═O)—R 17 , and —C(═O)—O—R 17 ; and

R 17 is chosen from hydrogen, C 1 -C 6 alkyl groups, C 3 -C 8 cycloalkyl groups, benzyl groups, and C 3 -C 8 heterocyclyl groups;

wherein R 1 , R 2 , R 3 , R 4 , R 10 , R 15 , R 16 , and R 17 are each independently substituted with 0 to 3 groups independently chosen from halogens, hydroxyl groups, C 1 -C 6 alkyl groups, —O—(C 1 -C 6 alkyl) groups, —NR 15 R 16 , C 3 -C 8 cycloalkyl groups, C 1 -C 6 alkylhydroxy groups, C 1 -C 6 alkylalkoxy groups, benzyl groups, and C 3 -C 8 heterocyclyl groups; and

wherein each R a is independently substituted with 0 to 3 groups independently chosen from halogens, hydroxyl groups, —NR 15 R 16 , C 1 -C 6 alkyl groups, —(C═O)—(C 1 -C 6 alkyl) groups, —(C═O)—(C 1 -C 6 alkyl)-(C 3 -C 10 heterocyclyl) groups, and C 1 -C 6 alkylcarboxylic acid groups, each of which is substituted with 0, 1, or 2 groups independently chosen from halogens, hydroxyl groups, —NR 15 R 16 , and C 1 -C 3 alkyl groups; and

w is 0, 1, or 2.

Also, in various embodiments, provided herein are therapeutic uses for the described ADC compounds and compositions, e.g., in treating a neoplastic disorder, e.g., a cancer. In certain aspects, the present disclosure provides methods of treating a neoplastic disorder, e.g., a cancer that expresses an antigen targeted by the antibody or antigen binding fragment of the ADC, such as HER2, CD138, EPHA2, MSLN, FOLH1, CDH6, CEACAM5, CFC1B, ENPP3, FOLR1, HAVCR1, KIT, MET, MUC16, SLC39A6, SLC44A4, or STEAP1.

In certain aspects, the present disclosure provides methods of treating a subject having or suspected of having a neoplastic disorder by administering to the subject a therapeutically effective amount and/or regimen of any one of the described ADCs or compositions. In some embodiments, the neoplastic disorder is a hematological malignancy or a solid tumor. In some embodiments, the neoplastic disorder is a hematological malignancy. In some embodiments, the hematological malignancy is selected from a B-cell malignancy, a leukemia, a lymphoma, and a myeloma. In some embodiments, the hematological malignancy is selected from acute myeloid leukemia and multiple myeloma. In some embodiments, the neoplastic disorder is a solid tumor.

›In some embodiments, the solid tumor is selected…

In some embodiments, the solid tumor is selected from breast cancer (e.g., HER2-positive breast cancer), gastric cancer (e.g., gastric adenocarcinoma), prostate cancer, ovarian cancer, lung cancer (e.g., lung adenocarcinoma), uterine cancer (e.g., uterine serous endometrial carcinoma), salivary duct carcinoma, melanoma, colon cancer, cervical cancer, pancreatic cancer, kidney cancer, colorectal cancer, and esophageal cancer. In some embodiments, the solid tumor is selected from HER2-positive breast cancer, gastric adenocarcinoma, prostate cancer, and osteosarcoma.

In some embodiments, treatment with the antibody-drug conjugate or composition induces bystander killing of neoplastic cells which do not express a target antigen but are adjacent to neoplastic cells which express a target antigen. In some embodiments, the subject has one or more neoplastic cells which express a target antigen.

In some embodiments, the target antigen is HER2. In some embodiments, the one or more neoplastic cells are derived from a HER2-expressing breast cancer, ovarian cancer, gastric cancer, lung cancer (e.g., lung adenocarcinoma), uterine cancer (e.g., uterine serous endometrial carcinoma), osteosarcoma, or salivary duct carcinoma. In some embodiments, the subject is non-responsive or poorly responsive to treatment with (a) an anti-HER2 antibody when administered alone and/or (b) a splicing modulator when administered alone. In some embodiments, the subject is intolerant, non-responsive, or poorly responsive to treatment with a splicing modulator when administered alone.

In some embodiments, the target antigen is CD138. In some embodiments, the one or more neoplastic cells are derived from a CD138-expressing multiple myeloma. In some embodiments, the subject is non-responsive or poorly responsive to treatment with (a) an anti-CD138 antibody when administered alone and/or (b) a splicing modulator when administered alone. In some embodiments, the subject is intolerant, non-responsive, or poorly responsive to treatment with a splicing modulator when administered alone.

In some embodiments, the target antigen is EPHA2. In some embodiments, the one or more neoplastic cells are derived from an EPHA2-expressing breast cancer, prostate cancer, ovarian cancer, lung cancer, melanoma, colon cancer, or esophageal cancer. In some embodiments, the subject is non-responsive or poorly responsive to treatment with (a) an anti-EPHA2 antibody when administered alone and/or (b) a splicing modulator when administered alone. In some embodiments, the subject is intolerant, non-responsive, or poorly responsive to treatment with a splicing modulator when administered alone.

In some embodiments, the target antigen is MSLN. In some embodiments, the one or more neoplastic cells are derived from a MSLN-expressing ovarian cancer, cervical cancer, pancreatic cancer, or lung cancer (e.g., lung adenocarcinoma). In some embodiments, the subject is non-responsive or poorly responsive to treatment with (a) an anti-MSLN antibody when administered alone and/or (b) a splicing modulator when administered alone. In some embodiments, the subject is intolerant, non-responsive, or poorly responsive to treatment with a splicing modulator when administered alone.

In some embodiments, the target antigen is FOLH1. In some embodiments, the one or more neoplastic cells are derived from a FOLH1-expressing prostate cancer. In some embodiments, the subject is non-responsive or poorly responsive to treatment with (a) an anti-FOLH1 antibody when administered alone and/or (b) a splicing modulator when administered alone. In some embodiments, the subject is intolerant, non-responsive, or poorly responsive to treatment with a splicing modulator when administered alone.

In some embodiments, the target antigen is CDH6. In some embodiments, the one or more neoplastic cells are derived from a CDH6-expressing kidney cancer. In some embodiments, the subject is non-responsive or poorly responsive to treatment with (a) an anti-CDH6 antibody when administered alone and/or (b) a splicing modulator when administered alone. In some embodiments, the subject is intolerant, non-responsive, or poorly responsive to treatment with a splicing modulator when administered alone.

In some embodiments, the target antigen is CEACAM5. In some embodiments, the one or more neoplastic cells are derived from a CEACAM5-expressing colorectal cancer. In some embodiments, the subject is non-responsive or poorly responsive to treatment with (a) an anti-CEACAM5 antibody when administered alone and/or (b) a splicing modulator when administered alone. In some embodiments, the subject is intolerant, non-responsive, or poorly responsive to treatment with a splicing modulator when administered alone.

In some embodiments, the target antigen is CFC1B. In some embodiments, the one or more neoplastic cells are derived from a CFC1B-expressing pancreatic cancer. In some embodiments, the subject is non-responsive or poorly responsive to treatment with (a) an anti-CFC1B antibody when administered alone and/or (b) a splicing modulator when administered alone. In some embodiments, the subject is intolerant, non-responsive, or poorly responsive to treatment with a splicing modulator when administered alone.

In some embodiments, the target antigen is ENPP3. In some embodiments, the one or more neoplastic cells are derived from an ENPP3-expressing kidney cancer. In some embodiments, the subject is non-responsive or poorly responsive to treatment with (a) an anti-ENPP3 antibody when administered alone and/or (b) a splicing modulator when administered alone. In some embodiments, the subject is intolerant, non-responsive, or poorly responsive to treatment with a splicing modulator when administered alone.

In some embodiments, the target antigen is FOLR1. In some embodiments, the one or more neoplastic cells are derived from a FOLR1-expressing ovarian cancer. In some embodiments, the subject is non-responsive or poorly responsive to treatment with (a) an anti-FOLR1 antibody when administered alone and/or (b) a splicing modulator when administered alone. In some embodiments, the subject is intolerant, non-responsive, or poorly responsive to treatment with a splicing modulator when administered alone.

›In some embodiments, the target antigen is HAVCR1…

In some embodiments, the target antigen is HAVCR1. In some embodiments, the one or more neoplastic cells are derived from a HAVCR1-expressing kidney cancer or esophageal cancer. In some embodiments, the subject is non-responsive or poorly responsive to treatment with (a) an anti-HAVCR1 antibody when administered alone and/or (b) a splicing modulator when administered alone. In some embodiments, the subject is intolerant, non-responsive, or poorly responsive to treatment with a splicing modulator when administered alone.

In some embodiments, the target antigen is KIT. In some embodiments, the one or more neoplastic cells are derived from a KIT-expressing kidney cancer. In some embodiments, the subject is non-responsive or poorly responsive to treatment with (a) an anti-KIT antibody when administered alone and/or (b) a splicing modulator when administered alone. In some embodiments, the subject is intolerant, non-responsive, or poorly responsive to treatment with a splicing modulator when administered alone.

In some embodiments, the target antigen is MET. In some embodiments, the one or more neoplastic cells are derived from a MET-expressing kidney cancer or esophageal cancer. In some embodiments, the subject is non-responsive or poorly responsive to treatment with (a) an anti-MET antibody when administered alone and/or (b) a splicing modulator when administered alone. In some embodiments, the subject is intolerant, non-responsive, or poorly responsive to treatment with a splicing modulator when administered alone.

In some embodiments, the target antigen is MUC16. In some embodiments, the one or more neoplastic cells are derived from a MUC16-expressing ovarian cancer, cervical cancer, or breast cancer. In some embodiments, the subject is non-responsive or poorly responsive to treatment with (a) an anti-MUC16 antibody when administered alone and/or (b) a splicing modulator when administered alone. In some embodiments, the subject is intolerant, non-responsive, or poorly responsive to treatment with a splicing modulator when administered alone.

In some embodiments, the target antigen is SLC39A6. In some embodiments, the one or more neoplastic cells are derived from a SLC39A6-expressing breast cancer or prostate cancer. In some embodiments, the subject is non-responsive or poorly responsive to treatment with (a) an anti-SLC39A6 antibody when administered alone and/or (b) a splicing modulator when administered alone. In some embodiments, the subject is intolerant, non-responsive, or poorly responsive to treatment with a splicing modulator when administered alone.

In some embodiments, the target antigen is SLC44A4. In some embodiments, the one or more neoplastic cells are derived from a SLC44A4-expressing prostate cancer. In some embodiments, the subject is non-responsive or poorly responsive to treatment with (a) an anti-SLC44A4 antibody when administered alone and/or (b) a splicing modulator when administered alone. In some embodiments, the subject is intolerant, non-responsive, or poorly responsive to treatment with a splicing modulator when administered alone.

In some embodiments, the target antigen is STEAP1. In some embodiments, the one or more neoplastic cells are derived from a STEAP1-expressing prostate cancer. In some embodiments, the subject is non-responsive or poorly responsive to treatment with (a) an anti-STEAP1 antibody when administered alone and/or (b) a splicing modulator when administered alone. In some embodiments, the subject is intolerant, non-responsive, or poorly responsive to treatment with a splicing modulator when administered alone.

In certain other aspects, the present disclosure provides methods of reducing or inhibiting growth of a tumor in a subject having or suspected of having a neoplastic disorder by administering to the subject a therapeutically effective amount and/or regimen of any one of the described ADCs or compositions.

In some embodiments, treatment with the antibody-drug conjugate or composition induces bystander killing of neoplastic tumor cells which do not express a target antigen but are adjacent to neoplastic tumor cells which express a target antigen. In some embodiments, the tumor comprises one or more neoplastic cells which express a target antigen.

In some embodiments, the target antigen is HER2. In some embodiments, the one or more neoplastic cells are derived from a HER2-expressing breast cancer, ovarian cancer, gastric cancer, lung cancer (e.g., lung adenocarcinoma), uterine cancer (e.g., uterine serous endometrial carcinoma), osteosarcoma, or salivary duct carcinoma. In some embodiments, the tumor is resistant or refractory to treatment with (a) an anti-HER2 antibody when administered alone and/or (b) a splicing modulator when administered alone.

In some embodiments, the target antigen is EPHA2. In some embodiments, the one or more neoplastic cells are derived from an EPHA2-expressing breast cancer, prostate cancer, ovarian cancer, lung cancer, melanoma, colon cancer, or esophageal cancer. In some embodiments, the tumor is resistant or refractory to treatment with (a) an anti-EPHA2 antibody when administered alone and/or (b) a splicing modulator when administered alone.

In some embodiments, the target antigen is MSLN. In some embodiments, the one or more neoplastic cells are derived from a MSLN-expressing ovarian cancer, cervical cancer, pancreatic cancer, or lung cancer (e.g., lung adenocarcinoma). In some embodiments, the tumor is resistant or refractory to treatment with (a) an anti-MSLN antibody when administered alone and/or (b) a splicing modulator when administered alone.

›In some embodiments, the target antigen is CDH6…

In some embodiments, the target antigen is KIT. In some embodiments, the one or more neoplastic cells are derived from a KIT-expressing kidney cancer. In some embodiments, the tumor is resistant or refractory to treatment with (a) an anti-KIT antibody when administered alone and/or (b) a splicing modulator when administered alone.

In still other aspects, the present disclosure provides methods of determining whether a subject having or suspected of having a neoplastic disorder will be responsive to treatment with any one of the described ADCs or compositions by providing a biological sample from the subject and contacting the biological sample with the ADC or composition. In some embodiments, the biological sample is a tumor sample. In some embodiments, the tumor sample is a tumor biopsy or blood sample. In some embodiments, the blood sample is selected from blood, a blood fraction, or a cell obtained from the blood or blood fraction. In some embodiments, the subject has one or more neoplastic cells which express a target antigen. In some embodiments, the target antigen is HER2. In some embodiments, the one or more neoplastic cells are derived from a HER2-expressing breast cancer, ovarian cancer, gastric cancer, lung cancer (e.g., lung adenocarcinoma), uterine cancer (e.g., uterine serous endometrial carcinoma), osteosarcoma, or salivary duct carcinoma. In some embodiments, the target antigen is CD138. In some embodiments, the one or more neoplastic cells are derived from a CD138-expressing multiple myeloma. In some embodiments, the target antigen is EPHA2. In some embodiments, the one or more neoplastic cells are derived from an EPHA2-expressing breast cancer, prostate cancer, ovarian cancer, lung cancer, melanoma, colon cancer, or esophageal cancer. In some embodiments, the target antigen is MSLN. In some embodiments, the one or more neoplastic cells are derived from a MSLN-expressing ovarian cancer, cervical cancer, pancreatic cancer, or lung cancer (e.g., lung adenocarcinoma). In some embodiments, the target antigen is FOLH1. In some embodiments, the one or more neoplastic cells are derived from a FOLH1-expressing prostate cancer. In some embodiments, the target antigen is CDH6. In some embodiments, the one or more neoplastic cells are derived from a CDH6-expressing kidney cancer. In some embodiments, the target antigen is CEACAM5. In some embodiments, the one or more neoplastic cells are derived from a CEACAM5-expressing colorectal cancer. In some embodiments, the target antigen is CFC1B. In some embodiments, the one or more neoplastic cells are derived from a CFC1B-expressing pancreatic cancer. In some embodiments, the target antigen is ENPP3. In some embodiments, the one or more neoplastic cells are derived from an ENPP3-expressing kidney cancer. In some embodiments, the target antigen is FOLR1. In some embodiments, the one or more neoplastic cells are derived from a FOLR1-expressing ovarian cancer. In some embodiments, the target antigen is HAVCR1. In some embodiments, the one or more neoplastic cells are derived from a HAVCR1-expressing kidney cancer or esophageal cancer. In some embodiments, the target antigen is KIT. In some embodiments, the one or more neoplastic cells are derived from a KIT-expressing kidney cancer. In some embodiments, the target antigen is MET. In some embodiments, the one or more neoplastic cells are derived from a MET-expressing kidney cancer or esophageal cancer. In some embodiments, the target antigen is MUC16. In some embodiments, the one or more neoplastic cells are derived from a MUC16-expressing ovarian cancer, cervical cancer, or breast cancer. In some embodiments, the target antigen is SLC39A6. In some embodiments, the one or more neoplastic cells are derived from a SLC39A6-expressing breast cancer or prostate cancer. In some embodiments, the target antigen is SLC44A4. In some embodiments, the one or more neoplastic cells are derived from a SLC44A4-expressing prostate cancer. In some embodiments, the target antigen is STEAP1. In some embodiments, the one or more neoplastic cells are derived from a STEAP1-expressing prostate cancer.

›Further provided herein, in various embodiments, are pharmaceutical…

Further provided herein, in various embodiments, are pharmaceutical compositions comprising an ADC and a pharmaceutically acceptable diluent, carrier, and/or excipient. Methods of producing the described ADC compounds and compositions are also disclosed.

›BRIEF DESCRIPTION OF THE DRAWINGS · 1 of 2

FIG. 1 shows dose response of exemplary payload compounds in a competitive binding assay. Nuclear extracts from 293F cells overexpressing wild type flag-tagged SF3B1 were immunoprecipitated with an anti-SF3B1 antibody and scintillation proximity assay (SPA) bead cocktail. Binding reactions contained the antibody-bead mixture and increasing concentrations of compound, followed by competition with an 3 H-labelled pladienolide B (PB) probe. The y-axis represents the percent change (% response) of specific binding relative to the DMSO control (0%). Data are represented as mean±standard deviation (SD).

FIG. 2 shows modulation of splicing by exemplary payload compounds in an in vitro splicing assay. Nuclear extracts from HeLa S3 cells were incubated with Ad2.2 pre-mRNA and increasing concentrations of compound, followed by quantification of splicing modulation via RT-PCR. The Ad2.2 sequence is derived from the adenoviral Ad2 pre-mRNA substrate with modifications around the branch point sequence. The y-axis represents the percent change (% response) of splicing relative to the DMSO control (0%). Data are represented as mean±SD.

FIG. 3 shows viability dose response of exemplary payload compounds in HER2-amplified breast cancer cells (HCC1954). Cells were incubated with compound for 144 hours (6 days) and viability was read in CellTiter-Glo® reagent Data are represented as mean±SD.

FIG. 4 shows the results of a cell binding assay. Binding of exemplary HER2-ADCs to JIMT1 cells was assessed by flow cytometry. Mean fluorescence intensity values were measured to determine binding of conjugates, followed by PE-labelled secondary. Data are represented as mean±SD.

FIG. 5 A shows viability dose response of exemplary HER2-ADCs in HER2-amplified breast cancer cells (HCC1954). FIG. 5 B shows viability dose response of exemplary HER2-ADCs in HER2-amplified gastric cancer cells (N87). FIG. 5 C shows viability dose response of exemplary HER2-ADCs in HER2-amplified breast cancer cells (SKBR3). Cells were incubated with conjugates for 144 hours (6 days) and viability was read in CellTiter-Glo® reagent. Data are represented as mean±SD.

FIG. 6 shows viability dose response of exemplary HER2-ADCs in non-HER2 expressing breast cancer cells (MCF7). Cells were incubated with conjugates for 144 hours (6 days) and viability was read in CellTiter-Glo® reagent. Data are represented as mean±SD.

FIG. 7 shows the results of a SLC25A19 splicing assay in HER2-amplified breast cancer cells (HCC1954). Cells were incubated with conjugates for 24 hours and splicing of SLC25A19 transcript was measured in a real time qPCR reaction with a specific Taqman primer-probe set. The y-axis represents the percent (%) response relative to the DMSO control (0%). Data are represented as mean±SD.

FIG. 8 shows the results of a bystander killing assay. H1568 cells overexpressing HER2 (target-positive) or H1568 cells tagged with luciferase (target-negative) plated either alone or incubated together in co-culture for 144 hours (6 days) were treated with exemplary HER2-ADCs. Plates were read with OneGlo® reagent. The y-axis represents the percent (%) response relative to the PBS control (100%). Data are represented as mean±SD.

FIG. 9 shows tumor growth kinetics for each group of HCC1954-implanted CB17-SCID mice treated with a single intravenous dose of an exemplary HER2-ADC or corresponding dose-matched payload (6-10 animals per group). Tumor volumes were measured twice weekly after treatment. Data are represented as mean±standard error of the mean (SEM).

FIG. 10 shows viability dose response of exemplary CD138-ADCs in a CD138-expressing multiple myeloma cell line. MOLP8 cells were incubated with conjugates for 144 hours (6 days) and viability was read in CellTiter-Glo® reagent. Data are represented as mean±SD.

FIG. 11 shows viability dose response of exemplary EPH2A-ADCs in an EPHA2-expressing prostate cancer cell line. PC3 cells were incubated with conjugates for 144 hours (6 days) and viability was read in CellTiter-Glo® reagent. Data are represented as mean±SD.

FIG. 12 A and FIG. 12 B show the results of an in vitro stability assay for an exemplary anti-HER2 ADC, AB185-ADL1-D1. The y-axis represents concentration of total antibody ( FIG. 12 A ) and conjugated (intact) payload ( FIG. 12 B ); the x-axis represents time as measured in hours at 37° C.

FIG. 13 shows plasma concentrations of an exemplary anti-HER2 ADC, AB185-ADL1-D1, following a single intravenous dose in CD17-SCID N87 tumor-bearing mice.

FIG. 14 shows a schematic diagram of an exemplary RNA sequencing and protein ligandome experiment.

FIG. 15 shows a schematic diagram of an exemplary T-cell priming experiment.

FIG. 16 shows the results of a FACS analysis. Monocytes were isolated from peripheral blood mononuclear cells (PBMC) and were induced to differentiate into dendritic cells (DC) through culturing in a cytokine cocktail. FACS was performed to validate the maturation of DC from monocytes.

FIG. 17 A-D show the results of an ELISpot assay. FIG. 17 A shows ELISpot plates indicating Neoantigen 1 priming of CD8+ T-cell activation. Stimulation of CD8+ T-cells was monitored by secretion of IFNγ. FIG. 17 B shows quantification of IFNγ spots (spot number) in Neoantigen 1 ELISpot plates ( FIG. 17 A ). FIG. 17 C shows ELISpot plates indicating Neoantigen 3 priming of CD8+ T-cell activation. Stimulation of CD8+ T-cells was monitored by secretion of IFNγ. FIG. 17 D shows quantification of IFNγ spots (fold change) in Neoantigen 3 ELISpot plates ( FIG. 17 C ).

FIG. 18 shows a plot comparing splicing potency (IC50 qPCR) against cellular potency (GI50 CTG) for exemplary anti-HER2 ADCs in HCC1954 breast cancer cells. Values shown are sized by cell lethality and shaded by depth of alternative splicing response.

FIG. 19 shows a plot comparing splicing potency (IC50 qPCR) against cellular potency (GI50 CTG) for exemplary anti-HER2 ADCs in N87 gastric cancer cells. Values shown are sized by cell lethality and shaded by depth of alternative splicing response.

›BRIEF DESCRIPTION OF THE DRAWINGS · 2 of 2

FIG. 20 shows a plot comparing potency and lethality of exemplary anti-HER2 ADCs (in HCC1954 breast cancer cells) against stability and permeability of corresponding payloads. Values shown are sized by payload stability and shaded by payload permeability.

FIG. 21 shows tumor growth kinetics for each group of N87-implanted CB17-SCID mice treated intravenously with vehicle or 10 mg/kg trastuzumab, TDM1, or an exemplary HER2-ADC Q7D for 2 cycles (N=8 per group). Data are represented as mean±standard error of the mean (SEM) (mm 3 ).

FIG. 22 shows body weight change for each group of N87-implanted CB17-SCID mice treated intravenously with vehicle or 10 mg/kg trastuzumab, TDM1, or an exemplary HER2-ADC Q7D for 2 cycles (N=8 per group). Data are represented as mean±standard error of the mean (SEM) (%).

FIG. 23 shows tumor growth kinetics (left) and body weight change (right) for each group of N87-implanted CB17-SCID mice treated intravenously with vehicle or 10 mg/kg trastuzumab, TDM1, or an exemplary HER2-ADC Q7D for 2 cycles (N=8 per group). Data are represented as mean±SEM (tumor volume, mm 3 ) or mean±SEM (body weight, %).

FIG. 24 A- 24 D show pharmacodynamics (PD) modulation of mRNA junctions in N87-implanted CB17-SCID mice treated intravenously with vehicle or 10 mg/kg trastuzumab, TDM1, or an exemplary HER2-ADC Q7D for 2 cycles. RT-qPCR of FBXW5 (mature mRNA transcript) was monitored and is shown in FIG. 24 A and FIG. 24 C . RT-qPCR of TAOK1 (neojunction transcript) was monitored and is shown in FIG. 24 B and FIG. 24 D . Animals (N=4 per group) were collected at either 48 hours ( FIG. 24 A and FIG. 24 B ) or at the times indicated ( FIG. 24 C and FIG. 24 D ). Tumors were isolated for RNA extraction and RT-qPCR.

FIG. 25 shows a schematic diagram of an exemplary target indication analysis.

FIG. 26 shows an exemplary bioconjugation scheme for preparation of ADCs using splicing modulators.

›DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The disclosed compositions and methods may be understood more readily by reference to the following detailed description taken in connection with the accompanying figures, which form a part of this disclosure.

Throughout this text, the descriptions refer to compositions and methods of using the compositions. Where the disclosure describes or claims a feature or embodiment associated with a composition, such a feature or embodiment is equally applicable to the methods of using the composition. Likewise, where the disclosure describes or claims a feature or embodiment associated with a method of using a composition, such a feature or embodiment is equally applicable to the composition.

When a range of values is expressed, it includes embodiments using any particular value within the range. Further, reference to values stated in ranges includes each and every value within that range. All ranges are inclusive of their endpoints and combinable. Men values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another embodiment. Reference to a particular numerical value includes at least that particular value, unless the context clearly dictates otherwise. The use of “or” will mean “and/or” unless the specific context of its use dictates otherwise. All references cited herein are incorporated by reference for any purpose. Mere a reference and the specification conflict, the specification will control.

It is to be appreciated that certain features of the disclosed compositions and methods, which are, for clarity, described herein in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the disclosed compositions and methods that are, for brevity, described in the context of a single embodiment, may also be provided separately or in any subcombination.

›Definitions · 1 of 64

Various terms relating to aspects of the description are used throughout the specification and claims. Such terms are to be given their ordinary meaning in the art unless otherwise indicated. Other specifically defined terms are to be construed in a manner consistent with the definitions provided herein.

As used herein, the singular forms “a,” “an,” and “the” include plural forms unless the context clearly dictates otherwise.

The terms “about” or “approximately” in the context of numerical values and ranges refers to values or ranges that approximate or are close to the recited values or ranges such that the embodiment may perform as intended, such as having a desired amount of nucleic acids or polypeptides in a reaction mixture, as is apparent to the skilled person from the teachings contained herein. In some embodiments, about means plus or minus 10% of a numerical amount.

The terms “antibody-drug conjugate,” “antibody conjugate,” “conjugate,” “immunoconjugate,” and “ADC” are used interchangeably, and refer to one or more therapeutic compounds (e.g., a splicing modulator) that is linked to one or more antibodies or antigen binding fragments and is defined by the generic formula: Ab-(L-D), (Formula I), wherein Ab=an antibody or antigen binding fragment, L=a linker moiety, D=a drug moiety (e.g., a splicing modulator drug moiety), and p=the number of drug moieties per antibody or antigen binding fragment. An ADC comprising a splicing modulator drug moiety may also be referred to herein more specifically as a “splicing modulator-loaded antibody” or a “SMLA.” In ADCs comprising a splicing modulator drug moiety, “p” refers to the number of splicing modulator compounds linked to the antibody or antigen binding fragment. In some embodiments, the linker L can include a cleavable moiety between the antibody or antigen binding fragment and the therapeutic compound. In some embodiments, the linker L can include a cleavable moiety that can be attached to either or both the antibody or antigen binding fragment and therapeutic compound by spacer unit(s). In some embodiments, when a spacer unit attaches the cleavable moiety to the therapeutic compound, it is a self-immolative spacer unit. In other embodiments, the linker L does not include a cleavable moiety, and is a non-cleavable linker. In some embodiments, the linker L can include at least one spacer unit that can directly attach to the antibody or antigen binding fragment and to the therapeutic compound. Exemplary cleavable and non-cleavable linkers are described and exemplified herein.

The term “antibody” is used in the broadest sense to refer to an immunoglobulin molecule that recognizes and specifically binds to a target, such as a protein, polypeptide, carbohydrate, polynucleotide, lipid, or combinations of the foregoing through at least one antigen recognition site within the variable region of the immunoglobulin molecule. The heavy chain of an antibody is composed of a heavy chain variable domain (V H ) and a heavy chain constant region (C H ). The light chain is composed of a light chain variable domain (V L ) and a light chain constant domain (C L ). For the purposes of this application, the mature heavy chain and light chain variable domains each comprise three complementarity determining regions (CDR1, CDR2 and CDR3) within four framework regions (FR1, FR2, FR3, and FR4) arranged from N-terminus to C-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. An “antibody” can be naturally occurring or man-made, such as monoclonal antibodies produced by conventional hybridoma technology. The term “antibody” includes full-length monoclonal antibodies and full-length polyclonal antibodies, as well as antibody fragments such as Fab, Fab′, F(ab′) 2 , Fv, and single chain antibodies. An antibody can be any one of the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses thereof (e.g., isotypes IgG1, IgG2, IgG3, IgG4). The term further encompasses human antibodies, chimeric antibodies, humanized antibodies and any modified immunoglobulin molecule containing an antigen recognition site, so long as it demonstrates the desired biological activity (e.g., binds the target antigen, internalizes within a target-antigen expressing cell).

The term “monoclonal antibody,” as used herein, refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic epitope. In contrast, conventional (polyclonal) antibody preparations typically include a multitude of antibodies directed against (or specific for) different epitopes. The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present disclosure may be made by the hybridoma method first described by Kohler et al. (1975) Nature 256:495, or may be made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). Monoclonal antibodies may also be isolated from phage antibody libraries using the techniques described in Clackson et al. (1991) Nature 352:624-8, and Marks et al. (1991) J Mol Biol. 222:581-97, for example.

The monoclonal antibodies described herein specifically include “chimeric” antibodies, in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they specifically bind the target antigen and/or exhibit the desired biological activity.

›Definitions · 2 of 64

The term “human antibody,” as used herein, refers an antibody produced by a human or an antibody having an amino acid sequence of an antibody produced by a human.

The term “chimeric antibody,” as used herein, refers to antibodies wherein the amino acid sequence of the immunoglobulin molecule is derived from two or more species. In some instances, the variable regions of both heavy and light chains correspond to the variable regions of antibodies derived from one species with the desired specificity, affinity, and activity while the constant regions are homologous to antibodies derived from another species (e.g., human) to minimize an immune response in the latter species.

As used herein, the term “humanized antibody” refers to forms of antibodies that contain sequences from non-human (e.g., murine) antibodies as well as human antibodies. Such antibodies are chimeric antibodies which contain minimal sequence derived from non-human immunoglobulin. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the framework (FR) regions are those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. The humanized antibody can be further modified by the substitution of residues, either in the Fv framework region and/or within the replaced non-human residues to refine and optimize antibody specificity, affinity, and/or activity.

The term “antigen binding fragment” or “antigen binding portion” of an antibody, as used herein, refers to one or more fragments of an antibody or protein that retain the ability to specifically bind to an antigen (e.g., HER2, CD138, EPHA2, MSLN, FOLH1, CDH6, CEACAM5, CFC1B, ENPP3, FOLR1, HAVCR1, KIT, MET, MUC16, SLC39A6, SLC44A4, STEAP1). Antigen binding fragments may also retain the ability to internalize into an antigen-expressing cell. In some embodiments, antigen binding fragments also retain immune effector activity. It has been shown that fragments of a full-length antibody can perform the antigen binding function of a full-length antibody. Examples of binding fragments encompassed within the term “antigen binding fragment” or “antigen binding portion” of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the V L , V H , C L , and C H1 domains; (ii) a F(ab′) 2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the V H and C H1 domains; (iv) a Fv fragment consisting of the V L and V H domains of a single arm of an antibody; (v) a dAb fragment, which comprises a single variable domain, e.g., a V H domain (see, e.g., Ward et al. (1989) Nature 341:544-6; and Intl. Pub. No. WO 1990/005144); and (vi) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, V L and V H , are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the V L and V H regions pair to form monovalent molecules (known as single chain Fv (scFv)). See, e.g., Bird et al. (1988) Science 242:423-6; and Huston et al. (1988) Proc Natl Acad Sci. USA 85:5879-83. Such single chain antibodies are also intended to be encompassed within the term “antigen binding fragment” or “antigen binding portion” of an antibody, and are known in the art as an exemplary type of binding fragment that can internalize into cells upon binding (see, e.g., Zhu et al. (2010) 9:2131-41; He et al. (2010) J Nucl Med. 51:427-32; and Fitting et al. (2015) MAbs 7:390-402). In certain embodiments, scFv molecules may be incorporated into a fusion protein. Other forms of single chain antibodies, such as diabodies are also encompassed. Diabodies are bivalent, bispecific antibodies in which V H and V L domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites (see e.g., Holliger et al. (1993) Proc Natl Acad Sci. USA 90:6444-8; and Poljak et al. (1994) Structure 2:1121-3). Antigen binding fragments are obtained using conventional techniques known to those of skill in the art, and the binding fragments are screened for utility (e.g., binding affinity, internalization) in the same manner as are intact antibodies. Antigen binding fragments may be prepared by cleavage of the intact protein, e.g., by protease or chemical cleavage.

“Internalizing” as used herein in reference to an antibody or antigen binding fragment refers to an antibody or antigen binding fragment that is capable of being taken through the cell's lipid bilayer membrane to an internal compartment (i.e., “internalized”) upon binding to the cell, preferably into a degradative compartment in the cell. For example, an internalizing anti-HER2 antibody is one that is capable of being taken into the cell after binding to HER2 on the cell membrane. In some embodiments, the antibody or antigen binding fragment used in the ADCs disclosed herein targets a cell surface antigen (e.g., HER2) and is an internalizing antibody or internalizing antigen binding fragment (i.e., the ADC transfers through the cellular membrane after antigen binding). In some embodiments, the internalizing antibody or antigen binding fragment binds a receptor on the cell surface. An internalizing antibody or internalizing antigen binding fragment that targets a receptor on the cell membrane may induce receptor-mediated endocytosis. In some embodiments, the internalizing antibody or internalizing antigen binding fragment is taken into the cell via receptor-mediated endocytosis.

›Definitions · 3 of 64

“Non-internalizing” as used herein in reference to an antibody or antigen binding fragment refers to an antibody or antigen binding fragment that remains at the cell surface upon binding to the cell. In some embodiments, the antibody or antigen binding fragment used in the ADCs disclosed herein targets a cell surface antigen and is a non-internalizing antibody or non-internalizing antigen binding fragment (i.e., the ADC remains at the cell surface and does not transfer through the cellular membrane after antigen binding). In some embodiments, the non-internalizing antibody or antigen binding fragment binds a non-internalizing receptor or other cell surface antigen. Exemplary non-internalizing cell surface antigens include but are not limited to CA125 and CEA, and antibodies that bind to non-internalizing antigen targets are also known in the art (see, e.g., Bast et al. (1981) J Clin Invest. 68(5):1331-7; Scholler and Urban (2007) Biomark Med. 1(4):513-23; and Boudousq et al. (2013) PLoS One 8(7):e69613).

The term “human epidermal growth factor receptor 2,” “HER2,” or “HER2/NEU,” as used herein, refers to any native form of human HER2. The term encompasses full-length HER2 (e.g., UniProt Reference Sequence: P04626; SEQ ID NO:31), as well as any form of human HER2 that may result from cellular processing. The term also encompasses functional variants or fragments of human HER2, including but not limited to splice variants, allelic variants, and isoforms that retain one or more biologic functions of human HER2 (i.e., variants and fragments are encompassed unless the context indicates that the term is used to refer to the wild-type protein only). HER2 can be isolated from human, or may be produced recombinantly or by synthetic methods.

The term “anti-HER2 antibody” or “antibody that binds to HER2” refers to any form of antibody or fragment thereof that binds, e.g., specifically binds, to HER2, and encompasses monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, and biologically functional antibody fragments so long as they bind, e.g., specifically bind, to HER2. U.S. Pat. No. 5,821,337 provides and is incorporated herein by reference for exemplary HER2-binding sequences, including exemplary anti-HER2 antibody sequences. In some embodiments, the anti-HER2 antibody used in the ADCs disclosed herein is an internalizing antibody or internalizing antibody fragment. Trastuzumab (U.S. Pat. No. 5,821,337; Molina et al. (2001) Cancer Res. 61(12):4744-9) is an exemplary anti-human HER2 antibody.

The term “syndecan-1,” “SDC1,” or “CD138,” as used herein, refers to any native form of human CD138. The term encompasses full-length CD138 (e.g., UniProt Reference Sequence: P18827; SEQ ID NO:32), as well as any form of human CD138 that may result from cellular processing. The term also encompasses functional variants or fragments of human CD138, including but not limited to splice variants, allelic variants, and isoforms that retain one or more biologic functions of human CD138 (i.e., variants and fragments are encompassed unless the context indicates that the term is used to refer to the wild-type protein only). CD138 can be isolated from a human, or may be produced recombinantly or by synthetic methods.

The term “anti-CD138 antibody” or “antibody that binds to CD138” refers to any form of antibody or fragment thereof that binds, e.g., specifically binds, to CD138, and encompasses monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, and biologically functional antibody fragments so long as they bind, e.g., specifically bind, to CD138. In some embodiments, the anti-CD138 antibody used in the ADCs disclosed herein is an internalizing antibody or internalizing antibody fragment. B-B4 (Tassone et al. (2004) Blood 104:3688-96) is an exemplary anti-human CD138 antibody.

The term “ephrin type-A receptor 2” or “EPHA2,” as used herein, refers to any native form of human EPHA2. The term encompasses full-length EPHA2 (e.g., UniProt Reference Sequence: P29317; SEQ ID NO:33), as well as any form of human EPHA2 that may result from cellular processing. The term also encompasses functional variants or fragments of human EPHA2, including but not limited to splice variants, allelic variants, and isoforms that retain one or more biologic functions of human EPHA2 (i.e., variants and fragments are encompassed unless the context indicates that the term is used to refer to the wild-type protein only). EPHA2 can be isolated from a human, or may be produced recombinantly or by synthetic methods.

The term “anti-EPHA2 antibody” or “antibody that binds to EPHA2” refers to any form of antibody or fragment thereof that binds, e.g., specifically binds, to EPHA2, and encompasses monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, and biologically functional antibody fragments so long as they bind, e.g., specifically bind, to EPHA2. WO 2007/030642 provides and is incorporated herein by reference for exemplary EPHA2-binding sequences, including exemplary anti-EPHA2 antibody sequences. In some embodiments, the anti-EPHA2 antibody used in the ADCs disclosed herein is an internalizing antibody or internalizing antibody fragment. 1C1 (WO 2007/030642; Jackson et al. (2008) Cancer Res. 68(22): 9367-74) is an exemplary anti-human EPHA2 antibody.

The term “mesothelin” or “MSLN,” as used herein, refers to any native form of human MSLN. The term encompasses full-length MSLN (e.g., UniProt Reference Sequence: Q13421; SEQ ID NO:43), as well as any form of human MSLN that may result from cellular processing. The term also encompasses functional variants or fragments of human MSLN, including but not limited to splice variants, allelic variants, and isoforms that retain one or more biologic functions of human MSLN (i.e., variants and fragments are encompassed unless the context indicates that the term is used to refer to the wild-type protein only). MSLN can be isolated from a human, or may be produced recombinantly or by synthetic methods.

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The term “anti-MSLN antibody” or “antibody that binds to MSLN” refers to any form of antibody or fragment thereof that binds, e.g., specifically binds, to MSLN, and encompasses monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, and biologically functional antibody fragments so long as they bind, e.g., specifically bind, to MSLN. WO 2011/074621 provides and is incorporated herein by reference for exemplary MSLN-binding sequences, including exemplary anti-MSLN antibody sequences. In some embodiments, the anti-MSLN antibody used in the ADCs disclosed herein is an internalizing antibody or internalizing antibody fragment. 11-25, IC14-30, IC7-4, IC17-35 and 2-9 are exemplary anti-human MSLN antibodies.

The term “glutamate carboxypeptidase 2” or “FOLH1,” as used herein, refers to any native form of human FOLH1. The term encompasses full-length FOLH1 (e.g., UniProt Reference Sequence: Q04609; SEQ ID NO:44), as well as any form of human FOLH1 that may result from cellular processing. The term also encompasses functional variants or fragments of human FOLH1, including but not limited to splice variants, allelic variants, and isoforms that retain one or more biologic functions of human FOLH1 (i.e., variants and fragments are encompassed unless the context indicates that the term is used to refer to the wild-type protein only). FOLH1 can be isolated from a human, or may be produced recombinantly or by synthetic methods.

The term “anti-FOLH1 antibody” or “antibody that binds to FOLH1” refers to any form of antibody or fragment thereof that binds, e.g., specifically binds, to FOLH1, and encompasses monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, and biologically functional antibody fragments so long as they bind, e.g., specifically bind, to FOLH1. WO 2019/012260 and WO 2017/212250 provide and are incorporated herein by reference for exemplary FOLH1-binding sequences, including exemplary anti-FOLH1 antibody sequences. In some embodiments, the anti-FOLH1 antibody used in the ADCs disclosed herein is an internalizing antibody or internalizing antibody fragment. J591 (deimmunized) is an exemplary anti-human FOLH1 antibody.

The term “cadherin-6” or “CDH6,” as used herein, refers to any native form of human CDH6. The term encompasses full-length CDH6 (e.g., UniProt Reference Sequence: P55285; SEQ ID NO:45), as well as any form of human CDH6 that may result from cellular processing. The term also encompasses functional variants or fragments of human CDH6, including but not limited to splice variants, allelic variants, and isoforms that retain one or more biologic functions of human CDH6 (i.e., variants and fragments are encompassed unless the context indicates that the term is used to refer to the wild-type protein only). CDH6 can be isolated from a human, or may be produced recombinantly or by synthetic methods.

The term “anti-CDH6 antibody” or “antibody that binds to CDH6” refers to any form of antibody or fragment thereof that binds, e.g., specifically binds, to CDH6, and encompasses monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, and biologically functional antibody fragments so long as they bind, e.g., specifically bind, to CDH6. WO 2018/185618 provides and is incorporated herein by reference for exemplary CDH6-binding sequences, including exemplary anti-CDH6 antibody sequences. In some embodiments, the anti-CDH6 antibody used in the ADCs disclosed herein is an internalizing antibody or internalizing antibody fragment.

The term “carcinoembryonic antigen-related cell adhesion molecule 5” or “CEACAM5,” as used herein, refers to any native form of human CEACAM5. The term encompasses full-length CEACAM5 (e.g., UniProt Reference Sequence: P06731; SEQ ID NO:46), as well as any form of human CEACAM5 that may result from cellular processing. The term also encompasses functional variants or fragments of human CEACAM5, including but not limited to splice variants, allelic variants, and isoforms that retain one or more biologic functions of human CEACAM5 (i.e., variants and fragments are encompassed unless the context indicates that the term is used to refer to the wild-type protein only). CEACAM5 can be isolated from a human, or may be produced recombinantly or by synthetic methods.

The term “anti-CEACAM5 antibody” or “antibody that binds to CEACAM5” refers to any form of antibody or fragment thereof that binds, e.g., specifically binds, to CEACAM5, and encompasses monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, and biologically functional antibody fragments so long as they bind, e.g., specifically bind, to CEACAM5. US 2015/0125386 provides and is incorporated herein by reference for exemplary CEACAM5-binding sequences, including exemplary anti-CEACAM5 antibody sequences. In some embodiments, the anti-CEACAM5 antibody used in the ADCs disclosed herein is an internalizing antibody or internalizing antibody fragment hMN14 is an exemplary anti-human CEACAM5 antibody.

The term “cryptic family protein 1B” or “CFC1B,” as used herein, refers to any native form of human CFC1B. The term encompasses full-length CFC1B (e.g., UniProt Reference Sequence: P0CG36; SEQ ID NO:47), as well as any form of human CFC1B that may result from cellular processing. The term also encompasses functional variants or fragments of human CFC1B, including but not limited to splice variants, allelic variants, and isoforms that retain one or more biologic functions of human CFC1B (i.e., variants and fragments are encompassed unless the context indicates that the term is used to refer to the wild-type protein only). CFC1B can be isolated from a human, or may be produced recombinantly or by synthetic methods.

The term “anti-CFC1B antibody” or “antibody that binds to CFC1B” refers to any form of antibody or fragment thereof that binds, e.g., specifically binds, to CFC1B, and encompasses monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, and biologically functional antibody fragments so long as they bind, e.g., specifically bind, to CFC1B. WO 2002/088170 provides and is incorporated herein by reference for exemplary CFC1B-binding sequences, including exemplary anti-CFC1B antibody sequences. In some embodiments, the anti-CFC1B antibody used in the ADCs disclosed herein is an internalizing antibody or internalizing antibody fragment.

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The term “ectonucleotide pyrophosphatase/phosphodiesterase family member 3” or “ENPP3,” as used herein, refers to any native form of human ENPP3. The term encompasses full-length ENPP3 (e.g., UniProt Reference Sequence: 014638; SEQ ID NO:48), as well as any form of human ENPP3 that may result from cellular processing. The term also encompasses functional variants or fragments of human ENPP3, including but not limited to splice variants, allelic variants, and isoforms that retain one or more biologic functions of human ENPP3 (i.e., variants and fragments are encompassed unless the context indicates that the term is used to refer to the wild-type protein only). ENPP3 can be isolated from a human, or may be produced recombinantly or by synthetic methods.

The term “anti-ENPP3 antibody” or “antibody that binds to ENPP3” refers to any form of antibody or fragment thereof that binds, e.g., specifically binds, to ENPP3, and encompasses monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, and biologically functional antibody fragments so long as they bind, e.g., specifically bind, to ENPP3. Donate et al. ((2016) Clin Cancer Res. 22(8):1989-99) provides and is incorporated herein by reference for exemplary ENPP3-binding sequences, including exemplary anti-ENPP3 antibody sequences. In some embodiments, the anti-ENPP3 antibody used in the ADCs disclosed herein is an internalizing antibody or internalizing antibody fragment.

The term “folate receptor alpha” or “FOLR1,” as used herein, refers to any native form of human FOLR1. The term encompasses full-length FOLR1 (e.g., UniProt Reference Sequence: P15328; SEQ ID NO:49), as well as any form of human FOLR1 that may result from cellular processing. The term also encompasses functional variants or fragments of human FOLR1, including but not limited to splice variants, allelic variants, and isoforms that retain one or more biologic functions of human FOLR1 (i.e., variants and fragments are encompassed unless the context indicates that the term is used to refer to the wild-type protein only). FOLR1 can be isolated from a human, or may be produced recombinantly or by synthetic methods.

The term “anti-FOLR1 antibody” or “antibody that binds to FOLR1” refers to any form of antibody or fragment thereof that binds, e.g., specifically binds, to FOLR1, and encompasses monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, and biologically functional antibody fragments so long as they bind, e.g., specifically bind, to FOLR1. WO 2005/080431 and Coney et al. ((1991) Cancer Res. 51(22):6125-32) provide and are incorporated herein by reference for exemplary FOLR1-binding sequences, including exemplary anti-FOLR1 antibody sequences. In some embodiments, the anti-FOLR1 antibody used in the ADCs disclosed herein is an internalizing antibody or internalizing antibody fragment. Farletuzumab and MOv19 are exemplary anti-human FOLR1 antibodies.

The term “hepatitis A virus cellular receptor 1” or “HAVCR1,” as used herein, refers to any native form of human HAVCR1. The term encompasses full-length HAVCR1 (e.g., UniProt Reference Sequence: Q96D42; SEQ ID NO:50), as well as any form of human HAVCR1 that may result from cellular processing. The term also encompasses functional variants or fragments of human HAVCR1, including but not limited to splice variants, allelic variants, and isoforms that retain one or more biologic functions of human HAVCR1 (i.e., variants and fragments are encompassed unless the context indicates that the term is used to refer to the wild-type protein only). HAVCR1 can be isolated from a human, or may be produced recombinantly or by synthetic methods.

The term “anti-HAVCR1 antibody” or “antibody that binds to HAVCR1” refers to any form of antibody or fragment thereof that binds, e.g., specifically binds, to HAVCR1, and encompasses monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, and biologically functional antibody fragments so long as they bind, e.g., specifically bind, to HAVCR1. Thomas et al. ((2016) Mol Cancer Ther. 15(12):2946-54) provides and is incorporated herein by reference for exemplary HAVCR1-binding sequences, including exemplary anti-HAVCR1 antibody sequences. In some embodiments, the anti-HAVCR1 antibody used in the ADCs disclosed herein is an internalizing antibody or internalizing antibody fragment.

The term “mast/stem cell growth factor receptor Kit” or “KIT,” as used herein, refers to any native form of human KIT. The term encompasses full-length KIT (e.g., UniProt Reference Sequence: P10721; SEQ ID NO:51), as well as any form of human KIT that may result from cellular processing. The term also encompasses functional variants or fragments of human KIT, including but not limited to splice variants, allelic variants, and isoforms that retain one or more biologic functions of human KIT (i.e., variants and fragments are encompassed unless the context indicates that the term is used to refer to the wild-type protein only). KIT can be isolated from a human, or may be produced recombinantly or by synthetic methods.

The term “anti-KIT antibody” or “antibody that binds to KIT” refers to any form of antibody or fragment thereof that binds, e.g., specifically binds, to KIT, and encompasses monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, and biologically functional antibody fragments so long as they bind, e.g., specifically bind, to KIT. Shi et al. ((2016) Proc Natl Acad Sci USA 113(33):E4784-93) and Abrams et al. ((2018) Clin Cancer Res. 24(17):4297-308) provide and are incorporated herein by reference for exemplary KIT-binding sequences, including exemplary anti-KIT antibody sequences. In some embodiments, the anti-KIT antibody used in the ADCs disclosed herein is an internalizing antibody or internalizing antibody fragment.

The term “hepatocyte growth factor receptor” or “MET,” as used herein, refers to any native form of human MET. The term encompasses full-length MET (e.g., UniProt Reference Sequence: P08581; SEQ ID NO:52), as well as any form of human MET that may result from cellular processing. The term also encompasses functional variants or fragments of human MET, including but not limited to splice variants, allelic variants, and isoforms that retain one or more biologic functions of human MET (i.e., variants and fragments are encompassed unless the context indicates that the term is used to refer to the wild-type protein only). MET can be isolated from a human, or may be produced recombinantly or by synthetic methods.

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The term “anti-MET antibody” or “antibody that binds to MET” refers to any form of antibody or fragment thereof that binds, e.g., specifically binds, to MET, and encompasses monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, and biologically functional antibody fragments so long as they bind, e.g., specifically bind, to MET. Yang et al. ((2019) Acta Pharmacol Sin.) provides and is incorporated herein by reference for exemplary MET-binding sequences, including exemplary anti-MET antibody sequences. In some embodiments, the anti-MET antibody used in the ADCs disclosed herein is an internalizing antibody or internalizing antibody fragment.

The term “mucin-16” or “MUC16,” as used herein, refers to any native form of human MUC16. The term encompasses full-length MUC16 (e.g., UniProt Reference Sequence: Q8WXI7; SEQ ID NO:53), as well as any form of human MUC16 that may result from cellular processing. The term also encompasses functional variants or fragments of human MUC16, including but not limited to splice variants, allelic variants, and isoforms that retain one or more biologic functions of human MUC16 (i.e., variants and fragments are encompassed unless the context indicates that the term is used to refer to the wild-type protein only). MUC16 can be isolated from a human, or may be produced recombinantly or by synthetic methods.

The term “anti-MUC16 antibody” or “antibody that binds to MUC16” refers to any form of antibody or fragment thereof that binds, e.g., specifically binds, to MUC16, and encompasses monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, and biologically functional antibody fragments so long as they bind, e.g., specifically bind, to MUC16. Liu et al. ((2016) Ann Oncol. 27(11):2124-30) provides and is incorporated herein by reference for exemplary MUC16-binding sequences, including exemplary anti-MUC16 antibody sequences. In some embodiments, the anti-MUC16 antibody used in the ADCs disclosed herein is an internalizing antibody or internalizing antibody fragment.

The term “zinc transporter ZIP6” or “SLC39A6,” as used herein, refers to any native form of human SLC39A6. The term encompasses full-length SLC39A6 (e.g., UniProt Reference Sequence:Q13433; SEQ ID NO:54), as well as any form of human SLC39A6 that may result from cellular processing. The term also encompasses functional variants or fragments of human SLC39A6, including but not limited to splice variants, allelic variants, and isoforms that retain one or more biologic functions of human SLC39A6 (i.e., variants and fragments are encompassed unless the context indicates that the term is used to refer to the wild-type protein only). SLC39A6 can be isolated from a human, or may be produced recombinantly or by synthetic methods.

The term “anti-SLC39A6 antibody” or “antibody that binds to SLC39A6” refers to any form of antibody or fragment thereof that binds, e.g., specifically binds, to SLC39A6, and encompasses monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, and biologically functional antibody fragments so long as they bind, e.g., specifically bind, to SLC39A6. Sussman et al. ((2014) Mol Cancer Ther. 13(12):2991-3000) provides and is incorporated herein by reference for exemplary SLC39A6-binding sequences, including exemplary anti-SLC39A6 antibody sequences. In some embodiments, the anti-SLC39A6 antibody used in the ADCs disclosed herein is an internalizing antibody or internalizing antibody fragment.

The term “choline transporter-like protein 4” or “SLC44A4,” as used herein, refers to any native form of human SLC44A4. The term encompasses full-length SLC44A4 (e.g., UniProt Reference Sequence: Q53GD3; SEQ ID NO:55), as well as any form of human SLC44A4 that may result from cellular processing. The term also encompasses functional variants or fragments of human SLC44A4, including but not limited to splice variants, allelic variants, and isoforms that retain one or more biologic functions of human SLC44A4 (i.e., variants and fragments are encompassed unless the context indicates that the term is used to refer to the wild-type protein only). SLC44A4 can be isolated from a human, or may be produced recombinantly or by synthetic methods.

The term “anti-SLC44A4 antibody” or “antibody that binds to SLC44A4” refers to any form of antibody or fragment thereof that binds, e.g., specifically binds, to SLC44A4, and encompasses monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, and biologically functional antibody fragments so long as they bind, e.g., specifically bind, to SLC44A4. Mattie et al. ((2016) Mol Cancer Ther. 15(11):2679-87) provides and is incorporated herein by reference for exemplary SLC44A4-binding sequences, including exemplary anti-SLC44A4 antibody sequences. In some embodiments, the anti-SLC44A4 antibody used in the ADCs disclosed herein is an internalizing antibody or internalizing antibody fragment.

The term “metalloreductase STEAP1” or “STEAP1,” as used herein, refers to any native form of human STEAP1. The term encompasses full-length STEAP1 (e.g., UniProt Reference Sequence: Q9UHE8; SEQ ID NO:56), as well as any form of human STEAP1 that may result from cellular processing. The term also encompasses functional variants or fragments of human STEAP1, including but not limited to splice variants, allelic variants, and isoforms that retain one or more biologic functions of human STEAP1 (i.e., variants and fragments are encompassed unless the context indicates that the term is used to refer to the wild-type protein only). STEAP1 can be isolated from a human, or may be produced recombinantly or by synthetic methods.

The term “anti-STEAP1 antibody” or “antibody that binds to STEAP1” refers to any form of antibody or fragment thereof that binds, e.g., specifically binds, to STEAP1, and encompasses monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, and biologically functional antibody fragments so long as they bind, e.g., specifically bind, to STEAP1. WO 2008/052187 provides and is incorporated herein by reference for exemplary STEAP1-binding sequences, including exemplary anti-STEAP1 antibody sequences. In some embodiments, the anti-STEAP1 antibody used in the ADCs disclosed herein is an internalizing antibody or internalizing antibody fragment.

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As used herein, the term “specific,” “specifically binds,” and “binds specifically” refers to a binding reaction between an antibody or antigen binding fragment (e.g., an anti-HER2 antibody) and a target antigen (e.g., HER2) in a heterogeneous population of proteins and other biologics. Antibodies can be tested for specificity of binding by comparing binding to an appropriate antigen to binding to an irrelevant antigen or antigen mixture under a given set of conditions. If the antibody binds to the appropriate antigen with at least 2, 5, 7, and preferably 10 or more times more affinity than to the irrelevant antigen or antigen mixture, then it is considered to be specific. A “specific antibody” or a “target-specific antibody” is one that only binds the target antigen (e.g., HER2), but does not bind (or exhibits minimal binding) to other antigens. In certain embodiments, an antibody or antigen binding fragment that specifically binds a target antigen (e.g., HER2) has a K D of less than 1×10 −6 M, less than 1×10 −7 M, less than 1×10 −8 M, less than 1×10 −9 M, less than 1×10 −10 M, less than 1×10 −11 M, less than 1×10 −12 M, or less than 1×10 −13 M. In certain embodiments, the K D is 1 pM to 500 pM. In some embodiments, the K D is between 500 pM to 1 μM, 1 μM to 100 nM, or 100 mM to 10 nM.

The term “epitope” refers to the portion of an antigen capable of being recognized and specifically bound by an antibody. When the antigen is a polypeptide, epitopes can be formed from contiguous amino acids or noncontiguous amino acids juxtaposed by tertiary folding of the polypeptide. The epitope bound by an antibody may be identified using any epitope mapping technique known in the art, including X-ray crystallography for epitope identification by direct visualization of the antigen-antibody complex, as well as monitoring the binding of the antibody to fragments or mutated variations of the antigen, or monitoring solvent accessibility of different parts of the antibody and the antigen. Exemplary strategies used to map antibody epitopes include, but are not limited to, array-based oligo-peptide scanning, limited proteolysis, site-directed mutagenesis, high-throughput mutagenesis mapping, hydrogen-deuterium exchange, and mass spectrometry (see, e.g., Gershoni et al. (2007) 21:145-56; and Hager-Braun and Tomer (2005) Expert Rev Proteomics 2:745-56).

Competitive binding and epitope binning can also be used to determine antibodies sharing identical or overlapping epitopes. Competitive binding can be evaluated using a cross-blocking assay, such as the assay described in “Antibodies, A Laboratory Manual,” Cold Spring Harbor Laboratory, Harlow and Lane (1 st edition 1988, 2 nd edition 2014). In some embodiments, competitive binding is identified when a test antibody or binding protein reduces binding of a reference antibody or binding protein to a target antigen such as HER2 (e.g., a binding protein comprising CDRs and/or variable domains selected from those identified in Tables 2-4), by at least about 50% in the cross-blocking assay (e.g., 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.5%, or more, or any percentage in between), and/or vice versa. In some embodiments, competitive binding can be due to shared or similar (e.g., partially overlapping) epitopes, or due to steric hindrance where antibodies or binding proteins bind at nearby epitopes (see, e.g., Tzartos, Methods in Molecular Biology (Morris, ed. (1998) vol. 66, pp. 55-66)). In some embodiments, competitive binding can be used to sort groups of binding proteins that share similar epitopes. For example, binding proteins that compete for binding can be “binned” as a group of binding proteins that have overlapping or nearby epitopes, while those that do not compete are placed in a separate group of binding proteins that do not have overlapping or nearby epitopes.

The term “k on ” or “k a ” refers to the on-rate constant for association of an antibody to the antigen to form the antibody/antigen complex. The rate can be determined using standard assays, such as a surface plasmon resonance, biolayer inferometry, or ELISA assay.

The term “k off ” or “k d ” refers to the off-rate constant for dissociation of an antibody from the antibody/antigen complex. The rate can be determined using standard assays, such as a surface plasmon resonance, biolayer inferometry, or ELISA assay.

The term “K D ” refers to the equilibrium dissociation constant of a particular antibody-antigen interaction. K D is calculated by k a /k d . The rate can be determined using standard assays, such as a surface plasmon resonance, biolayer inferometry, or ELISA assay.

The term “p” or “drug loading” or “drug:antibody ratio” or “drug-to-antibody ratio” or “DAR” refers to the number of drug moieties per antibody or antigen binding fragment, i.e., drug loading, or the number of -L-D moieties per antibody or antigen binding fragment (Ab) in ADCs of Formula (I). In ADCs comprising a splicing modulator drug moiety, “p” refers to the number of splicing modulator compounds linked to the antibody or antigen binding fragment. For example, if two splicing modulator compounds (e.g., two compounds each having the structure of D1) are linked to an antibody or antigen binding fragment, p=2. In compositions comprising multiple copies of ADCs of Formula (I), “average p” refers to the average number of -L-D moieties per antibody or antigen binding fragment, also referred to as “average drug loading.”

A “linker” or “linker moiety” is used herein to refer to any chemical moiety that is capable of covalently joining a compound, usually a drug moiety such as a splicing modulator drug moiety, to another moiety such as an antibody or antigen binding fragment. Linkers can be susceptible to or substantially resistant to acid-induced cleavage, peptidase-induced cleavage, light-based cleavage, esterase-induced cleavage, and/or disulfide bond cleavage, at conditions under which the compound or the antibody remains active.

The term “agent” is used herein to refer to a chemical compound, a mixture of chemical compounds, a biological macromolecule, or an extract made from biological materials. The term “therapeutic agent” or “drug” refers to an agent that is capable of modulating a biological process and/or has biological activity. The splicing modulator compounds described herein are exemplary therapeutic agents.

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The term “chemotherapeutic agent” or “anti-cancer agent” is used herein to refer to all agents that are effective in treating cancer regardless of mechanism of action. Inhibition of metastasis or angiogenesis is frequently a property of a chemotherapeutic agent. Chemotherapeutic agents include antibodies, biological molecules, and small molecules, and encompass the splicing modulator compounds described herein. A chemotherapeutic agent may be a cytotoxic or cytostatic agent. The term “cytostatic agent” refers to an agent that inhibits or suppresses cell growth and/or multiplication of cells. The term “cytotoxic agent” refers to a substance that causes cell death primarily by interfering with a cell's expression activity and/or functioning.

As used herein, the terms “splicing modulator,” “spliceosome modulator,” or “splice modulator” refer to compounds that have anti-cancer activity by interacting with components of the spliceosome. In some embodiments, a splicing modulator alters the rate or form of splicing in a target cell. Splicing modulators that function as inhibitory agents, for example, are capable of decreasing uncontrolled cellular proliferation. In some embodiments, the splicing modulators may act by binding to the SF3b spliceosome complex. Such modulators may be natural compounds or synthetic compounds. Non-limiting examples of splicing modulators and categories of such modulators include pladienolide (e.g., pladienolide D or pladienolide B), pladienolide derivatives (e.g., pladienolide D or pladienolide B derivatives), herboxidiene, herboxidiene derivatives, spliceostatin, spliceostatin derivatives, sudemycin, or sudemycin derivatives. As used herein, the terms “derivative” and “analog” when referring to a splicing modulator, or the like, means any such compound that retains essentially the same, similar, or enhanced biological function or activity as the original compound but has an altered chemical or biological structure. In some embodiments, the splicing modulator is a pladienolide or pladienolide derivative.

As used herein, a “pladienolide derivative” refers to a compound which is structurally related to a member of the family of natural products known as the pladienolides and which retains one or more biological functions of the starting compound. Pladienolides were first identified in the bacteria Streptomyces platensis (Mizui et al. (2004) J Antibiot. 57:188-96) as being potently cytotoxic and resulting in cell cycle arrest in the G1 and G2/M phases of the cell cycle (e.g., Bonnal et al. (2012) Nat Rev Drug Dis 11:847-59). There are seven naturally occurring pladienolides, pladienolide A-G (Mizui et al. (2004) J Antibiot. 57:188-96; Sakai et al. (2004) J Antibiotics 57:180-7). U.S. Pat. Nos. 7,884,128 and 7,816,401 describe exemplary methods of synthesizing pladienolide B and D and are each incorporated herein by reference for such methods. Synthesis of pladienolide B and D may also be performed using the exemplary methods described in Kanada et al. ((2007) Angew Chem Int Ed. 46:4350-5). Kanada et al. and Intl. Pub. No. WO 2003/099813 describe exemplary methods for synthesizing E7107 (D11) (Compound 45 of WO 2003/099813) from Pladienolide D (11107D of WO 2003/099813). A corresponding U.S. Pat. No. 7,550,503 to Kotake et al. Each of these references is incorporated herein for the described synthesis methods.

As used herein, a “splicing modulator drug moiety” refers to the component of an ADC or composition that provides the structure of a splicing modulator compound, e.g., the splicing modulator (D) component in an ADC of Formula (I), or in a composition comprising -L-D.

As used herein, a “spliceosome” refers to a ribonucleoprotein complex that removes introns from one or more RNA segments, such as pre-mRNA segments.

The term “homolog” refers to a molecule which exhibits homology to another molecule, by for example, having sequences of chemical residues that are the same or similar at corresponding positions.

The term “inhibit” or “inhibition of,” as used herein, means to reduce by a measurable amount, and can include but does not require complete prevention or inhibition.

The term “target-negative,” “target antigen-negative,” or “antigen-negative” refers to the absence of target antigen expression by a cell or tissue. The term “target-positive,” “target antigen-positive,” or “antigen-positive” refers to the presence of target antigen expression. For example, a cell or a cell line that does not express a target antigen may be described as target-negative, whereas a cell or cell line that expresses a target antigen may be described as target-positive.

The term “bystander killing” or “bystander effect” refers to the killing of target-negative cells in the presence of target-positive cells, wherein killing of target-negative cells is not observed in the absence of target-positive cells. Cell-to-cell contact, or at least proximity between target-positive and target-negative cells, enables bystander killing. This type of killing is distinguishable from “off-target killing,” which refers to the indiscriminate killing of target-negative cells. “Off-target killing” may be observed in the absence of target-positive cells.

The terms “neoplastic disorder” and “cancer” are used herein interchangeably to refer to the presence of cells possessing characteristics typical of cancer-causing cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and/or certain morphological features. Often, cancer cells can be in the form of a tumor or mass, but such cells may exist alone within a subject, or may circulate in the blood stream as independent cells, such as leukemic or lymphoma cells. The terms “neoplastic disorder” and “cancer” includes all types of cancers and cancer metastases, including hematological malignancy, solid tumors, sarcomas, carcinomas and other solid and non-solid tumor cancers. Hematological malignancies may include B-cell malignancies, cancers of the blood (leukemias), cancers of plasma cells (myelomas, e.g., multiple myeloma), or cancers of the lymph nodes (lymphomas). Exemplary B-cell malignancies include chronic lymphocytic leukemia (CLL), follicular lymphoma, mantle cell lymphoma, and diffuse large B-cell lymphoma. Leukemias may include acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), chronic myelomonocytic leukemia (CMML), acute monocytic leukemia (AMoL), etc. Lymphomas may include Hodgkin's lymphoma and non-Hodgkin's lymphoma. Other hematologic malignancies may include myelodysplasia syndrome (MDS). Solid tumors may include carcinomas such as adenocarcinoma, e.g., breast cancer, pancreatic cancer, prostate cancer, colon or colorectal cancer, lung cancer, gastric cancer, cervical cancer, endometrial cancer, ovarian cancer, cholangiocarcinoma, glioma, melanoma, etc.

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The terms “tumor” and “neoplasm” refer to any mass of tissue that results from excessive cell growth or proliferation, either benign or malignant, including precancerous lesions.

The terms “tumor cell” and “neoplastic cell” are used interchangeably and refer to individual cells or the total population of cells derived from a tumor or neoplasm, including both non-tumorigenic cells and cancer stem cells. As used herein, the term “tumor cell” will be modified by the term “non-tumorigenic” when referring solely to those tumor cells lacking the capacity to renew and differentiate to distinguish those tumor cells from cancer stem cells.

The terms “subject” and “patient” are used interchangeably herein to refer to any animal, such as any mammal, including but not limited to, humans, non-human primates, rodents, and the like. In some embodiments, the mammal is a mouse. In some embodiments, the mammal is a human. In some embodiments, the subject is a mouse. In some embodiments, the subject is a human.

The term “co-administration” or administration “in combination with” one or more therapeutic agents includes concurrent administration and consecutive administration in any order.

A “pharmaceutical composition” refers to a preparation which is in such form as to permit administration and subsequently provide the intended biological activity of the active ingredient(s) and/or to achieve a therapeutic effect, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered. The pharmaceutical composition may be sterile.

A “pharmaceutical excipient” comprises a material such as an adjuvant, a carrier, pH-adjusting and buffering agents, tonicity adjusting agents, wetting agents, preservative, and the like.

“Pharmaceutically acceptable” means approved or approvable by a regulatory agency of the Federal or a state government, or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia, for use in animals, and more particularly in humans

A “pharmaceutically acceptable salt” is a salt that retains the desired biological activity of the parent compound and does not impart undesired toxicological effects. Examples of such salts are: (a) acid addition salts formed with inorganic acids, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and the like; and salts formed with organic acids, for example, acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic acid, polygalacturonic acid, and the like; and (b) salts formed from elemental anions such as chlorine, bromine, and iodine. See, e.g., Haynes et al. “Commentary: Occurrence of Pharmaceutically Acceptable Anions and Cations in the Cambridge Structural Database,” J Pharmaceutical Sciences, vol. 94, no. 10 (2005), and Berge et al. “Pharmaceutical Salts,” J Pharmaceutical Sciences, vol. 66, no. 1 (1977), which are incorporated by reference herein.

The term “effective amount,” as used herein, refers to the amount of a compound, ADC, or composition described herein (e.g., a splicing modulator or an ADC) that is sufficient to perform a specifically stated purpose, for example to produce a therapeutic effect after administration, such as a reduction in tumor growth rate or tumor volume, a reduction in a symptom of cancer, or some other indicia of treatment efficacy. An effective amount can be determined in a routine manner in relation to the stated purpose. The term “therapeutically effective amount” refers to an amount of a compound, an ADC, or composition described herein effective for detectable killing, reduction, and/or inhibition of the growth or spread of tumor cells, the size or number of tumors, and/or other measure of the level, stage, progression and/or severity of the cancer. The therapeutically effective amount can vary depending upon the intended application (in vitro or in vivo), or the subject and disease condition being treated, e.g., the weight and age of the subject, the severity of the disease condition, the manner of administration and the like, which can readily be determined by one of ordinary skill in the art. The term also applies to a dose that will induce a particular response in target cells, e.g., inhibition of cell growth. The specific dose may vary depending on, for example, the particular pharmaceutical composition, the subject and their age and existing health conditions or risk for health conditions, the dosing regimen to be followed, the severity of the disease, whether it is administered in combination with other agents, timing of administration, the tissue to which it is administered, and the physical delivery system in which it is carried. In the case of cancer, a therapeutically effective amount of ADC can reduce the number of cancer cells, reduce tumor size, inhibit (e.g., slow or stop) tumor metastasis, inhibit (e.g., slow or stop) tumor growth, and/or relieve one or more symptoms.

A “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.

As used herein, “to treat” or “therapeutic” and grammatically related terms, refer to any improvement of any consequence of disease, such as prolonged survival, less morbidity, and/or a lessening of side effects which result from an alternative therapeutic modality. As is readily appreciated in the art, full eradication of disease is encompassed but not required for a treatment act. “Treatment” or “treat,” as used herein, refers to the administration of a described ADC or composition to a subject, e.g., a patient. The treatment can be to cure, heal, alleviate, relieve, alter, remedy, ameliorate, palliate, improve or affect the disorder, the symptoms of the disorder or the predisposition toward the disorder, e.g., a cancer. In some embodiments, in addition to treating a subject with a condition, a composition disclosed herein can also be provided prophylactically to prevent or reduce the likelihood of developing that condition.

›Definitions · 10 of 64

In some embodiments, a labeled ADC is used. Suitable “labels” include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent moieties, chemiluminescent moieties, magnetic particles, and the like.

By “protein,” as used herein, is meant at least two covalently attached amino acids. The term encompasses polypeptides, oligopeptides, and peptides. In some embodiments, the two or more covalently attached amino acids are attached by a peptide bond. The protein may be made up of naturally occurring amino acids and peptide bonds, for example when the protein is made recombinantly using expression systems and host cells. Alternatively, the protein may include synthetic amino acids (e.g., homophenylalanine, citrulline, omithine, and norieucine), or peptidomimetic structures, i.e., “peptide or protein analogs,” such as peptoids. Peptoids are an exemplary class of peptidomimetics whose side chains are appended to the nitrogen atom of the peptide backbone, rather than to the α-carbons (as they are in amino acids), and have different hydrogen bonding and conformational characteristics in comparison to peptides (see, e.g., Simon et al. (1992) Proc Natl Acad Sci. USA 89:9367). As such, peptoids can be resistant to proteolysis or other physiological or storage conditions, and effective at permeating cell membranes. Such synthetic amino acids may be incorporated in particular when the antibody is synthesized in vitro by conventional methods well known in the art. In addition, any combination of peptidomimetic, synthetic and naturally occurring residues/structures can be used. “Amino acid” also includes imino acid residues, such as proline and hydroxyproline. The amino acid “R group” or “side chain” may be in either the (L)- or the (S)-configuration. In a specific embodiment, the amino acids are in the (L)- or (S)-configuration.

A “recombinant protein” is a protein made using recombinant techniques using any techniques and methods known in the art, i.e., through the expression of a recombinant nucleic acid. Methods and techniques for the production of recombinant proteins are well known in the art.

An “isolated” protein is unaccompanied by at least some of the material with which it is normally associated in its natural state, for example constituting at least about 5%, or at least about 50% by weight of the total protein in a given sample. It is understood that the isolated protein may constitute from 5% to 99.9% by weight of the total protein content depending on the circumstances. For example, the protein may be made at a significantly higher concentration through the use of an inducible promoter or high expression promoter, such that the protein is made at increased concentration levels. The definition includes the production of an antibody in a wide variety of organisms and/or host cells that are known in the art.

For amino acid sequences, sequence identity and/or similarity may be determined using standard techniques known in the art, including, but not limited to, the local sequence identity algorithm of Smith and Waterman (1981) Adv Appl Math. 2:482, the sequence identity alignment algorithm of Needleman and Wunsch (1970) J Mol Biol. 48:443, the search for similarity method of Pearson and Lipman (1988) Proc Nat Acad Sci. USA 85:2444, computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.), the Best Fit sequence program described by Devereux et al. (1984) Nucl Acid Res. 12:387-95, preferably using the default settings, or by inspection. Preferably, percent identity is calculated by FastDB based upon the following parameters: mismatch penalty of 1; gap penalty of 1; gap size penalty of 0.33; and joining penalty of 30 (“Current Methods in Sequence Comparison and Analysis,” Macromolecule Sequencing and Synthesis, Selected Methods and Applications, pp. 127-149 (1988), Alan R. Liss, Inc).

An example of a useful algorithm is PILEUP. PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pairwise alignments. It can also plot a tree showing the clustering relationships used to create the alignment. PILEUP uses a simplification of the progressive alignment method of Feng & Doolittle (1987) J Mol Evol. 35:351-60; the method is similar to that described by Higgins and Sharp (1989) CABIOS 5:151-3. Useful PILEUP parameters including a default gap weight of 3.00, a default gap length weight of 0.10, and weighted end gaps.

Another example of a useful algorithm is the BLAST algorithm, described in: Altschul et al. (1990) J Mol Biol. 215:403-10; Altschul et al. (1997) Nucl Acid Res. 25:3389-402; and Karin et al. (1993) Proc Natl Acad Sci. USA 90:5873-87. A particularly useful BLAST program is the WU-BLAST-2 program which was obtained from Altschul et al. (1996) Methods in Enzymology 266:460-80. WU-BLAST-2 uses several search parameters, most of which are set to the default values. The adjustable parameters are set with the following values: overlap span=I, overlap fraction=0.125, word threshold (T)=II. The HSP S and HSP S2 parameters are dynamic values and are established by the program itself depending upon the composition of the particular sequence and composition of the particular database against which the sequence of interest is being searched; however, the values may be adjusted to increase sensitivity.

An additional useful algorithm is gapped BLAST as reported by Altschul et al. (1997) Nucl Acid Res. 25:3389-402. Gapped BLAST uses BLOSUM-62 substitution scores; threshold T parameter set to 9; the two-hit method to trigger ungapped extensions, charges gap lengths of k a cost of 10+k; Xu set to 16, and Xg set to 40 for database search stage and to 67 for the output stage of the algorithms. Gapped alignments are triggered by a score corresponding to about 22 bits.

Generally, the amino acid homology, similarity, or identity between proteins disclosed herein and variants thereof, including variants of target antigens (such as HER2, CD138, EPHA2, MSLN, FOLH1, CDH6, CEACAM5, CFC1B, ENPP3, FOLR1, HAVCR1, KIT, MET, MUC16, SLC39A6, SLC44A4, or STEAP1) and variants of antibody variable domains (including individual variant CDRs), are at least 80% to the sequences depicted herein, e.g., homologies or identities of at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, almost 100%, or 100%.

›Definitions · 11 of 64

In a similar manner, “percent (%) nucleic acid sequence identity” with respect to the nucleic acid sequence of the antibodies and other proteins identified herein is defined as the percentage of nucleotide residues in a candidate sequence that are identical with the nucleotide residues in the coding sequence of the antigen binding protein. A specific method utilizes the BLASTN module of WU-BLAST-2 set to the default parameters, with overlap span and overlap fraction set to 1 and 0.125, respectively.

While the site or region for introducing an amino acid sequence variation is predetermined, the mutation per se need not be predetermined. For example, in order to optimize the performance of a mutation at a given site, random mutagenesis may be conducted at the target codon or region and the expressed antigen binding protein CDR variants screened for the optimal combination of desired activity. Techniques for making substitution mutations at predetermined sites in DNA having a known sequence are well known, for example, MI3 primer mutagenesis and PCR mutagenesis

“Alkyl” or “alkyl group,” as used herein, means a straight-chain, branched, or cyclic hydrocarbon chain that is completely saturated. In certain embodiments, alkyl groups may contain 1-8 carbon atoms (“C 1 -C 8 alkyl”). In certain embodiments, alkyl groups may contain 1-6 carbon atoms (“C 1 -C 6 alkyl”). In certain embodiments, alkyl groups contain 1-3 carbon atoms. In still other embodiments, alkyl groups contain 2-3 carbon atoms, and in yet other embodiments alkyl groups contain 1-2 carbon atoms.

“Alkylalkoxy,” as used herein, means an alkyl group substituted with an alkoxy group. “Alkoxy”, as used herein, refers to an alkyl group, as previously defined, attached to the principal carbon chain through an oxygen (“alkoxy”) atom.

“Alkylamino,” as used herein, means an alkyl group substituted with an amino group. “Amino,” as used herein, refers to —NH 2 , —NH(alkyl), or —N(alkyl)(alkyl).

“Alkylhydroxy,” as used herein, means an alkyl group substituted with an amino group. “Hydroxy” or “hydroxyl,” as used herein, refers to —OH.

“Alkylene” refers to a divalent radical of an alkyl group. For example, —CH 2 —, —CH 2 CH 2 —, —CH 2 CH 2 CH 2 —, —CH 2 CH 2 CH 2 CH 2 —, —CH 2 CH 2 CH 2 CH 2 CH 2 —, and —CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 — refer to methylene, ethylene, n-propylene, n-butylene, n-pentylene, and n-hexylene, respectively.

“Carbocycle,” as used herein, includes both aromatic (e.g., aryl) and non-aromatic (e.g., cycloalkyl) groups. In certain embodiments, carbocycle groups contain 3-10 carbon atoms (“3 to 10 membered carbocycle”). In certain embodiments, carbocycle groups contain 3-8 carbon atoms (“3 to 8 membered carbocycle”). In certain embodiments, carbocycle groups contain 3-6 carbon atoms (“3 to 6 membered carbocycle”). In certain embodiments, carbocycle groups contain 3-5 carbon atoms (“3 to 5 membered carbocycle”).

“Halogen” refers to a radical of any halogen, e.g., —F, —Cl, —Br, or —I.

The terms “heterocycle”, “heterocyclyl”, and “heterocyclic” as used herein, mean a monocyclic heterocycle, a bicyclic heterocycle, or a tricyclic heterocycle containing at least one heteroatom in the ring.

The monocyclic heterocycle is a 3-, 4-, 5-, 6-, 7, or 8-membered ring containing at least one heteroatom independently chosen from O, N, and S. In some embodiments, the heterocycle is a 3- or 4-membered ring containing one heteroatom chosen from O, N and S. In some embodiments, the heterocycle is a 5-membered ring containing zero or one double bond and one, two or three heteroatoms chosen from O, N and S. In some embodiments, the heterocycle is a 6-, 7-, or 8-membered ring containing zero, one or two double bonds and one, two or three heteroatoms chosen from O, N and S. Representative examples of monocyclic heterocycle include, but are not limited to, azetidinyl, azepanyl, aziridinyl, diazepanyl, 1,3-dioxanyl, 1,3-dioxolanyl, dihydropyranyl (including 3,4-dihydro-2H-pyran-6-yl), 1,3-dithiolanyl, 1,3-dithianyl, imidazolinyl, imidazolidinyl, isothiazolinyl, isothiazolidinyl, isoxazolinyl, isoxazolidinyl, morpholinyl, oxadiazolinyl, oxadiazolidinyl, oxazolinyl, oxazolidinyl, piperazinyl, piperidinyl, pyranyl, pyrazolinyl, pyrazolidinyl, pyrrolinyl, pyrrolidinyl, tetrahydrofuranyl, tetrahydropyranyl (including tetrahydro-2H-pyran-4-yl), tetrahydrothienyl, thiadiazolinyl, thiadiazolidinyl, thiazolinyl, thiazolidinyl, thiomorpholinyl, 1,1-dioxidothiomorpholinyl (thiomorpholine sulfone), thiopyranyl, and trithianyl.

The bicyclic heterocycles of the present disclosure may include a monocyclic heterocycle fused to an aryl group, or a monocyclic heterocycle fused to a monocyclic cycloalkyl, or a monocyclic heterocycle fused to a monocyclic cycloalkenyl, or a monocyclic heterocycle fused to a monocyclic heterocycle having a total of 5 to 12 ring atoms. Examples of bicyclic heterocycles include, but are not limited to, 3,4-dihydro-2H-pyranyl, 1,3-benzodioxolyl, 1,3-benzodithiolyl, 2,3-dihydro-1,4-benzodioxinyl, 2,3-dihydro-1-benzofuranyl, 2,3-dihydro-1-benzothienyl, 2,3-dihydro-1H-indolyl, and 1,2,3,4-tetrahydroquinolinyl.

The terms “heterocycle”, “heterocyclyl”, and “heterocyclic” encompass heteroaryls. “Heteroaryl” refers to a cyclic moiety having one or more closed rings, with one or more heteroatoms (oxygen, nitrogen or sulfur) in at least one of the rings, wherein at least one of the rings is aromatic, and wherein the ring or rings may independently be fused, and/or bridged. Examples include without limitation phenyl, thiophenyl, triazolyl, pyridinyl, pyrimidinyl, pyridazinyl, and pyrazinyl.

As described herein, compounds of the disclosure may contain “optionally substituted” moieties. In general, the term “substituted,” whether preceded by the term “optionally” or not, means that one or more hydrogens of the designated moiety are replaced with a suitable substituent. Unless otherwise indicated, an “optionally substituted” group may have a suitable substituent at each substitutable position of the group, and when more than one position in any given structure may be substituted with more than one substituent chosen from a specified group, the substituent may be either the same or different at each position. Combinations of substituents envisioned under this disclosure are preferably those that result in the formation of stable or chemically feasible compounds.

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One skilled in the art will be understand that “substitution” or “substituted with” or “absent” includes the implicit proviso that such substitution or absence is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution or absence results in a stable compound, e.g., which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc. For purposes of this disclosure, the heteroatoms such as nitrogen may have hydrogen substituents, and/or any permissible substituents of organic compounds described herein which satisfy the valences of the heteroatoms.

“Stable” refers to compounds that are not substantially altered chemically and/or physically when subjected to conditions to allow for their production, detection, and, in certain embodiments, their recovery, purification, and use for one or more of the purposes disclosed herein. In some embodiments, a stable compound or chemically feasible compound is one that is not substantially altered when kept at a temperature of 40° C. or less, in the absence of moisture or other chemically reactive conditions, for at least a week. In some embodiments, the compounds disclosed herein are stable.

Enantiomers taught herein may include “enantiomerically pure” isomers that comprise substantially a single enantiomer, for example, greater than or equal to 90%, 92%, 95%, 98%, or 99%, or equal to 100% of a single enantiomer, at a particular asymmetric center or centers. An “asymmetric center” or “chiral center” refers to a tetrahedral carbon atom that comprises four different substituents.

The compounds described herein may also contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute such compounds. For example, the compounds may be radiolabeled with radioactive isotopes, such as, for example, deuterium ( 2 H), tritium ( 3 H), carbon-13 ( 13 C), or carbon-14 ( 14 C). All isotopic variations of the compounds disclosed herein, whether radioactive or not, are intended to be encompassed within the scope of the present disclosure. In addition, all tautomeric forms of the compounds described herein are intended to be within the scope of the claimed disclosure.

Antibody-Drug Conjugates

The antibody-drug conjugate (ADC) compounds of the present disclosure include those with anti-cancer activity. In particular, the ADC compounds include an antibody or antigen binding fragment (including an antigen binding fragment thereof) conjugated (i.e., covalently attached by a linker) to a drug moiety (e.g., a splicing modulator), wherein the drug moiety when not conjugated to an antibody or antigen binding fragment has a cytotoxic or cytostatic effect. In various embodiments, the drug moiety when not conjugated to an antibody or antigen binding fragment is capable of binding to and/or interacting with the SF3b spliceosome complex. In various embodiments, the drug moiety when not conjugated to an antibody or antigen binding fragment is capable of modulating in vitro and/or in vivo RNA splicing. By targeting RNA splicing, in various embodiments, the drug moieties and ADCs disclosed herein are potent antiproliferative agents. In various embodiments, the drug moieties and ADCs disclosed herein can target both actively dividing and quiescent cells.

In various embodiments, the present disclosure is based, at least in part, on the discovery that certain biologically active splicing modulators may provide improved properties when used in ADCs. While a splicing modulator may show desirably improved features (e.g., robust SF3b spliceosome complex binding, potent modulation of RNA splicing) when used on its own, in various embodiments, the splicing modulator may exhibit fewer of the same desirably improved features when conjugated to an antibody or antigen binding fragment. Thus, the development and production of an ADC for use as a human therapeutic agent, e.g., as an oncologic agent, may require more than the identification of an antibody capable of binding to a desired target or targets and attaching to a drug used on its own to treat cancer. Linking the antibody to the drug may have significant effects on the activity of one or both of the antibody and the drug, effects which will vary depending on the type of linker and/or drug chosen. In some embodiments, therefore, the components of the ADC are selected to (i) retain one or more therapeutic properties exhibited by the antibody and drug moieties in isolation, (ii) maintain the specific binding properties of the antibody or antigen binding fragment; (iii) optimize drug loading and drug-to-antibody ratios; (iv) allow delivery, e.g., intracellular delivery, of the drug moiety via stable attachment to the antibody or antigen binding fragment; (v) retain ADC stability as an intact conjugate until transport or delivery to a target site; (vi) minimize aggregation of the ADC prior to or after administration; (vii) allow for the therapeutic effect, e.g., cytotoxic effect, of the drug moiety after cleavage or other release mechanism in the cellular environment; (viii) exhibit in vivo anti-cancer treatment efficacy comparable to or superior to that of the antibody and drug moieties in isolation; (ix) minimize off-target killing by the drug moiety; and/or (x) exhibit desirable pharmacokinetic and pharmacodynamics properties, formulatability, and toxicologic/immunologic profiles. Each of these properties may be needed to identify an improved ADC for therapeutic use (Ab et al. (2015) Mol Cancer Ther. 14:1605-13).

In various embodiments, the ADCs disclosed herein exhibit unexpectedly favorable properties in some or each of the categories listed above. For instance, in some embodiments, the ADC constructs disclosed herein exhibit surprisingly favorable drug loading, aggregation, and/or stability profiles, and/or preserve antibody binding function, drug activity, and/or improved bystander killing, while reducing off-target killing, as compared to ADCs comprising an alternate linker and/or drug moiety (e.g., an alternate splicing modulator). In some embodiments, ADC constructs disclosed herein demonstrate superior stability, activity, potency, or other effect (measured in vivo or in vitro) as compared to ADCs using an alternate linker and/or drug moiety (e.g., an alternate splicing modulator). In some embodiments, the ADC constructs disclosed herein exhibit in vivo treatment efficacy when administered as a single dose. In some embodiments, the ADC constructs disclosed herein are surprisingly stable as compared to ADCs using an alternate linker and/or drug moiety (e.g., an alternate splicing modulator).

›Definitions · 13 of 64

The ADC compounds of the present disclosure may selectively deliver an effective dose of a cytotoxic or cytostatic agent to cancer cells or to tumor tissue. It has been discovered that the disclosed ADCs have potent cytotoxic and/or cytostatic activity against cells expressing the respective target antigen (e.g., HER2, CD138, EPHA2, MSLN, FOLH1, CDH6, CEACAM5, CFC1B, ENPP3, FOLR1, HAVCR1, KIT, MET, MUC16, SLC39A6, SLC44A4, STEAP1). In some embodiments, the cytotoxic and/or cytostatic activity of the ADC is dependent on target antigen expression in a cell. In some embodiments, the disclosed ADCs are particularly effective at killing cancer cells expressing a target antigen while minimizing off-target killing. In some embodiments, the disclosed ADCs do not exhibit a cytotoxic and/or cytostatic effect on cancer cells that do not express a target antigen.

Exemplary HER2-expressing cancers include but are not limited to breast cancer, gastric cancer, bladder cancer, urothelial cell carcinoma, esophageal cancer, lung cancer (e.g., lung adenocarcinoma), uterine cancer (e.g., uterine serous endometrial carcinoma), salivary duct carcinoma, cervical cancer, endometrial cancer, and ovarian cancer (English et al. (2013) Mol Diagn Ther. 17:85-99).

Exemplary CD138-expressing cancers include but are not limited to intrathoracic cancer (e.g., lung cancer, mesothelioma), skin cancer (e.g., basal cell carcinoma, squamous cell carcinoma), head and neck cancer (e.g., laryngeal, hypopharynx, nasopharyngeal), breast cancer, urogenital cancer (e.g., cervical cancer, ovarian cancer, endometrial cancer, prostate cancer, bladder cancer, urothelial cancer), hematological malignancies (e.g., myeloma such as multiple myeloma, B-cell malignancies, Hodgkin's lymphoma), and thyroid cancer (Szatmdri et al. (2015) Dis Markers 2015:796052).

Exemplary EPHA2-expressing cancers include breast cancer, brain cancer, ovarian cancer, bladder cancer, pancreatic cancer, esophageal cancer, lung cancer, prostate cancer, melanoma, esophageal cancer, and gastric cancer (Tandon et al. (2011) Expert Opin Ther Targets 15(1):31-51).

In some embodiments, cleavage of an ADC releases the splicing modulator from the antibody or antigen binding fragment and linker. In some embodiments, the linker and/or splicing modulator is designed to facilitate bystander killing (the killing of neighboring cells). In some embodiments, the linker and/or splicing modulator is designed to facilitate bystander killing through cleavage after cellular internalization and diffusion of the linker-drug moiety and/or the drug moiety alone to neighboring cells. In some embodiments, the linker promotes cellular internalization. In some embodiments, the linker is designed to minimize cleavage in the extracellular environment and thereby reduce toxicity to off-target tissue (e.g., non-cancerous tissue), while preserving ADC binding to target tissue and bystander killing of cancerous tissue that does not express an antigen targeted by the antibody or antigen binding fragment of an ADC, but surrounds target cancer tissue expressing that antigen. In some embodiments, the drug moiety, or the catabolite of the drug moiety produced by cleavage of an ADC, is designed to facilitate uptake by target cells or by neighboring cells (i.e., cell permeable). Such drug moieties and catabolites may be referred to herein as “bystander active,” whereas drug moieties or catabolites with reduced cell permeability may be referred to as “bystander inactive.”

In some embodiments, the disclosed ADCs also demonstrate bystander killing activity, but low off-target cytotoxicity. Without being bound by theory, the bystander killing activity of an ADC may be particularly beneficial where its penetration into a solid tumor is limited and/or target antigen expression among tumor cells is heterogeneous. In some embodiments, an ADC comprising a cleavable linker is particularly effective at bystander killing and/or demonstrates improved bystander killing activity, relative to comparable treatment with an ADC comprising a non-cleavable linker. In some embodiments, the ADCs disclosed herein exhibit improved solubility and target cell penetrance over the drug moieties on their own. In some embodiments, the ADCs disclosed herein exhibit improved cytotoxicity over that of the drug moiety on its own. In some embodiments, ADCs disclosed herein use drug moieties that exhibit lower cytotoxicity, when evaluated as a stand-alone drug, yet are surprisingly better than ADCs comprising other drug moieties which have higher cytotoxicity when evaluated as a stand-alone drug. In some embodiments, cleavage and release of the splicing modulator improves cytotoxicity of the ADC, relative to comparable treatment with an ADC comprising a non-cleavable linker. In other embodiments, cleavage and release of the splicing modulator is not required for an ADC to possess a desirable biological activity. In some embodiments, an ADC comprising a non-cleavable linker having increased spacer length (e.g., ADL12) provides the same or similar cytotoxicity relative to comparable treatment with an ADC comprising a cleavable linker (e.g., ADL1, ADL5) and surprisingly superior cytotoxicity relative to comparable treatment with an ADC comprising a shorter non-cleavable linker. In some embodiments, an ADC comprising a non-cleavable linker having increased spacer length without a carbonyl group (e.g., ADL12) provides the same or similar cytotoxicity relative to comparable treatment with an ADC comprising a cleavable linker (e.g., ADL1, ADL5) and surprisingly superior cytotoxicity relative to comparable treatment with an ADC comprising a non-cleavable linker having the same or similar spacer length with a carbonyl group (e.g., ADL10). In some embodiments, the removal of a carbonyl group from a non-cleavable MC linker (e.g., ADL12) can result in a greater than 50-fold, greater than 75-fold, greater than 100-fold, greater than 150-fold, or greater than 200-fold increase in cytotoxicity, relative to comparable treatment with an ADC comprising an unmodified non-cleavable MC linker (e.g., ADL10). In some embodiments, the removal of a carbonyl group from a non-cleavable MC linker (e.g., ADL12) and increased spacer length (e.g., the addition of at least one spacer unit) can result in a greater than 50-fold, greater than 75-fold, greater than 100-fold, greater than 150-fold, or greater than 200-fold increase in cytotoxicity, relative to comparable treatment with an ADC comprising an unmodified non-cleavable MC linker (e.g., ADL10).

›Definitions · 14 of 64

Provided herein are ADC compounds comprising an antibody or antigen binding fragment thereof (Ab) which targets a tumor cell, a splicing modulator drug moiety (D), and a linker moiety (L) that covalently attaches Ab to D. In certain aspects, the antibody or antigen binding fragment is able to bind to a tumor-associated antigen (e.g., HER2, CD138, EPHA2, MSLN, FOLH1, CDH6, CEACAM5, CFC1B, ENPP3, FOLR1, HAVCR1, KIT, MET, MUC16, SLC39A6, SLC44A4, STEAP1) with high specificity and high affinity. In certain embodiments, the antibody or antigen binding fragment is internalized into a target cell upon binding, e.g., into a degradative compartment in the cell. In various embodiments, ADCs that internalize upon binding to a target cell, undergo degradation, and release the splicing modulator drug moiety to kill cancer cells may be used. The splicing modulator drug moiety may be released from the antibody and/or the linker moiety of the ADC by enzymatic action, hydrolysis, oxidation, or any other mechanism.

An exemplary ADC has Formula (I):

Ab-(L-D) p (I)

wherein Ab=an antibody or antigen binding fragment, L=a linker moiety, D=a splicing modulator drug moiety, and p=the number of splicing modulator drug moieties per antibody or antigen binding fragment.

In certain preferred embodiments, the drug-targeting moiety for use in the described ADCs and compositions is an antibody or antigen binding fragment. Other exemplary drug-targeting moieties for use in the described ADCs and compositions are also provided and described herein. In some embodiments, a drug-targeting moiety can be any one of a variety of cell-binding agents and non-antibody scaffolds. In some embodiments, the drug-targeting moiety is a cell-binding agent. As used herein, the term “cell-binding agent” refers to any agent that is capable of binding to an animal (e.g., human) cell and delivering a drug moiety (e.g., a splicing modulator drug moiety as disclosed herein). The term encompasses the exemplary antibodies and antigen binding fragments disclosed herein (e.g., monoclonal antibodies and fragments thereof such as Fabs and scFVs). The term further encompasses exemplary cell-binding agents such as DARPins, duobodies, bicyclic peptides, nanobodies, centyrins, MSH (melanocyte-stimulating hormone), receptor-Fc fusion molecules, T-cell receptor structures, steroid hormones such as androgens and estrogens, growth factors, colony-stimulating factors such as EGF, and other non-antibody scaffolds. In various embodiments, non-antibody scaffolds can broadly fall into two structural classes, namely domain-sized compounds (approximately 6-20 kDa) and constrained peptides (approximately 2-4 kDa). Exemplary domain-sized scaffolds include but are not limited to affibodies, affilins, anticalins, atrimers, DARPins, FN3 scaffolds (e.g., adnectins and centyrins), fynomers, Kunitz domains, pronectins, O-bodies, and receptor-Fc fusion proteins, whereas exemplary constrained peptides include avimers, bicyclic peptides, and Cys-knots. In some embodiments, the drug-targeting moiety used in the described ADCs and compositions is selected from an affibody, an affilin, an anticalin, an atrimer, a DARPin, a FN3 scaffold such as an adnectin or a centyrin, a fynomer, a Kunitz domain, a pronectin, an O-body, an avimer, a bicyclic peptide, and a Cys-knot. In some embodiments, the drug-targeting moiety used in the described ADCs and compositions is a receptor-Fc fusion protein, e.g., a HER2-Fc chimeric fusion protein. Non-antibody scaffolds are reviewed, e.g., in Vazquez-Lombardi et al. (2015) Drug Dis Today 20(10):1271-83.

Antibodies

The antibody or antigen binding fragment (Ab) of Formula (I) includes within its scope any antibody or antigen binding fragment that specifically binds to a target antigen on a cancer cell. The antibody or antigen binding fragment may bind to a target antigen with a dissociation constant (K D ) of ≤1 mM, ≤100 nM or ≤10 nM, or any amount in between, as measured by, e.g., BIAcore® analysis. In certain embodiments, the K D is 1 pM to 500 pM. In some embodiments, the K D is between 500 pM to 1 μM, 1 μM to 100 nM, or 100 mM to 10 nM.

In some embodiments, the antibody or antigen binding fragment is a four-chain antibody (also referred to as an immunoglobulin or a full-length or intact antibody), comprising two heavy chains and two light chains. In some embodiments, the antibody or antigen binding fragment is a two-chain half body (one light chain and one heavy chain), or an antigen binding fragment of an immunoglobulin. In some embodiments, the antibody or antigen binding fragment is an antigen binding fragment of an immunoglobulin that retains the ability to bind a target cancer antigen and/or provide a function of an immunoglobulin.

In some embodiments, the antibody or antigen binding fragment is an antibody or antigen binding fragment thereof. In some embodiments, the antibody or antigen binding fragment is an internalizing antibody or internalizing antigen binding fragment thereof. In some embodiments, the internalizing antibody or internalizing antigen binding fragment thereof binds to a target cancer antigen expressed on the surface of a cell and enters the cell upon binding. In some embodiments, the splicing modulator drug moiety of the ADC is released from the antibody or antigen binding fragment of the ADC after the ADC enters and is present in a cell expressing the target cancer antigen (i.e., after the ADC has been internalized), e.g., by cleavage, by degradation of the antibody or antigen binding fragment, or by any other suitable release mechanism.

Amino acid sequences of exemplary antibodies of the present disclosure are set forth in Tables 2-4.

In various embodiments, an ADC disclosed herein may comprise any set of heavy and light chain variable domains listed in the tables above, or the set of six CDR sequences from the heavy and light chain set, e.g., by transplanting the six CDRs into a chosen human donor antibody framework. In various embodiments, an ADC disclosed herein may comprise amino acid sequences that are homologous to the sequences listed in the tables above, so long as the ADC retains the ability to bind to its target cancer antigen (e.g., with a K D of less than 1×10 −8 M) and retains one or more functional properties of the ADCs disclosed herein (e.g., ability to internalize, modulate RNA splicing, inhibit cell growth, etc.).

›Definitions · 15 of 64

In some embodiments, the ADC further comprises human heavy and light chain constant domains or fragments thereof. For instance, the ADC may comprise a human IgG heavy chain constant domain (such as an IgG1) and a human kappa or lambda light chain constant domain. In various embodiments, the antibody or antigen binding fragment of the described ADCs comprises a human immunoglobulin G subtype 1 (IgG1) heavy chain constant domain with a human Ig kappa light chain constant domain.

In various other embodiments, the target cancer antigen for an ADC is human epidermal growth factor receptor 2 (HER2).

In various embodiments, the anti-HER2 antibody or antigen binding fragment thereof comprises three heavy chain CDRs and three light chain CDRs as follows: heavy chain CDR1 (HCDR1) consisting of SEQ ID NO:1, heavy chain CDR2 (HCDR2) consisting of SEQ ID NO:2, heavy chain CDR3 (HCDR3) consisting of SEQ ID NO:3; light chain CDR1 (LCDR1) consisting of SEQ ID NO:4, light chain CDR2 (LCDR2) consisting of SEQ ID NO:5, and light chain CDR3 (LCDR3) consisting of SEQ ID NO:6, as defined by the Kabat numbering system.

In various embodiments, the anti-HER2 antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:19, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:20. In some embodiments, the anti-HER2 antibody or antigen binding fragment thereof comprises the heavy chain variable region amino acid sequence of SEQ ID NO:19 and the light chain variable region amino acid sequence of SEQ ID NO:20, or sequences that are at least 95% identical to the disclosed sequences. In some embodiments, the anti-HER2 antibody or antigen binding fragment thereof has a heavy chain variable region amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:19 and/or a light chain variable region amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:20.

In various embodiments, the anti-HER2 antibody or antigen binding fragment thereof is an internalizing antibody or internalizing antigen binding fragment. In various embodiments, the anti-HER2 antibody comprises a human IgG1 heavy chain constant domain and a human Ig kappa light chain constant domain.

In various embodiments, the anti-HER2 antibody comprises the heavy chain amino acid sequence of SEQ ID NO:19 or a sequence that is at least 95% identical to SEQ ID NO:19, and the light chain amino acid sequence of SEQ ID NO:20 or a sequence that is at least 95% identical to SEQ ID NO:20. In particular embodiments, the anti-HER2 antibody comprises the heavy chain amino acid sequence of SEQ ID NO:19 and the light chain amino acid sequence of SEQ ID NO:20, or sequences that are at least 95% identical to the disclosed sequences. In some embodiments, the anti-HER2 antibody has a heavy chain amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:19 and a light chain amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:20. In various embodiments, the anti-HER2 antibody is trastuzumab, or an antigen binding fragment thereof.

In various embodiments, the anti-HER2 antibody or antigen binding fragment thereof comprises the three heavy chain CDRs and three light chain CDRs of trastuzumab or wherein the CDRs include no more than one, two, three, four, five, or six amino acid additions, deletions or substitutions of HCDR1 (SEQ ID NO:1), HCDR2 (SEQ ID NO:2), HCDR3 (SEQ ID NO:3); LCDR1 (SEQ ID NO:4), LCDR2 (SEQ ID NO:5), and LCDR3 (SEQ ID NO:6).

In various other embodiments, the target cancer antigen for an ADC is human syndecan-1 (CD138).

In various embodiments, the anti-CD138 antibody or antigen binding fragment thereof comprises three heavy chain CDRs and three light chain CDRs as follows: heavy chain CDR1 (HCDR1) consisting of SEQ ID NO:7, heavy chain CDR2 (HCDR2) consisting of SEQ ID NO:8, heavy chain CDR3 (HCDR3) consisting of SEQ ID NO:9; light chain CDR1 (LCDR1) consisting of SEQ ID NO:10, light chain CDR2 (LCDR2) consisting of SEQ ID NO:11, and light chain CDR3 (LCDR3) consisting of SEQ ID NO:12, as defined by the Kabat numbering system.

In various embodiments, the anti-CD138 antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:21, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:22. In some embodiments, the anti-CD138 antibody or antigen binding fragment thereof comprises the heavy chain variable region amino acid sequence of SEQ ID NO:21 and the light chain variable region amino acid sequence of SEQ ID NO:22, or sequences that are at least 95% identical to the disclosed sequences. In some embodiments, the anti-CD138 antibody or antigen binding fragment thereof has a heavy chain variable region amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:21 and/or a light chain variable region amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:22.

In various embodiments, the anti-CD138 antibody or antigen binding fragment thereof is an internalizing antibody or internalizing antigen binding fragment. In various embodiments, the anti-CD138 antibody comprises a murine IgG2a heavy chain constant domain and a murine Ig kappa light chain constant domain. In various embodiments, the anti-CD138 antibody comprises a human IgG2a heavy chain constant domain and a human Ig kappa light chain constant domain.

In various embodiments, the anti-CD138 antibody comprises the heavy chain amino acid sequence of SEQ ID NO:21 or a sequence that is at least 95% identical to SEQ ID NO:21, and the light chain amino acid sequence of SEQ ID NO:22 or a sequence that is at least 95% identical to SEQ ID NO:22. In particular embodiments, the anti-CD138 antibody comprises the heavy chain amino acid sequence of SEQ ID NO:21 and the light chain amino acid sequence of SEQ ID NO:22, or sequences that are at least 95% identical to the disclosed sequences. In some embodiments, the anti-CD138 antibody has a heavy chain amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:21 and a light chain amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:22. In various embodiments, the anti-CD138 antibody is B-B4, or an antigen binding fragment thereof.

›Definitions · 16 of 64

In various embodiments, the anti-CD138 antibody or antigen binding fragment thereof comprises the three heavy chain CDRs and three light chain CDRs of B-B4 or wherein the CDRs include no more than one, two, three, four, five, or six amino acid additions, deletions or substitutions of HCDR1 (SEQ ID NO:7), HCDR2 (SEQ ID NO:8), HCDR3 (SEQ ID NO:9); LCDR1 (SEQ ID NO:10), LCDR2 (SEQ ID NO:11), and LCDR3 (SEQ ID NO:12).

In various other embodiments, the target cancer antigen for an ADC is human ephrin type-A receptor 2 (EPHA2).

In various embodiments, the anti-EPHA2 antibody or antigen binding fragment thereof comprises three heavy chain CDRs and three light chain CDRs as follows: heavy chain CDR1 (HCDR1) consisting of SEQ ID NO:13, heavy chain CDR2 (HCDR2) consisting of SEQ ID NO:14, heavy chain CDR3 (HCDR3) consisting of SEQ ID NO:15; light chain CDR1 (LCDR1) consisting of SEQ ID NO:16, light chain CDR2 (LCDR2) consisting of SEQ ID NO:17, and light chain CDR3 (LCDR3) consisting of SEQ ID NO:18, as defined by the Kabat numbering system.

In various embodiments, the anti-EPHA2 antibody or antigen binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:23, and a light chain variable region comprising the amino acid sequence of SEQ ID NO:24. In some embodiments, the anti-EPHA2 antibody or antigen binding fragment thereof comprises the heavy chain variable region amino acid sequence of SEQ ID NO:23 and the light chain variable region amino acid sequence of SEQ ID NO:24, or sequences that are at least 95% identical to the disclosed sequences. In some embodiments, the anti-EPHA2 antibody or antigen binding fragment thereof has a heavy chain variable region amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:23 and/or a light chain variable region amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:24.

In various embodiments, the anti-EPHA2 antibody or antigen binding fragment thereof is an internalizing antibody or internalizing antigen binding fragment. In various embodiments, the anti-EPHA2 antibody comprises a human IgG1 heavy chain constant domain and a human Ig kappa light chain constant domain.

In various embodiments, the anti-EPHA2 antibody comprises the heavy chain amino acid sequence of SEQ ID NO:23 or a sequence that is at least 95% identical to SEQ ID NO:23, and the light chain amino acid sequence of SEQ ID NO:24 or a sequence that is at least 95% identical to SEQ ID NO:24. In particular embodiments, the anti-EPHA2 antibody comprises the heavy chain amino acid sequence of SEQ ID NO:23 and the light chain amino acid sequence of SEQ ID NO:24, or sequences that are at least 95% identical to the disclosed sequences. In some embodiments, the anti-EPHA2 antibody has a heavy chain amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:23 and a light chain amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:24. In some embodiments, the anti-EPHA2 antibody comprises a heavy chain encoded by the nucleotide sequence of SEQ ID NO:23; and a light chain encoded by the nucleotide sequence of SEQ ID NO:24. In various embodiments, the anti-EPHA2 antibody is 1C1, or an antigen binding fragment thereof.

In various embodiments, the anti-EPHA2 antibody or antigen binding fragment thereof comprises the three heavy chain CDRs and three light chain CDRs of 1C1 or wherein the CDRs include no more than one, two, three, four, five, or six amino acid additions, deletions or substitutions of HCDR1 (SEQ ID NO:13), HCDR2 (SEQ ID NO:14), HCDR3 (SEQ ID NO:15); LCDR1 (SEQ ID NO:16), LCDR2 (SEQ ID NO:17), and LCDR3 (SEQ ID NO:18).

In various embodiments, amino acid substitutions are of single residues. Insertions usually will be on the order of from about 1 to about 20 amino acid residues, although considerably larger insertions may be tolerated as long as biological function is retained (e.g., binding to a target antigen). Deletions usually range from about 1 to about 20 amino acid residues, although in some cases deletions may be much larger. Substitutions, deletions, insertions, or any combination thereof may be used to arrive at a final derivative or variant. Generally, these changes are done on a few amino acids to minimize the alteration of the molecule, particularly the immunogenicity and specificity of the antigen binding protein. However, larger changes may be tolerated in certain circumstances. Conservative substitutions are generally made in accordance with the following chart depicted as Table 6.

Substantial changes in function or immunological identity are made by selecting substitutions that are less conservative than those shown in Table 6. For example, substitutions may be made which more significantly affect the structure of the polypeptide backbone in the area of the alteration, for example the alpha-helical or beta-sheet structure; the charge or hydrophobicity of the molecule at the target site; or the bulk of the side chain. The substitutions which in general may produce the greatest changes in the polypeptide's properties are those in which (a) a hydrophilic residue, e.g., seryl or threonyl, is substituted for (or by) a hydrophobic residue, e.g., leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine or proline is substituted for (or by) any other residue; (c) a residue having an electropositive side chain, e.g., lysyl, arginyl, or histidyl, is substituted for (or by) an electronegative residue, e.g., glutamyl or aspartyl; or (d) a residue having a bulky side chain, e.g., phenylalanine, is substituted for (or by) one not having a side chain, e.g., glycine.

In various embodiments where variant antibody sequences are used in an ADC, the variants typically exhibit the same qualitative biological activity and will elicit the same immune response, although variants may also be selected to modify the characteristics of the antigen binding proteins as needed. Alternatively, the variant may be designed such that the biological activity of the antigen binding protein is altered. For example, glycosylation sites may be altered or removed.

›Definitions · 17 of 64

Various antibodies may be used with the ADCs used herein to target cancer cells. As shown below, the linker-payloads in the ADCs disclosed herein are surprisingly effective with different tumor antigen-targeting antibodies. Suitable antigens expressed on tumor cells but not healthy cells, or expressed on tumor cells at a higher level than on healthy cells, are known in the art, as are antibodies directed against them. These antibodies may be used with the linkers and splicing modulator payloads disclosed herein. In some embodiments, the antibody or antigen binding fragment targets HER2, and the HER2-targeting antibody or antigen binding fragment is trastuzumab. In some embodiments, the antibody or antigen binding fragment targets CD138, and the CD138-targeting antibody or antigen binding fragment is B-B4. In some embodiments, the antibody or antigen binding fragment targets EPHA2, and the EPHA2-targeting antibody or antigen binding fragment is 1C1. In some embodiments, while the disclosed linkers and splicing modulator payloads are surprisingly effective with several different tumor-targeting antibodies, HER2-targeting antibodies such as trastuzumab, CD138-targeting antibodies such as B-B4, and EPHA2-targeting antibodies such as 1C1 provided particularly improved drug:antibody ratio, aggregation level, stability (i.e., in vitro and in vivo stability), tumor targeting (i.e., cytotoxicity, potency), and/or treatment efficacy. Improved treatment efficacy can be measured in vitro or in vivo, and may include reduced tumor growth rate and/or reduced tumor volume.

In certain embodiments, alternate antibodies to the same targets or antibodies to different antigen targets are used and provide at least some of the favorable functional properties described above (e.g., improved stability, improved tumor targeting, improved treatment efficacy, etc.). In some embodiments, some or all of these favorable functional properties are observed when the disclosed linkers and splicing modulator payloads are conjugated to an alternate HER2-, CD138-, or EPHA2-targeting antibody or antigen binding fragment. In some other embodiments, some or all of these favorable functional properties are observed when the disclosed linkers and splicing modulator payloads are conjugated to a HER2-targeting antibody or antigen binding fragment. In some embodiments, the antibody or antigen binding fragment targets HER2. In some embodiments, the HER2-targeting antibody or antigen binding fragment is trastuzumab. In some other embodiments, some or all of these favorable functional properties are observed when the disclosed linkers and splicing modulator payloads are conjugated to a CD138-targeting antibody or antigen binding fragment. In some embodiments, the antibody or antigen binding fragment targets CD138. In some embodiments, the CD138-targeting antibody or antigen binding fragment is B-B4. In some other embodiments, some or all of these favorable functional properties are observed when the disclosed linkers and splicing modulator payloads are conjugated to an EPHA2-targeting antibody or antigen binding fragment. In some embodiments, the antibody or antigen binding fragment targets EPHA2. In some embodiments, the EPHA2-targeting antibody or antigen binding fragment is 1C1.

Linkers

In various embodiments, the linker in an ADC is stable extracellularly in a sufficient manner to be therapeutically effective. In some embodiments, the linker is stable outside a cell, such that the ADC remains intact when present in extracellular conditions (e.g., prior to transport or delivery into a cell). The term “intact,” used in the context of an ADC, means that the antibody or antigen binding fragment remains attached to the drug moiety (e.g., the splicing modulator). As used herein, “stable,” in the context of a linker or ADC comprising a linker, means that no more than 20%, no more than about 15%, no more than about 10%, no more than about 5%, no more than about 3%, or no more than about 1% of the linkers (or any percentage in between) in a sample of ADC are cleaved (or in the case of an overall ADC are otherwise not intact) when the ADC is present in extracellular conditions. In some embodiments, the linkers and/or ADCs disclosed herein are surprisingly stable compared to alternate linkers and/or ADCs with alternate linkers and/or splicing modulator payloads. In some embodiments, the ADCs disclosed herein can remain intact for more than about 48 hours, more than 60 hours, more than about 72 hours, more than about 84 hours, or more than about 96 hours.

Whether a linker is stable extracellularly can be determined, for example, by including an ADC in plasma for a predetermined time period (e.g., 2, 4, 6, 8, 16, 24, 48, or 72 hours) and then quantifying the amount of free drug moiety present in the plasma. Stability may allow the ADC time to localize to target tumor cells and prevent the premature release of the drug moiety, which could lower the therapeutic index of the ADC by indiscriminately damaging both normal and tumor tissues. In some embodiments, the linker is stable outside of a target cell and releases the drug moiety from the ADC once inside of the cell, such that the drug can bind to its target (e.g., to the SF3b spliceosome complex). Thus, an effective linker will: (i) maintain the specific binding properties of the antibody or antigen binding fragment; (ii) allow delivery, e.g., intracellular delivery, of the drug moiety via stable attachment to the antibody or antigen binding fragment; (iii) remain stable and intact until the ADC has been transported or delivered to its target site; and (iv) allow for the therapeutic effect, e.g., cytotoxic effect, of the drug moiety after cleavage or alternate release mechanism.

Linkers may impact the physico-chemical properties of an ADC. As many cytotoxic agents are hydrophobic in nature, linking them to the antibody with an additional hydrophobic moiety may lead to aggregation. ADC aggregates are insoluble and often limit achievable drug loading onto the antibody, which can negatively affect the potency of the ADC. Protein aggregates of biologics, in general, have also been linked to increased immunogenicity. As shown below, linkers disclosed herein result in ADCs with low aggregation levels and desirable levels of drug loading.

›Definitions · 18 of 64

A linker may be “cleavable” or “non-cleavable” (Ducry and Stump (2010) Bioconjugate Chem. 21:5-13). Cleavable linkers are designed to release the drug moiety (e.g., the splicing modulator) when subjected to certain environment factors, e.g., when internalized into the target cell, whereas non-cleavable linkers generally rely on the degradation of the antibody or antigen binding fragment itself.

In some embodiments, the linker is a non-cleavable linker. In some embodiments, the splicing modulator drug moiety of the ADC is released by degradation of the antibody or antigen binding fragment. Non-cleavable linkers tend to remain covalently associated with at least one amino acid of the antibody and the drug upon internalization by and degradation within the target cell. Numerous exemplary non-cleavable linkers are described herein, and others are known in the art. Exemplary non-cleavable linkers may comprise thioether, cyclohexyl, N-succinimidyl 4-(N-maleimidomethyl) cyclohexane-1 carboxylate (SMCC), or N-hydroxysuccinimide (NHS), one or more polyethylene glycol (PEG) moieties, e.g., 1, 2, 3, 4, 5, or 6 PEG moieties, or one or more alkyl moieties.

In some embodiments, the linker is a cleavable linker. A cleavable linker refers to any linker that comprises a cleavable moiety. As used herein, the term “cleavable moiety” refers to any chemical bond that can be cleaved. Suitable cleavable chemical bonds are well known in the art and include, but are not limited to, acid labile bonds, protease/peptidase labile bonds, photolabile bonds, disulfide bonds, and esterase labile bonds. Linkers comprising a cleavable moiety can allow for the release of the splicing modulator drug moiety from the ADC via cleavage at a particular site in the linker.

In some embodiments, the linker is cleavable under intracellular conditions, such that cleavage of the linker sufficiently releases the splicing modulator drug moiety from the antibody or antigen binding fragment in the intracellular environment to activate the drug and/or render the drug therapeutically effective. In some embodiments, the splicing modulator drug moiety is not cleaved from the antibody or antigen binding fragment until the ADC enters a cell that expresses an antigen specific for the antibody or antigen binding fragment of the ADC, and the splicing modulator drug moiety is cleaved from the antibody or antigen binding fragment upon entering the cell. In some embodiments, the linker comprises a cleavable moiety that is positioned such that no part of the linker or the antibody or antigen binding fragment remains bound to the splicing modulator drug moiety upon cleavage. Exemplary cleavable linkers include acid labile linkers, protease/peptidase-sensitive linkers, photolabile linkers, dimethyl-, disulfide-, or sulfonamide-containing linkers.

In some embodiments, the linker is a pH-sensitive linker, and is sensitive to hydrolysis at certain pH values. Typically, the pH-sensitive linker is cleavable under acidic conditions. This cleavage strategy generally takes advantage of the lower pH in the endosomal (pH ˜5-6) and lysosomal (pH ˜4.8) intracellular compartments, as compared to the cytosol (pH ˜7.4), to trigger hydrolysis of an acid labile group in the linker, such as a hydrazone (Jain et al. (2015) Pharm Res 32:3526-40). In some embodiments, the linker is an acid labile and/or hydrolyzable linker. For example, an acid labile linker that is hydrolyzable in the lysosome, and contains an acid labile group (e.g., a hydrazone, a semicarbazone, a thiosemicarbazone, a cis-aconitic amide, an orthoester, an acetal, a ketal, or the like) can be used. See, e.g., U.S. Pat. Nos. 5,122,368; 5,824,805; 5,622,929; Dubowchik and Walker (1999) Pharm Therapeutics 83:67-123; Neville et al. (1989) Biol Chem. 264:14653-61. Such linkers are relatively stable under neutral pH conditions, such as those in the blood, but are unstable at below pH 5.5 or 5.0, the approximate pH of the lysosome. In certain embodiments, the hydrolyzable linker is a thioether linker (such as, e.g., a thioether attached to the therapeutic agent via an acylhydrazone bond) (see, e.g., U.S. Pat. No. 5,622,929).

In some embodiments, the linker is cleavable under reducing conditions. In some embodiments, the linker is cleavable in the presence of a reducing agent, such as glutathione or dithiothreitol. In some embodiments, the linker is a cleavable disulfide linker or a cleavable sulfonamide linker.

In some embodiments, the linker is a cleavable disulfide linker. A variety of disulfide linkers are known in the art, including, for example, those that can be formed using SATA (N-succinimidyl-5-acetylthioacetate), SPDP (N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB (N-succinimidyl-3-(2-pyridyldithio)butyrate) and SMPT (N-succinimidyloxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)toluene), SPDB and SMPT. See, e.g., Thorpe et al. (1987) Cancer Res. 47:5924-31; Wawrzynczak et al., In Immunoconjugates: Antibody Conjugates in Radioimagery and Therapy of Cancer (C. W. Vogel ed., Oxford U. Press, 1987). See also U.S. Pat. No. 4,880,935. Disulfide linkers are typically used to exploit the abundance of intracellular thiols, which can facilitate the cleavage of their disulfide bonds. The intracellular concentrations of the most abundance intracellular thiol, reduced glutathione, are generally in the range of 1-10 nM, which is about 1,000-fold higher than that of the most abundant low-molecular thiol in the blood (i.e., cysteine) at about 5 μM (Goldmacher et al., In Cancer Drug Discovery and Development: Antibody-Drug Conjugates and Immunotoxins (G. L. Phillips ed., Springer, 2013)). The intracellular enzymes of the protein disulfide isomerase family may also contribute to the intracellular cleavage of a disulfide linker. As used herein, a cleavable disulfide linker refers to any linker that comprises a cleavable disulfide moiety. The term “cleavable disulfide moiety” refers to a disulfide bond that can be cleaved and/or reduced, e.g., by a thiol or enzyme.

›Definitions · 19 of 64

In some embodiments, the linker is a cleavable sulfonamide linker. As used herein, a cleavable sulfonamide linker refers to any linker that comprises a cleavable sulfonamide moiety. The term “cleavable sulfonamide moiety” refers to a sulfonamide group, i.e., sulfonyl group connected to an amine group, wherein the sulfur-nitrogen bond can be cleaved.

In some embodiments, the linker may be a dendritic type linker for covalent attachment of more than one drug moiety to an antibody or antigen binding fragment through a branching, multifunctional linker moiety. See, e.g., Sun et al. (2002) Bioorg Med Chem Lett. 12:2213-5; Sun et al. (2003) Bioorg Med Chem. 11:1761-8. Dendritic linkers can increase the molar ratio of drug to antibody, i.e., drug loading, which is related to the potency of the ADC. Thus, where an antibody or antigen binding fragment bears only one reactive cysteine thiol group, for example, a multitude of splicing modulator drug moieties may be attached through a dendritic linker. In some embodiments, the linker moiety or linker-drug moiety may be attached to the antibody or antigen binding fragment via reduced disulfide bridging chemistry or limited lysine utilization technology. See, e.g., Intl. Publ. Nos. WO 2013/173391 and VO 2013/173393.

In some embodiments, the linker is cleavable by a cleaving agent, e.g., an enzyme, that is present in the intracellular environment (e.g., within a lysosome or endosome or caveola). The linker can be, e.g., a peptide linker that is cleaved by an intracellular peptidase or protease enzyme, including, but not limited to, a lysosomal or endosomal protease.

In some embodiments, the linker is a cleavable peptide linker. As used herein, a cleavable peptide linker refers to any linker that comprises a cleavable peptide moiety. The term “cleavable peptide moiety” refers to any chemical bond linking amino acids (natural or synthetic amino acid derivatives) that can be cleaved by an agent that is present in the intracellular environment. For instance, a linker may comprise a valine-alanine (Val-Ala) sequence, or a valine-citrulline (Val-Cit) sequence that is cleavable by a peptidase such as cathepsin, e.g., cathepsin B. In some embodiments, a linker may comprise a glutamic acid-valine-citrulline (Glu-Val-Cit) sequence. In some embodiments, the linker is an enzyme-cleavable linker and a cleavable peptide moiety in the linker is cleavable by the enzyme. In some embodiments, the cleavable peptide moiety is cleavable by a lysosomal enzyme, e.g., cathepsin. In some embodiments, the linker is a cathepsin-cleavable linker. In some embodiments, the cleavable peptide moiety in the linker is cleavable by a lysosomal cysteine cathepsin, such as cathepsin B, C, F, H, K, L, O, S, V, X, or W. In some embodiments, the cleavable peptide moiety is cleavable by cathepsin B. An exemplary dipeptide that may be cleaved by cathepsin B is valine-citrulline (Val-Cit) (Dubowchik et al. (2002) Bioconjugate Chem. 13:855-69).

In some embodiments, the linker or the cleavable peptide moiety in the linker comprises an amino acid unit. In some embodiments, the amino acid unit allows for cleavage of the linker by a protease, thereby facilitating release of the splicing modulator drug moiety from the ADC upon exposure to one or more intracellular proteases, such as one or more lysosomal enzymes (Doronina et al. (2003) Nat Biotechnol. 21:778-84; Dubowchik and Walker (1999) Pharm Therapeutics 83:67-123). Exemplary amino acid units include, but are not limited to, dipeptides, tripeptides, tetrapeptides, and pentapeptides. Exemplary dipeptides include, but are not limited to, valine-alanine (Val-Ala), valine-citrulline (Val-Cit), alanine-asparagine (Ala-Asn), alanine-phenylalanine (Ala-Phe), phenylalanine-lysine (Phe-Lys), alanine-lysine (Ala-Lys), alanine-valine (Ala-Val), valine-lysine (Val-Lys), lysine-lysine (Lys-Lys), phenylalanine-citrulline (Phe-Cit), leucine-citrulline (Leu-Cit), isoleucine-citrulline (Ile-Cit), tryptophan-citrulline (Trp-Cit), and phenylalanine-alanine (Phe-Ala). Exemplary tripeptides include, but are not limited to, alanine-alanine-asparagine (Ala-Ala-Asn), glycine-valine-citrulline (Gly-Val-Cit), glycine-glycine-glycine (Gly-Gly-Gly), phenylalanine-phenylalanine-lysine (Phe-Phe-Lys), glutamic acid-valine-citrulline (Glu-Val-Cit) (see, e.g., Anami et al. (2018) Nat Comm. 9:2512, which is incorporated herein by reference for exemplary linkers comprising Glu-Val-Cit), and glycine-phenylalanine-lysine (Gly-Phe-Lys). Other exemplary amino acid units include, but are not limited to, Gly-Phe-Gly-Gly (SEQ ID NO:34), Gly-Phe-Leu-Gly (SEQ ID NO:35), Ala-Leu-Ala-Leu (SEQ ID NO:36), Phe-N 9 -tosyl-Arg, and Phe-N 9 -Nitro-Arg, as described in, e.g., U.S. Pat. No. 6,214,345. In some embodiments, the amino acid unit in the linker comprises Val-Ala. In some embodiments, the amino acid unit in the linker comprises Val-Cit. In some embodiments, the amino acid unit in the linker comprises Glu-Val-Cit. An amino acid unit may comprise amino acid residues that occur naturally and/or minor amino acids and/or non-naturally occurring amino acid analogs, such as citrulline. Amino acid units can be designed and optimized for enzymatic cleavage by a particular enzyme, for example, a tumor-associated protease, a lysosomal protease such as cathepsin B, C, D, or S, or a plasmin protease.

In some embodiments, the linker is a cleavable β-glucuronide linker. As used herein, a cleavable β-glucuronide linker refers to any linker that comprises a cleavable β-glucuronide moiety. An exemplary cleavable β-glucuronide linker comprises the structure:

The term “cleavable β-glucuronide moiety” refers to a glycosidic bond that can be cleaved by an agent having β-glucuronidase activity. In some embodiments, the linker comprises a glycosidic bond that can be cleaved by a β-glucuronidase. A β-glucuronidase is a UDP-glucuronosyl transferase that catalyzes the hydrolysis of the glycosidic bond of glucuronides with β-configuration.

›Definitions · 20 of 64

In some embodiments, an ADC disclosed herein comprises a cleavable β-glucuronide moiety in the linker that is cleavable by the enzyme. In some embodiments, the cleavable β-glucuronide moiety in the linker is cleavable by a lysosomal enzyme, e.g., a β-glucuronidase. In some embodiments, the linker is a β-glucuronidase-cleavable linker. In some embodiments, the cleavable β-glucuronide moiety in the linker allows for cleavage of the linker by a β-glucuronidase after internalization of the ADC, thereby facilitating release of the drug moiety from the ADC in the cellular environment.

In some embodiments, the linker in any of the ADCs disclosed herein may comprise at least one spacer unit joining the antibody or antigen binding fragment to the drug moiety (e.g., the splicing modulator drug moiety). In some embodiments, a spacer unit between the antibody or antigen binding fragment and cleavable moiety, when present, joins a cleavage site (e.g., a cleavable peptide moiety) in the linker to the antibody or antigen binding fragment. In some embodiments, a spacer unit between the drug moiety and cleavable moiety, when present, joins a cleavage site (e.g., a cleavable peptide moiety) in the linker to the drug moiety. In some embodiments, no cleavage site is present, and the spacer unit is used to link the antibody or antigen binding fragment to the drug moiety.

In some embodiments, the linker, and/or spacer unit in the linker, is substantially hydrophilic. A hydrophilic linker may be used to reduce the extent to which the drug may be pumped out of resistant cancer cells through multiple drug resistance (MDR) or functionally similar transporters. In some embodiments, a hydrophilic linker may include one or more polyethylene glycol (PEG) moieties, e.g., 1, 2, 3, 4, 5, or 6 PEG moieties. In some embodiments, the linker comprises 2 PEG moieties.

In some embodiments, the spacer unit in the linker comprises one or more PEG moieties. In some embodiments, the spacer unit comprises one or more -(PEG) m -, and m is an integer from 1 to 10 (i.e., m may be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In some embodiments, m ranges from 1 to 10; from 2 to 8; from 2 to 6; from 2 to 5; from 2 to 4; or from 2 to 3. In some embodiments, m is 2. In some embodiments, the spacer unit comprises (PEG) 2 , (PEG) 3 , (PEG) 4 , (PEG) 5 , (PEG) 6 , (PEG) 7 , (PEG) 8 , (PEG) 9 , or (PEG) 10 . In some embodiments, the spacer unit comprises (PEG) 2 .

In some embodiments, the spacer unit in the linker comprises an alkyl moiety. In some embodiments, the spacer unit comprises one or more —(CH 2 ) n —, and n is an integer from 1 to 10 (i.e., n may be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In some embodiments, n ranges from 1 to 10; from 2 to 8; from 2 to 6; from 2 to 5; from 2 to 4; or from 2 to 3. In some embodiments, n is 2. In some embodiments, n is 5. In some embodiments, n is 6. In some embodiments, the spacer unit comprises (CH 2 ) 2 , (CH 2 ) 3 , (CH 2 ) 4 , (CH 2 ) 5 , (CH 2 ) 6 , (CH 2 ) 7 , (CH 2 ) 8 , (CH 2 ) 9 , or (CH 2 ) 10 . In some embodiments, the spacer unit comprises (CH 2 ) 2 (“Et”). In some embodiments, the spacer unit comprises (CH 2 ) 6 (“Hex”). In some embodiments, the spacer unit comprises (CH 2 ) 2 —O—(CH 2 ) 2 (“Et-O-Et”).

A spacer unit may be used, for example, to link the antibody or antigen binding fragment to the drug moiety, either directly or indirectly. In some embodiments, the spacer unit links the antibody or antigen binding fragment to the splicing modulator drug moiety directly. In some embodiments, the antibody or antigen binding fragment and the splicing modulator drug moiety are attached via a spacer unit comprising one or more PEG moieties (e.g., (PEG) 2 ), or one or more alkyl moieties (e.g., (CH 2 ) 2 , (CH 2 ) 6 , or (CH 2 ) 2 —O—(CH 2 ) 2 ). In some embodiments, the spacer unit links the antibody or antigen binding fragment to the splicing modulator drug moiety indirectly. In some embodiments, the spacer unit links the antibody or antigen binding fragment to the splicing modulator drug moiety indirectly through a cleavable moiety (e.g., a cleavable peptide or a cleavable β-glucuronide) and/or an attachment moiety to join the spacer unit to the antibody or antigen binding fragment, e.g., a maleimide moiety.

The spacer unit, in various embodiments, attaches to the antibody or antigen binding fragment (i.e., the antibody or antigen binding fragment) via a maleimide (Mal) moiety.

A spacer unit that attaches to the antibody or antigen binding fragment via a Mal is referred to herein as a “Mal-spacer unit.” The term “Mal” or “maleimide moiety,” as used herein, means a compound that contains a maleimide group and that is reactive with a sulfhydryl group, e.g., a sulfhydryl group of a cysteine residue on the antibody or antigen binding fragment. Other functional groups that are reactive with sulfhydryl groups (thiols) include, but are not limited to, iodoacetamide, bromoacetamide, vinyl pyridine, disulfide, pyridyl disulfide, isocyanate, and isothiocyanate. In some embodiments, the Mal-spacer unit is reactive with a cysteine residue on the antibody or antigen binding fragment. In some embodiments, the Mal-spacer unit is joined to the antibody or antigen binding fragment via the cysteine residue. In some embodiments, the Mal-spacer unit comprises a PEG moiety. In some embodiments, the Mal-spacer unit comprises an alkyl moiety.

In certain embodiments, the linker comprises the Mal-spacer unit and a cleavable peptide moiety. In some embodiments, the cleavable peptide moiety comprises an amino acid unit. In some embodiments, the amino acid unit comprises Val-Cit. In some embodiments, the amino acid unit comprises Val-Ala. In some embodiments, the amino acid unit comprises Glu-Val-Cit. In some embodiments, the linker comprises the Mal-spacer unit and Val-Cit. In some embodiments, the linker comprises the Mal-spacer unit and Val-Ala. In some embodiments, the linker comprises the Mal-spacer unit and Val-Cit, wherein the Mal-spacer unit comprises a maleimidocaproyl (MC). In some embodiments, the linker comprises the Mal-spacer unit and Val-Ala, wherein the Mal-spacer unit comprises a maleimidocaproyl (MC). In some embodiments, the linker comprises the Mal-spacer unit and a cleavable β-glucuronide moiety.

›Definitions · 21 of 64

In some embodiments, the linker comprises the structure: Mal-spacer unit. In some embodiments, the Mal-spacer unit comprises a maleimidocaproyl (MC). In some embodiments, the linker comprises the structure: MC. In some embodiments, the linker comprises the structure: Mal-(CH 2 ) 2 (“Mal-Et”). In some embodiments, the linker comprises the structure: Mal-(CH 2 ) 6 (“Mal-Hex”). In some embodiments, the linker comprises the structure: Mal-(CH 2 ) 2 —O—(CH 2 ) 2 (“Mal-Et-O-Et”). In some embodiments, the linker comprises the structure: Mal-(PEG) 2 . In some embodiments, the linker comprises the structure: Mal-(PEG) 2 -CO.

In various embodiments, the Mal-spacer unit attaches the antibody or antigen binding fragment to a cleavable peptide moiety. In some embodiments, the linker comprises Mal-spacer unit-peptide. In some embodiments, the linker comprises the structure: Mal-spacer unit-Val-Cit. In some embodiments, the Mal-spacer unit comprises a maleimidocaproyl (MC). In some embodiments, the linker comprises the structure: MC-Val-Cit.

In some embodiments, the linker comprises the structure: Mal-spacer unit-Val-Ala. In some embodiments, the Mal-spacer unit comprises a maleimidocaproyl (MC). In some embodiments, the linker comprises the structure: MC-Val-Ala.

In various embodiments, the Mal-spacer unit attaches the antibody or antigen binding fragment to a cleavable β-glucuronide moiety. In some embodiments, the linker comprises Mal-spacer unit-β-glucuronide. In some embodiments, the linker comprises MC-β-glucuronide.

In various embodiments, the cleavable moiety in the linker is joined directly to the splicing modulator drug moiety. In other embodiments, a spacer unit is used to attach the cleavable moiety in the linker to the splicing modulator drug moiety. In various embodiments, the splicing modulator is attached to the cleavable moiety in the linker by a spacer unit.

A spacer unit may be “self-immolative” or “non-self-immolative.” A “non-self-immolative” spacer unit is one in which part or all of the spacer unit remains bound to the splicing modulator drug moiety upon cleavage of the linker. Examples of non-self-immolative spacer units include, but are not limited to, a glycine spacer unit and a glycine-glycine spacer unit. Non-self-immolative spacer units may eventually degrade over time but do not readily release a linked native drug moiety entirely under cellular conditions. A “self-immolative” spacer unit allows for release of the native drug moiety under intracellular conditions. A “native drug” or “native drug moiety” is one where no part of the spacer unit or other chemical modification remains after cleavage/degradation of the spacer unit.

Self-immolation chemistry is known in the art and could be readily selected for the disclosed ADCs. In various embodiments, the spacer unit attaching the cleavable moiety in the linker to the splicing modulator drug moiety is self-immolative, and undergoes self-immolation concurrently with or shortly before/after cleavage of the cleavable moiety under intracellular conditions. In some embodiments, the splicing modulator is attached to the cleavable moiety in the linker by a self-immolative spacer unit. In certain embodiments, the splicing modulator is attached to the cleavable moiety in the linker by a self-immolative spacer unit, the cleavable moiety comprises Val-Cit, and a maleimidocaproyl (MC) joins the cleavable moiety to the antibody or antigen binding fragment. In certain embodiments, the splicing modulator is attached to the cleavable moiety in the linker by a self-immolative spacer unit, the cleavable moiety comprises Val-Ala, and a maleimidocaproyl (MC) joins the cleavable moiety to the antibody or antigen binding fragment. In certain embodiments, the splicing modulator is attached to the cleavable moiety in the linker by a self-immolative spacer unit, the cleavable moiety comprises Glu-Val-Cit, and a maleimidocaproyl (MC) joins the cleavable moiety to the antibody or antigen binding fragment. In certain embodiments, the splicing modulator is joined to the antibody or antigen binding fragment via a Mal-spacer unit (e.g., MC) in the linker joined to a Val-Cit cleavable moiety and a pABC or pAB self-immolative spacer unit. In certain other embodiments, the splicing modulator is joined to the antibody or antigen binding fragment via a Mal-spacer unit (e.g., MC) in the linker joined to a Val-Ala cleavable moiety and a pABC or pAB self-immolative spacer unit. In certain other embodiments, the splicing modulator is joined to the antibody or antigen binding fragment via a Mal-spacer unit (e.g., MC) in the linker joined to a Glu-Val-Cit cleavable moiety and a pABC or pAB self-immolative spacer unit.

In certain embodiments, the self-immolative spacer unit in the linker comprises a p-aminobenzyl unit. In some embodiments, a p-aminobenzyl alcohol (pABOH) is attached to an amino acid unit or other cleavable moiety in the linker via an amide bond, and a carbamate, methylcarbamate, or carbonate is made between the pABOH and the drug moiety (Hamann et al. (2005) Expert Opin Ther Patents 15:1087-103). In some embodiments, the self-immolative spacer unit is or comprises p-aminobenzyloxycarbonyl (pABC). Without being bound by theory, it is thought that the self-immolation of pABC involves a spontaneous 1,6-elimination reaction (Jain et al. (2015) Pharm Res. 32:3526-40).

In various embodiments, the structure of the p-aminobenzyloxycarbonyl (pABC) used in the disclosed ADCs is shown below:

In various embodiments, the self-immolative spacer unit attaches the cleavable moiety in the linker to the splicing modulator. In some embodiments, the self-immolative spacer unit is pABC. In some embodiments, the pABC attaches the cleavable moiety in the linker to the splicing modulator. In some embodiments, the pABC undergoes self-immolation upon cleavage of the cleavable moiety, and the splicing modulator is released from the ADC in its native, active form.

In some embodiments, an anti-HER2 antibody or antigen binding fragment is joined to the splicing modulator by a linker comprising MC-Val-Cit-pABC. In other embodiments, an anti-HER2 antibody or antigen binding fragment is joined to the splicing modulator by a linker comprising MC-Val-Ala-pABC.

›Definitions · 22 of 64

In some embodiments, an anti-CD138 antibody or antigen binding fragment is joined to the splicing modulator by a linker comprising MC-Val-Cit-pABC. In other embodiments, an anti-CD138 antibody or antigen binding fragment is joined to the splicing modulator by a linker comprising MC-Val-Ala-pABC.

In some embodiments, an anti-EPHA2 antibody or antigen binding fragment is joined to the splicing modulator by a linker comprising MC-Val-Cit-pABC. In other embodiments, an anti-EPHA2 antibody or antigen binding fragment is joined to the splicing modulator by a linker comprising MC-Val-Ala-pABC.

In some embodiments, the pABC undergoes self-immolation upon cleavage of a cleavable peptide moiety in the linker. In some embodiments, the cleavable peptide moiety comprises an amino acid unit. In some embodiments, the linker comprises amino acid unit-pABC. In some embodiments, the amino acid unit is Val-Cit. In some embodiments, the linker comprises Val-Cit-pABC. In some embodiments, the amino acid unit is Val-Ala. In some embodiments, the linker comprises Val-Ala-pABC. In some embodiments, the amino acid unit is Glu-Val-Cit. In some embodiments, the linker comprises Glu-Val-Cit-pABC. In some embodiments, the amino acid unit is Ala-Ala-Asn. In some embodiments, the linker comprises Ala-Ala-Asn-pABC.

In some embodiments, the pABC undergoes self-immolation upon cleavage of a cleavable β-glucuronide moiety in the linker. In some embodiments, the linker comprises β-glucuronide-pABC.

In certain embodiments, the self-immolative spacer unit in the linker comprises a p-aminobenzyl unit. In some embodiments, the self-immolative spacer unit in the linker comprises a p-aminobenzyl (pAB). In some embodiments, the self-immolation of pAB involves a spontaneous 1,6-elimination reaction.

In various embodiments, the structure of the p-aminobenzyl (pAB) used in the disclosed ADCs is shown below:

In various embodiments, the self-immolative spacer unit attaches the cleavable moiety in the linker to the splicing modulator. In some embodiments, the self-immolative spacer unit is pAB. In some embodiments, the pAB attaches the cleavable moiety in the linker to the splicing modulator. In some embodiments, the pAB undergoes self-immolation upon cleavage of the cleavable moiety, and the splicing modulator is released from the ADC in its native, active form.

In some embodiments, an anti-HER2 antibody or antigen binding fragment is joined to the splicing modulator by a linker comprising MC-Val-Cit-pAB. In other embodiments, an anti-HER2 antibody or antigen binding fragment is joined to the splicing modulator by a linker comprising MC-Val-Ala-pAB.

In some embodiments, an anti-CD138 antibody or antigen binding fragment is joined to the splicing modulator by a linker comprising MC-Val-Cit-pAB. In other embodiments, an anti-CD138 antibody or antigen binding fragment is joined to the splicing modulator by a linker comprising MC-Val-Ala-pAB.

In some embodiments, an anti-EPHA2 antibody or antigen binding fragment is joined to the splicing modulator by a linker comprising MC-Val-Cit-pAB. In other embodiments, an anti-EPHA2 antibody or antigen binding fragment is joined to the splicing modulator by a linker comprising MC-Val-Ala-pAB.

In some embodiments, the pAB undergoes self-immolation upon cleavage of a cleavable peptide moiety in the linker. In some embodiments, the cleavable peptide moiety comprises an amino acid unit. In some embodiments, the linker comprises amino acid unit-pAB. In some embodiments, the amino acid unit is Val-Cit. In some embodiments, the linker comprises Val-Cit-pAB. In some embodiments, the amino acid unit is Val-Ala. In some embodiments, the linker comprises Val-Ala-pAB. In some embodiments, the amino acid unit is Glu-Val-Cit. In some embodiments, the linker comprises Glu-Val-Cit-pAB. In some embodiments, the amino acid unit is Ala-Ala-Asn. In some embodiments, the linker comprises Ala-Ala-Asn-pAB.

In some embodiments, the pAB undergoes self-immolation upon cleavage of a cleavable β-glucuronide moiety in the linker. In some embodiments, the linker comprises β-glucuronide-pAB.

In some other embodiments, the splicing modulator is attached to the cleavable moiety in the linker by a non-self-immolative spacer unit. In certain embodiments, the splicing modulator is attached to the cleavable moiety in the linker by a non-self-immolative spacer unit, the cleavable moiety comprises Val-Cit, and a maleimidocaproyl (MC) joins the cleavable moiety to the antibody or antigen binding fragment. In certain embodiments, the splicing modulator is attached to the cleavable moiety in the linker by a non-self-immolative spacer unit, the cleavable moiety comprises Val-Ala, and a maleimidocaproyl (MC) joins the cleavable moiety to the antibody or antigen binding fragment.

In various aspects, the antibody or antigen binding fragment of the ADC is conjugated to the splicing modulator drug moiety via a linker, wherein the linker comprises a Mal-spacer unit (e.g., MC), a cleavable amino acid unit, and a pABC. In some embodiments, the spacer unit comprises an alkyl moiety. In some embodiments, the Mal-spacer unit comprises a maleimidocaproyl (MC). In some embodiments, the linker comprises Mal-spacer unit-amino acid unit-pABC. In some embodiments, the linker comprises MC-amino acid unit-pABC. In some embodiments, the linker comprises MC-Val-Cit-pABC. In some embodiments, the linker comprises MC-Val-Ala-pABC. In some embodiments, the linker comprises MC-Glu-Val-Cit-pABC. In some embodiments, the linker comprises MC-Ala-Ala-Asn-pABC.

In various other aspects, the antibody or antigen binding fragment of the ADC is conjugated to the splicing modulator drug moiety via a linker, wherein the linker comprises a Mal-spacer unit (e.g., MC), a cleavable amino acid unit, and a pAB. In some embodiments, the spacer unit comprises an alkyl moiety. In some embodiments, the Mal-spacer unit comprises a maleimidocaproyl (MC). In some embodiments, the linker comprises Mal-spacer unit-amino acid unit-pAB. In some embodiments, the linker comprises MC-amino acid unit-pAB. In some embodiments, the linker comprises MC-Val-Cit-pAB. In some embodiments, the linker comprises MC-Val-Ala-pAB. In some embodiments, the linker comprises MC-Glu-Val-Cit-pAB. In some embodiments, the linker comprises MC-Ala-Ala-Asn-pAB.

›Definitions · 23 of 64

In still other aspects, the antibody or antigen binding fragment of the ADC is conjugated to the splicing modulator drug moiety via a linker, wherein the linker comprises a Mal-spacer unit (e.g., MC), a cleavable β-glucuronide, and a pAB. In some embodiments, the linker comprises Mal-spacer unit-β-glucuronide-pAB. In some embodiments, the linker comprises MC-β-glucuronide-pAB.

In various embodiments, the ADC compound has Formula (I):

Ab-(L-D) p (I)

wherein Ab is an antibody or antigen binding fragment which targets a neoplastic cell;

D is a splicing modulator;

L is a linker that covalently attaches Ab to D; and

p is an integer from 1 to 15.

In some embodiments, the antibody or antigen binding fragment (Ab) of the ADC is conjugated to the splicing modulator drug moiety via a linker, wherein the linker is any of the linkers disclosed or incorporated by reference herein, or comprises one or more components of any of the linkers disclosed or incorporated by reference herein.

In some embodiments, the linker comprises a cleavable moiety that is positioned such that no part of the linker or the antibody or antigen binding fragment remains bound to the splicing modulator after cleavage. In some embodiments, the cleavable moiety is a cleavable peptide moiety, e.g., an amino acid unit such as Val-Cit or Val-Ala. In some embodiments, the amino acid unit or linker comprises Val-Cit. In some embodiments, the amino acid unit or linker comprises Val-Ala. In some embodiments, the amino acid unit or linker comprises Glu-Val-Cit.

In some embodiments, the linker comprises at least one spacer unit joining the antibody or antigen binding fragment to the cleavable moiety. In some embodiments, the linker comprises at least one spacer unit joining the antibody or antigen binding fragment to the drug moiety. In some embodiments, the spacer unit or linker comprises at least one alkyl moiety.

In some embodiments, a spacer unit in the linker attaches to the antibody or antigen binding fragment via a Mal moiety (“Mal-spacer unit”). In some embodiments, the Mal-spacer unit comprises at least one alkyl moiety. In some embodiments, the linker comprises a maleimidocaproyl (MC). In some embodiments, the linker comprises Mal-(CH 2 ) 2 (“Mal-Et”). In some embodiments, the linker comprises Mal-(CH 2 ) 6 (“Mal-Hex”). In some embodiments, the linker comprises Mal-(CH 2 ) 2 —O—(CH 2 ) 2 (“Mal-Et-O-Et”). In some embodiments, the linker comprises Mal-(PEG) 2 -CO. In some embodiments, the Mal-spacer unit attaches the antibody or antigen binding fragment to the drug moiety.

In some embodiments, the Mal-spacer unit or linker comprises Mal-(PEG) 2 , Mal-(PEG) 3 , Mal-(PEG) 4 , Mal-(PEG) 5 , Mal-(PEG) 6 , Mal-(PEG) 7 , or Mal-(PEG) 8 . In some embodiments, the Mal-spacer unit or linker comprises Mal-(PEG) 2 . In some embodiments, the Mal-spacer unit or linker comprises Mal-(PEG) 2 -CO, Mal-(PEG) 3 -CO, Mal-(PEG) 4 -CO, Mal-(PEG) 5 -CO, Mal-(PEG) 6 -CO, Mal-(PEG) 7 -CO, or Mal-(PEG) 8 -CO.

In some embodiments, the Mal-spacer unit or linker comprises Mal-(PEG) 2 -CO. In some embodiments, the Mal-spacer unit or linker comprises Mal-(PEG) 2 -CO and at least one additional spacer unit. In some embodiments, the Mal-(PEG) 2 -CO attaches the antibody or antigen binding fragment to the drug moiety. In some embodiments, linker comprises or consists of Mal-(PEG) 2 -CO. An example of a “Mal-(PEG) 2 -CO” linker is also referred to herein as “ADL2” or an “ADL2” linker.

In some embodiments, the Mal-spacer unit or linker comprises MC. In some embodiments, the Mal-spacer unit or linker comprises MC and at least one additional spacer unit. In some embodiments, the MC attaches the antibody or antigen binding fragment to the drug moiety. In some embodiments, the linker comprises or consists of MC. An example of an “MC” linker is also referred to herein as “ADL10” or an “ADL10” linker.

In some embodiments, the Mal-spacer unit or linker comprises Mal-(CH 2 ) 6 (“Mal-Hex”). In some embodiments, the Mal-spacer unit or linker comprises Mal-Hex and at least one additional spacer unit. In some embodiments, the Mal-Hex attaches the antibody or antigen binding fragment to the drug moiety. In some embodiments, the linker comprises Mal-Hex. An example of a “Mal-Hex” linker is also referred to herein as “ADL12” or an “ADL12” linker.

In some embodiments, the Mal-spacer unit or linker comprises Mal-(CH 2 ) 2 (“Mal-Et”). In some embodiments, the Mal-spacer unit or linker comprises Mal-Et and at least one additional spacer unit. In some embodiments, the Mal-Et attaches the antibody or antigen binding fragment to the drug moiety. In some embodiments, the linker comprises Mal-Et. An example of a “Mal-Et” linker is also referred to herein as “ADL14” or an “ADL14” linker.

In some embodiments, the Mal-spacer unit or linker comprises Mal-(CH 2 ) 2 —O—(CH 2 ) 2 (“Mal-Et-O-Et”). In some embodiments, the Mal-spacer unit or linker comprises Mal-Et-O-Et and at least one additional spacer unit. In some embodiments, the Mal-Et-O-Et attaches the antibody or antigen binding fragment to the drug moiety. In some embodiments, the linker comprises Mal-Et-O-Et. An example of a “Mal-Et-O-Et” linker is also referred to herein as “ADL15” or an “ADL15” linker.

In some other embodiments, the Mal-spacer unit attaches the antibody or antigen binding fragment to the cleavable moiety in the linker. In some embodiments, the cleavable moiety in the linker is a cleavable peptide moiety, e.g., an amino acid unit. In some embodiments, the cleavable peptide moiety is Val-Cit or Val-Ala. In some embodiments, the Mal-spacer unit or linker comprises MC. In some embodiments, the linker comprises MC-Val-Cit. In some embodiments, the linker comprises MC-Val-Ala. In some embodiments, the linker comprises MC-Glu-Val-Cit. In some embodiments, the linker comprises MC-Ala-Ala-Asn.

›Definitions · 24 of 64

In some embodiments, a spacer unit attaches the cleavable moiety in the linker to the splicing modulator. In some embodiments, the spacer unit that attaches the cleavable moiety to the splicing modulator is self-immolative.

In some embodiments, the spacer unit comprises pABC. In some embodiments, the pABC attaches the cleavable moiety to the splicing modulator. In some embodiments, the cleavable moiety is a cleavable peptide moiety, e.g., an amino acid unit. In some embodiments, the linker comprises amino acid unit-pABC.

In some embodiments, the linker comprises Val-Cit-pABC. In some embodiments, the linker comprises Val-Cit-pABC and a MC Mal-spacer unit joining the linker to the antibody or antigen binding fragment. In some embodiments, the linker comprises MC-Val-Cit-pABC. In some embodiments, the linker comprises MC-Val-Cit-pABC and at least one additional spacer unit. An example of an MC-Val-Cit-pABC linker is also referred to herein as “ADL1” or an “ADL1” linker.

In some embodiments, the linker comprises Val-Ala-pABC. In some embodiments, the linker comprises Val-Ala-pABC and a MC Mal-spacer unit joining the linker to the antibody or antigen binding fragment. In some embodiments, the linker comprises MC-Val-Ala-pABC. In some embodiments, the linker comprises MC-Val-Ala-pABC and at least one additional spacer unit. An example of an MC-Val-Ala-pABC linker is also referred to herein as “ADL6” or an “ADL6” linker.

In some embodiments, the linker comprises Glu-Val-Cit-pABC. In some embodiments, the linker comprises Glu-Val-Cit-pABC and a MC Mal-spacer unit joining the linker to the antibody or antigen binding fragment. In some embodiments, the linker comprises MC-Glu-Val-Cit-pABC. In some embodiments, the linker comprises MC-Glu-Val-Cit-pABC and at least one additional spacer unit. An example of an MC-Glu-Val-Cit-pABC linker is also referred to herein as “ADL23” or an “ADL23” linker.

In some embodiments, the linker comprises Ala-Ala-Asn-pABC. In some embodiments, the linker comprises Ala-Ala-Asn-pABC and a MC Mal-spacer unit joining the linker to the antibody or antigen binding fragment. In some embodiments, the linker comprises MC-Ala-Ala-Asn-pABC. In some embodiments, the linker comprises MC-Ala-Ala-Asn-pABC and at least one additional spacer unit. An example of an MC-Ala-Ala-Asn-pABC linker is also referred to herein as “ADL21” or an “ADL21” linker.

In some other embodiments, the spacer unit comprises pAB. In some embodiments, the pAB attaches the cleavable moiety to the splicing modulator. In some embodiments, the cleavable moiety is a cleavable peptide moiety, e.g., an amino acid unit. In some embodiments, the linker comprises amino acid unit-pAB.

In some embodiments, the linker comprises Val-Ala-pAB. In some embodiments, the linker comprises Val-Ala-pAB and an MC Mal-spacer unit joining the linker to the antibody or antigen binding fragment. In some embodiments, the linker comprises MC-Val-Ala-pAB. In some embodiments, the linker comprises MC-Val-Ala-pAB and at least one additional spacer unit. An example of an MC-Val-Ala-pAB linker is also referred to herein as “ADL5” or an “ADL5” linker.

In some embodiments, the linker comprises Val-Cit-pAB. In some embodiments, the linker comprises Val-Cit-pAB and an MC Mal-spacer unit joining the linker to the antibody or antigen binding fragment. In some embodiments, the linker comprises MC-Val-Cit-pAB. In some embodiments, the linker comprises MC-Val-Cit-pAB and at least one additional spacer unit. An example of an MC-Val-Cit-pAB linker is also referred to herein as “ADL7” or an “ADL7” linker.

In some embodiments, the linker comprises β-glucuronide-pABC. In some embodiments, the linker comprises β-glucuronide-pABC and an MC Mal-spacer unit joining the linker to the antibody or antigen binding fragment. In some embodiments, the linker comprises MC-β-glucuronide-pABC. In some embodiments, the linker comprises MC-β-glucuronide-pABC and at least one additional spacer unit. An example of an MC-β-glucuronide-pABC is also referred to herein as “ADL13” or an “ADL13” linker.

In some embodiments, the linker comprises β-glucuronide-pAB. In some embodiments, the linker comprises β-glucuronide-pAB and an MC Mal-spacer unit joining the linker to the antibody or antigen binding fragment. In some embodiments, the linker comprises MC-β-glucuronide-pAB.

In some embodiments, the antibody or antigen binding fragment is conjugated to the splicing modulator drug moiety via an ADL1, ADL2, ADL5, ADL6, ADL7, ADL12, ADL13, ADL14, ADL21, ADL23, or ADL15 linker. It has been discovered, in various embodiments, that ADCs comprising an ADL1, ADL2, ADL5, ADL6, ADL7, ADL12, ADL13, ADL14, ADL21, ADL23, or ADL15 linker and a splicing modulator drug moiety disclosed herein demonstrate desirable properties for a therapeutic ADC. In various embodiments, these properties include, but are not limited to, effective levels of drug loading, low aggregation levels, stability under storage conditions or when in circulation in the body (e.g., serum stability), retained affinity for target-expressing cells comparable to unconjugated antibody, potent cytotoxicity against target-expressing cells, low levels of off-target cell killing, high levels of bystander killing, and/or effective in vivo anti-cancer activity, all as compared to ADCs using other linker-payloads. For instance, in various embodiments, ADCs comprising an ADL1, ADL2, ADL5, ADL6, ADL7, ADL12, ADL13, ADL14, ADL21, ADL23, or ADL15 linker and a splicing modulator drug moiety disclosed herein exhibit an increased ability to inhibit growth and/or proliferation in target-expressing cells, as compared to ADCs using other linker-payloads (e.g., an ADL10 linker and a splicing modulator drug moiety). In various embodiments, ADCs comprising an ADL1, ADL2, ADL5, ADL6, ADL7, ADL12, ADL13, ADL14, ADL21, ADL23, or ADL15 linker and a splicing modulator drug moiety disclosed herein exhibit surprisingly increased in vivo stability (e.g., plasma stability), as compared to other splicing modulator-based ADCs (e.g., a thailanstatin A-based ADC, for example, as reported in Puthenveetil et al. Bioconjugate Chem. (2016) 27:1880-8).

›Definitions · 25 of 64

In some embodiments, the good or superior functional properties provided by the particular combination of an ADL1, ADL2, ADL5, ADL6, ADL7, ADL12, ADL13, ADL14, ADL21, ADL23, or ADL15 linker and a splicing modulator drug moiety disclosed herein may be observed with the linker-payload conjugated to, e.g., an anti-HER2 antibody such as trastuzumab; an anti-CD138 antibody such as B-B4; or an anti-EPHA2 antibody such as 1C1.

In some embodiments, the ADC comprises ADL1-splicing modulator and an antibody or antigen binding fragment comprising an antibody or an antigen binding fragment thereof that retains the ability to target and internalize in a neoplastic cell. In some embodiments, the ADC comprises ADL2-splicing modulator and an antibody or antigen binding fragment comprising an antibody or an antigen binding fragment thereof that retains the ability to target and internalize in a neoplastic cell. In some embodiments, the ADC comprises ADL5-splicing modulator and an antibody or antigen binding fragment comprising an antibody or an antigen binding fragment thereof that retains the ability to target and internalize in a neoplastic cell. In some embodiments, the ADC comprises ADL6-splicing modulator and an antibody or antigen binding fragment comprising an antibody or an antigen binding fragment thereof that retains the ability to target and internalize in a neoplastic cell. In some embodiments, the ADC comprises ADL7-splicing modulator and an antibody or antigen binding fragment comprising an antibody or an antigen binding fragment thereof that retains the ability to target and internalize in a neoplastic cell. In some embodiments, the ADC comprises ADL12-splicing modulator and an antibody or antigen binding fragment comprising an antibody or an antigen binding fragment thereof that retains the ability to target and internalize in a neoplastic cell. In some embodiments, the ADC comprises ADL13-splicing modulator and an antibody or antigen binding fragment comprising an antibody or an antigen binding fragment thereof that retains the ability to target and internalize in a neoplastic cell. In some embodiments, the ADC comprises ADL14-splicing modulator and an antibody or antigen binding fragment comprising an antibody or an antigen binding fragment thereof that retains the ability to target and internalize in a neoplastic cell. In some embodiments, the ADC comprises ADL15-splicing modulator and an antibody or antigen binding fragment comprising an antibody or an antigen binding fragment thereof that retains the ability to target and internalize in a neoplastic cell.

In some embodiments, the ADC comprises ADL1-splicing modulator and an antibody or antigen binding fragment thereof that targets a HER2-expressing neoplastic cell. In some embodiments, the ADC comprises ADL2-splicing modulator and an antibody or antigen binding fragment thereof that targets a HER2-expressing neoplastic cell. In some embodiments, the ADC comprises ADL5-splicing modulator and an antibody or antigen binding fragment thereof that targets a HER2-expressing neoplastic cell. In some embodiments, the ADC comprises ADL6-splicing modulator and an antibody or antigen binding fragment thereof that targets a HER2-expressing neoplastic cell. In some embodiments, the ADC comprises ADL7-splicing modulator and an antibody or antigen binding fragment thereof that targets a HER2-expressing neoplastic cell. In some embodiments, the ADC comprises ADL12-splicing modulator and an antibody or antigen binding fragment thereof that targets a HER2-expressing neoplastic cell. In some embodiments, the ADC comprises ADL13-splicing modulator and an antibody or antigen binding fragment thereof that targets a HER2-expressing neoplastic cell. In some embodiments, the ADC comprises ADL14-splicing modulator and an antibody or antigen binding fragment thereof that targets a HER2-expressing neoplastic cell. In some embodiments, the ADC comprises ADL15-splicing modulator and an antibody or antigen binding fragment thereof that targets a HER2-expressing neoplastic cell.

In some embodiments, the antibody or antigen binding fragment thereof that targets a HER2-expressing neoplastic cell is an internalizing antibody or internalizing antigen binding fragment. In some embodiments, the antibody or antigen binding fragment thereof that targets a HER2-expressing neoplastic cell comprises three heavy chain complementarity determining regions (HCDRs) comprising amino acid sequences of SEQ ID NO:1 (HCDR1), SEQ ID NO:2 (HCDR2), and SEQ ID NO:3 (HCDR3); and three light chain complementarity determining regions (LCDRs) comprising amino acid sequences of SEQ ID NO:4 (LCDR1), SEQ ID NO:5 (LCDR2), and SEQ ID NO:6 (LCDR3).

In some embodiments, the ADC has Formula (I):

Ab-(L-D) p (I)

wherein:

(i) Ab is an anti-HER2 antibody or antigen binding fragment thereof comprising three heavy chain complementarity determining regions (HCDRs) comprising amino acid sequences of SEQ ID NO:1 (HCDR1), SEQ ID NO:2 (HCDR2), and SEQ ID NO:3 (HCDR3); and three light chain complementarity determining regions (LCDRs) comprising amino acid sequences of SEQ ID NO:4 (LCDR1), SEQ ID NO:5 (LCDR2), and SEQ ID NO:6 (LCDR3); (ii) D is a splicing modulator; (iii) L is a linker comprising ADL1, ADL2, ADL5, ADL6, ADL7, ADL12, ADL13, ADL14, ADL21, ADL23, or ADL15; and (iv) p is an integer from 1 to 15.

In some embodiments, the antibody or antigen binding fragment thereof that targets a HER2-expressing neoplastic cell comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO:19, and a light chain variable region comprising an amino acid sequence of SEQ ID NO:20. In some embodiments, the antibody or antigen binding fragment thereof that targets a HER2-expressing neoplastic cell comprises a human IgG1 heavy chain constant domain and a human Ig kappa light chain constant domain. In some embodiments, the antibody is trastuzumab. In some embodiments, p is an integer from 1 to 10, from 2 to 8, or from 4 to 8. In some embodiments, p is 4. In some embodiments, p is 8.

›Definitions · 26 of 64

In some embodiments, the ADC comprises ADL1-splicing modulator and an antibody or antigen binding fragment thereof that targets a CD138-expressing neoplastic cell. In some embodiments, the ADC comprises ADL2-splicing modulator and an antibody or antigen binding fragment thereof that targets a CD138-expressing neoplastic cell. In some embodiments, the ADC comprises ADL5-splicing modulator and an antibody or antigen binding fragment thereof that targets a CD138-expressing neoplastic cell. In some embodiments, the ADC comprises ADL6-splicing modulator and an antibody or antigen binding fragment thereof that targets a CD138-expressing neoplastic cell. In some embodiments, the ADC comprises ADL7-splicing modulator and an antibody or antigen binding fragment thereof that targets a CD138-expressing neoplastic cell. In some embodiments, the ADC comprises ADL12-splicing modulator and an antibody or antigen binding fragment thereof that targets a CD138-expressing neoplastic cell. In some embodiments, the ADC comprises ADL13-splicing modulator and an antibody or antigen binding fragment thereof that targets a CD138-expressing neoplastic cell. In some embodiments, the ADC comprises ADL14-splicing modulator and an antibody or antigen binding fragment thereof that targets a CD138-expressing neoplastic cell. In some embodiments, the ADC comprises ADL15-splicing modulator and an antibody or antigen binding fragment thereof that targets a CD138-expressing neoplastic cell.

In some embodiments, the antibody or antigen binding fragment thereof that targets a CD138-expressing neoplastic cell is an internalizing antibody or internalizing antigen binding fragment. In some embodiments, the antibody or antigen binding fragment thereof that targets a CD138-expressing neoplastic cell comprises three heavy chain complementarity determining regions (HCDRs) comprising amino acid sequences of SEQ ID NO:7 (HCDR1), SEQ ID NO:8 (HCDR2), and SEQ ID NO:9 (HCDR3); and three light chain complementarity determining regions (LCDRs) comprising amino acid sequences of SEQ ID NO:10 (LCDR1), SEQ ID NO:11 (LCDR2), and SEQ ID NO:12 (LCDR3).

In some embodiments, the ADC has Formula (I):

Ab-(L-D) p (I)

wherein:

(i) Ab is an anti-CD138 antibody or antigen binding fragment thereof comprising three heavy chain complementarity determining regions (HCDRs) comprising amino acid sequences of SEQ ID NO:7 (HCDR1), SEQ ID NO:8 (HCDR2), and SEQ ID NO:9 (HCDR3); and three light chain complementarity determining regions (LCDRs) comprising amino acid sequences of SEQ ID NO:10 (LCDR1), SEQ ID NO:11 (LCDR2), and SEQ ID NO:12 (LCDR3); (ii) D is a splicing modulator; (iii) L is a linker comprising ADL1, ADL2, ADL5, ADL6, ADL7, ADL12, ADL13, ADL14, ADL21, ADL23, or ADL15; and (iv) p is an integer from 1 to 15.

In some embodiments, the antibody or antigen binding fragment thereof that targets a CD138-expressing neoplastic cell comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO:21, and a light chain variable region comprising an amino acid sequence of SEQ ID NO:22. In some embodiments, the antibody or antigen binding fragment thereof that targets a CD138-expressing neoplastic cell comprises a murine IgG2a heavy chain constant domain and a murine Ig kappa light chain constant domain. In some embodiments, the antibody or antigen binding fragment thereof that targets a CD138-expressing neoplastic cell comprises a human IgG2a heavy chain constant domain and a human Ig kappa light chain constant domain. In some embodiments, the antibody is B-B4. In some embodiments, p is an integer from 1 to 10, from 2 to 8, or from 4 to 8. In some embodiments, p is 4. In some embodiments, p is 8.

In some embodiments, the ADC comprises ADL1-splicing modulator and an antibody or antigen binding fragment thereof that targets an EPHA2-expressing neoplastic cell. In some embodiments, the ADC comprises ADL2-splicing modulator and an antibody or antigen binding fragment thereof that targets an EPHA2-expressing neoplastic cell. In some embodiments, the ADC comprises ADL5-splicing modulator and an antibody or antigen binding fragment thereof that targets an EPHA2-expressing neoplastic cell. In some embodiments, the ADC comprises ADL6-splicing modulator and an antibody or antigen binding fragment thereof that targets an EPHA2-expressing neoplastic cell. In some embodiments, the ADC comprises ADL7-splicing modulator and an antibody or antigen binding fragment thereof that targets an EPHA2-expressing neoplastic cell. In some embodiments, the ADC comprises ADL12-splicing modulator and an antibody or antigen binding fragment thereof that targets an EPHA2-expressing neoplastic cell. In some embodiments, the ADC comprises ADL13-splicing modulator and an antibody or antigen binding fragment thereof that targets an EPHA2-expressing neoplastic cell. In some embodiments, the ADC comprises ADL14-splicing modulator and an antibody or antigen binding fragment thereof that targets an EPHA2-expressing neoplastic cell. In some embodiments, the ADC comprises ADL15-splicing modulator and an antibody or antigen binding fragment thereof that targets an EPHA2-expressing neoplastic cell.

In some embodiments, the antibody or antigen binding fragment thereof that targets an EPHA2-expressing neoplastic cell is an internalizing antibody or internalizing antigen binding fragment. In some embodiments, the antibody or antigen binding fragment thereof that targets an EPHA2-expressing neoplastic cell comprises three heavy chain complementarity determining regions (HCDRs) comprising amino acid sequences of SEQ ID NO:13 (HCDR1), SEQ ID NO:14 (HCDR2), and SEQ ID NO:15 (HCDR3); and three light chain complementarity determining regions (LCDRs) comprising amino acid sequences of SEQ ID NO:16 (LCDR1), SEQ ID NO:17 (LCDR2), and SEQ ID NO:18 (LCDR3).

In some embodiments, the ADC has Formula (I):

Ab-(L-D) p (I)

wherein:

(i) Ab is an anti-EPHA2 antibody or antigen binding fragment thereof comprising three heavy chain complementarity determining regions (HCDRs) comprising amino acid sequences of SEQ ID NO:13 (HCDR1), SEQ ID NO:14 (HCDR2), and SEQ ID NO:15 (HCDR3); and three light chain complementarity determining regions (LCDRs) comprising amino acid sequences of SEQ ID NO:16 (LCDR1), SEQ ID NO:17 (LCDR2), and SEQ ID NO:18 (LCDR3); (ii) D is a splicing modulator; (iii) L is a linker comprising ADL1, ADL2, ADL5, ADL6, ADL7, ADL12, ADL13, ADL14, ADL21, ADL23, or ADL15; and (iv) p is an integer from 1 to 15.

›Definitions · 27 of 64

In some embodiments, the antibody or antigen binding fragment thereof that targets an EPHA2-expressing neoplastic cell comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO:23, and a light chain variable region comprising an amino acid sequence of SEQ ID NO:24. In some embodiments, the antibody or antigen binding fragment thereof that targets an EPHA2-expressing neoplastic cell comprises a human IgG1 heavy chain constant domain and a human Ig kappa light chain constant domain. In some embodiments, the antibody is 1C1. In some embodiments, p is an integer from 1 to 10, from 2 to 8, or from 4 to 8. In some embodiments, p is 4. In some embodiments, p is 8.

Drug Moieties

The drug moiety (D) of the ADCs described herein can be any chemotherapeutic agent. Useful classes of chemotherapeutic agents include, for example, modulators of RNA splicing. In certain preferred embodiments, the drug moiety is a splicing modulator. Exemplary splicing modulator compounds are described and exemplified herein.

In various embodiments, the drug moiety is a splicing modulator compound of Formula (II):

or a pharmaceutically acceptable salt thereof, wherein:

R 4 , R 5 , and R 8 are each independently chosen from hydrogen, hydroxyl groups, —O—(C 1 -C 6 alkyl) groups, —O—C(═O)—(C 1 -C 6 alkyl) groups, and C 1 -C 6 alkyl groups;

R 6 and R 7 are each independently chosen from hydrogen, —O—R 17 , —O—C(═O)—R 17 , —O—C(═O)—NR 15 R 16 , C 1 -C 6 alkyl groups, and —NR 15 R 16 ; R 15 and R 16 are each independently chosen from hydrogen, R 17 , —C(═O)—R 17 , and —C(═O)—O—R 17 ;

R 17 is chosen from hydrogen, C 1 -C 6 alkyl groups, C 3 -C 8 cycloalkyl groups, benzyl groups, and C 3 -C 8 heterocyclyl groups; and

Z is chosen from

wherein R 1 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 15 , R 16 , and R 17 are each independently substituted with 0 to 3 groups independently chosen from halogens, hydroxyl groups, C 1 -C 6 alkyl groups, —O—(C 1 -C 6 alkyl) groups, —NR 15 R 16 , C 3 -C 8 cycloalkyl groups, C 1 -C 6 alkylhydroxy groups, C 1 -C 6 alkylalkoxy groups, benzyl groups, and C 3 -C 8 heterocyclyl groups,

wherein at least one of R 6 and R 7 is hydrogen.

In some embodiments, R 1 is chosen from hydrogen, C 1 -C 4 alkyl groups, C 1 -C 4 alkylcarboxylic acid groups, and C 3 -C 8 cycloalkyl groups. In some embodiments, R 1 is hydrogen. In some embodiments, R 1 is a C 1 -C 4 alkyl group. In some embodiments, R, is methyl. In some embodiments, R 1 is ethyl. In some embodiments, R 1 is a C 1 -C 4 alkylcarboxylic acid group. In some embodiments, R 1 is —CH 2 CH 2 CH 2 CO 2 H. In some embodiments, R 1 is a C 3 -C 8 cycloalkyl group. In some embodiments, R 1 is cycloheptyl.

In some embodiments, R 3 is chosen from hydrogen, C 1 -C 4 alkyl groups, C 1 -C 4 alkylalkoxy groups, C 1 -C 4 alkylcarboxylic acid groups, and C 1 -C 4 alkylhydroxy groups. In some embodiments, R 3 is chosen from hydrogen and C 1 -C 4 alkylcarboxylic acid groups. In some embodiments, R 3 is hydrogen. In some embodiments, R 3 is a C 1 -C 4 alkylcarboxylic acid group. In some embodiments, R 3 is —CH 2 CH 2 CO 2 H.

In some embodiments, R 4 is chosen from hydrogen, hydroxyl groups, —O—(C 1 -C 4 alkyl) groups, —O—C(═O)—(C 1 -C 4 alkyl) groups, and C 1 -C 4 alkyl groups. In some embodiments, R 4 is hydrogen. In some embodiments, R 4 is hydroxyl. In some embodiments, R 4 is a —O—(C 1 -C 4 alkyl) group. In some embodiments, R 4 is —OCH 3 . In some embodiments, R 4 is —OCH 2 CH 3 . In some embodiments, R 4 is a —O—C(═O)—(C 1 -C 4 alkyl) group. In some embodiments, R 4 is —O—C(═O)—CH 3 . In some embodiments, R 4 is —O—C(═O)—CH 2 CH 3 . In some embodiments, R 4 is a C 1 -C 4 alkyl group. In some embodiments, R 4 is methyl. In some embodiments, R 4 is ethyl.

In some embodiments, R 5 is chosen from hydrogen, hydroxyl groups, —O—(C 1 -C 4 alkyl) groups, and C 1 -C 4 alkyl groups. In some embodiments, R 5 is hydrogen. In some embodiments, R 5 is hydroxyl. In some embodiments, R 5 is a —O—(C 1 -C 4 alkyl) group. In some embodiments, R 5 is a C 1 -C 4 alkyl group.

In some embodiments, R 6 is hydrogen. In some embodiments, R 7 is hydrogen. In some embodiments, R 6 is hydrogen and R 7 is —O—R 17 . In some embodiments, R 6 is hydrogen and R 7 is —OR 17 , wherein R 17 is chosen from hydrogen and C 1 -C 4 alkyl groups. In some embodiments, R 6 is hydrogen and R 7 is —O—R 17 , wherein R 17 is hydrogen. In some embodiments, R 6 is —O—R 17 and R 7 is hydrogen. In some embodiments, R 6 is —O—R 17 and R 7 is hydrogen, wherein R 17 is chosen from hydrogen and C 1 -C 4 alkyl groups. In some embodiments, R 6 is —O—R 17 and R 7 is hydrogen, wherein R 17 is hydrogen. In some embodiments, R 6 is hydrogen and R 7 is —NR 15 R 16 . In some embodiments, R 6 is hydrogen and R 7 is —NR 15 R 16 , wherein R 15 is H and R 16 is chosen from hydrogen, R 17 , —C(═O)—R 17 , and —C(═O)—O—R 17 . In some embodiments, R 6 is hydrogen and R 7 is —NR 15 R 16 , wherein R 15 is H and R 16 is chosen from hydrogen, R 17 , —C(═O)—R 17 , and —C(═O)—O—R 17 , and wherein R 17 is chosen from hydrogen, C 1 -C 6 alkyl groups, C 3 -C 8 cycloalkyl groups, and C 3 -C 8 heterocyclyl groups. In some embodiments, R 6 is —O—R 17 . In some embodiments, R 6 is —O—C(═O)—R 17 . In some embodiments, R 6 is C 1 -C 6 alkyl. In some embodiments, R 6 is C 1 -C 4 alkyl. In some embodiments, R 6 is C 1 alkyl. In some embodiments, R 6 is —NR 15 R 16 . In some embodiments, R 7 is —O—R 17 . In some embodiments, R 7 is —O—C(═O)—R 17 . In some embodiments, R 7 is C 1 -C 6 alkyl. In some embodiments, R 7 is C 1 -C 4 alkyl. In some embodiments, R 7 is C 1 alkyl. In some embodiments, R 7 is —NR 15 R 16 .

›Definitions · 28 of 64

In some embodiments, R 8 is chosen from hydrogen, hydroxyl groups, —O—(C 1 -C 4 alkyl) groups, and (C 1 -C 4 alkyl). In some embodiments, R 8 is hydrogen. In some embodiments, R 8 is a hydroxyl group. In some embodiments, R 8 is an —O—(C 1 -C 4 alkyl) group. In some embodiments, R 8 is an —O—(C 1 alkyl) group.

In some embodiments, R 15 is hydrogen. In some embodiments, R 15 is R 17 . In some embodiments, R 15 is —C(═O)—R 17 . In some embodiments, R 15 is —C(═O)—O—R 17 .

In some embodiments, R 16 is hydrogen. In some embodiments, R 16 is R 17 . In some embodiments, R 16 is —C(═O)—R 17 . In some embodiments, R 16 is —C(═O)—O—R 17 .

In some embodiments, R 17 is chosen from hydrogen, C 1 -C 4 alkyl groups, C 3 -C 8 cycloalkyl groups, and C 3 -C 8 heterocyclyl groups. In some embodiments, R 17 is hydrogen. In some embodiments, R 17 is a C 1 -C 4 alkyl group. In some embodiments, R 17 is a C 1 alkyl group. In some embodiments, R 17 is a C 3 -C 8 cycloalkyl group. In some embodiments, R 17 is a C 3 cycloalkyl group. In some embodiments, R 17 is a C 4 cycloalkyl group. In some embodiments, R 17 is a C 5 cycloalkyl group. In some embodiments, R 17 is a C 6 cycloalkyl group. In some embodiments, R 17 is a C 3 -C 8 heterocyclyl group. In some embodiments, R 17 is a C 3 heterocyclyl group. In some embodiments, R 17 is a C 4 heterocyclyl group. In some embodiments, R 17 is a C 5 heterocyclyl group. In some embodiments, R 17 is a C 6 heterocyclyl group. In some embodiments, R 17 is a C 7 heterocyclyl group. In some embodiments, R 17 is a C 8 heterocyclyl group.

In some embodiments, Z is

In some embodiments, the splicing modulator compound of Formula (II) attaches to the linker L, e.g., in an ADC of Formula (I), as shown in Formula (II-A):

wherein Z′ is chosen from

and

wherein all other variables are as defined for Formula (II).

In various other embodiments, the drug moiety is a splicing modulator compound of Formula (IV):

or a pharmaceutically acceptable salt thereof, wherein:

R 4 , R 5 , and R 8 are each independently chosen from hydrogen, hydroxyl groups, —O—(C 1 -C 6 alkyl) groups, —O—C(═O)—(C 1 -C 6 alkyl) groups, and C 1 -C 6 alkyl groups;

R 6 and R 7 are each independently chosen from hydrogen, —O—R 17 , —O—C(═O)—R 17 , —O—C(═O)—NR 15 R 16 , C 1 -C 6 alkyl groups, and —NR 15 R 16 ;

R 15 and R 16 are each independently chosen from hydrogen, R 17 , —C(═O)—R 17 , and —C(═O)—O—R 17 ; and

R 17 is chosen from hydrogen, C 1 -C 6 alkyl groups, C 3 -C 8 cycloalkyl groups, benzyl groups, and C 3 -C 8 heterocyclyl groups;

wherein at least one of R 6 and R 7 is hydrogen.

In some embodiments, R 1 is chosen from hydrogen, C 1 -C 4 alkyl groups, C 1 -C 4 alkylcarboxylic acid groups, and C 3 -C 6 cycloalkyl groups. In some embodiments, R 1 is hydrogen. In some embodiments, R 1 is a C 1 -C 4 alkyl group. In some embodiments, R 1 is methyl. In some embodiments, R 1 is ethyl. In some embodiments, R 1 is a C 1 -C 4 alkylcarboxylic acid group. In some embodiments, R 1 is —CH 2 CH 2 CH 2 CO 2 H. In some embodiments, R 1 is a C 3 -C 8 cycloalkyl group. In some embodiments, R 1 is cycloheptyl.

In some embodiments, R 6 is hydrogen. In some embodiments, R 6 is hydrogen. In some embodiments, R 6 is hydrogen and R 7 is —O—R 17 . In some embodiments, R 6 is hydrogen and R 7 is —OR 17 , wherein R 17 is chosen from hydrogen and C 1 -C 4 alkyl groups. In some embodiments, R 6 is hydrogen and R 7 is —O—R 17 , wherein R 17 is hydrogen. In some embodiments, R 6 is —O—R 17 and R 7 is hydrogen. In some embodiments, R 6 is —O—R 17 and R 7 is hydrogen, wherein R 17 is chosen from hydrogen and C 1 -C 4 alkyl groups. In some embodiments, R 6 is —O—R 17 and R 7 is hydrogen, wherein R 17 is hydrogen. In some embodiments, R 6 is hydrogen and R 7 is —NR 15 R 16 . In some embodiments, R 6 is hydrogen and R 7 is —NR 15 R 16 , wherein R 15 is H and R 16 is chosen from hydrogen, R 17 , —C(═O)—R 17 , and —C(═O)—O—R 17 . In some embodiments, R 6 is hydrogen and R 7 is —NR 15 R 16 , wherein R 15 is H and R 16 is chosen from hydrogen, R 17 , —C(═O)—R 17 , and —C(═O)—O—R 17 , and wherein R 17 is chosen from hydrogen, C 1 -C 6 alkyl groups, C 3 -C 8 cycloalkyl groups, and C 3 -C 8 heterocyclyl groups. In some embodiments, R 6 is —O—R 17 . In some embodiments, R 6 is —O—C(═O)—R 17 . In some embodiments, R 6 is C 1 -C 6 alkyl. In some embodiments, R 6 is C 1 -C 4 alkyl. In some embodiments, R 6 is C 1 alkyl. In some embodiments, R 6 is —NR 15 R 16 .

›Definitions · 29 of 64

In some embodiments, R 7 is —O—R 17 . In some embodiments, R 7 is —O—C(═O)—R 17 . In some embodiments, R 7 is C 1 -C 6 alkyl. In some embodiments, R 7 is C 1 -C 4 alkyl. In some embodiments, R 7 is C 1 alkyl. In some embodiments, R 7 is —NR 15 R 16 .

In some embodiments, R 15 is hydrogen. In some embodiments, R 15 is R 17 . In some embodiments, R 15 is —C(═O)—R 17 . In some embodiments, R 15 is —C(═O)—O—R 17 .

In some embodiments, R 16 is hydrogen. In some embodiments, R 16 is R 17 . In some embodiments, R 16 is —C(═O)—R 17 . In some embodiments, R 16 is —C(═O)—O—R 17 .

In some embodiments, the splicing modulator compound of Formula (IV) attaches to the linker L, e.g., in an ADC of Formula (I), as shown in Formula (IV-A):

In various other embodiments, the drug moiety is a splicing modulator compound of Formula (VI):

or a pharmaceutically acceptable salt thereof, wherein:

R 4 , R 5 , and R 8 are each independently chosen from hydrogen, hydroxyl groups, —O—(C 1 -C 6 alkyl) groups, —O—C(═O)—(C 1 -C 6 alkyl) groups, and C 1 -C 6 alkyl groups;

R 6 and R 7 are each independently chosen from hydrogen, —O—R 17 , —O—C(═O)—R 17 , —O—C(═O)—NR 15 R 16 , C 1 -C 6 alkyl groups, —NR 15 R 16 , and a linker;

R 10 is chosen from hydrogen, C 1 -C 6 alkyl groups, —C(═O)—(C 1 -C 6 alkyl) groups, and —CD 3 ;

R 15 and R 16 are each independently chosen from hydrogen, R 17 , —C(═O)—R 17 , and —C(═O)—O—R 17 ;

R 17 is chosen from hydrogen, C 1 -C 6 alkyl groups, C 3 -C 8 cycloalkyl groups, benzyl groups, and C 3 -C 8 heterocyclyl groups; and

a is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

wherein at least one of R 6 and R 7 is hydrogen; and

wherein R 1 and R 9 cannot both be absent.

In some embodiments, R 1 is chosen from hydrogen, C 1 -C 4 alkyl groups, C 1 -C 4 alkylcarboxylic acid groups, and C 3 -C 8 cycloalkyl groups. In some embodiments, R 1 is hydrogen. In some embodiments, R 1 is a C 1 -C 4 alkyl group. In some embodiments, R 1 is methyl. In some embodiments, R 1 is ethyl. In some embodiments, R 1 is a C 1 -C 4 alkylcarboxylic acid group. In some embodiments, R 1 is —CH 2 CH 2 CH 2 CO 2 H. In some embodiments, R 1 is a C 3 -C 8 cycloalkyl group. In some embodiments, R 1 is cycloheptyl.

In some embodiments, R 9 is chosen from absent, hydrogen, C 1 -C 4 alkyl groups, —(C═O)—(C 1 -C 4 alkyl) groups, and —CD 3 . In some embodiments, R 9 is absent. In some embodiments, R 9 is hydrogen. In some embodiments, R 9 is a C 1 -C 4 alkyl group. In some embodiments, the C 1 -C 4 alkyl group is methyl. In some embodiments, the C 1 -C 4 alkyl group is ethyl. In some embodiments, R 9 is a —(C═O)—(C 1 -C 4 alkyl) group. In some embodiments, the —(C═O)—(C 1 -C 4 alkyl) group is —(C═O)-methyl. In some embodiments, R 9 is —CD 3 .

›Definitions · 30 of 64

In some embodiments, R 10 is chosen from hydrogen, C 1 -C 4 alkyl groups, —(C═O)—(C 1 -C 4 alkyl) groups, and —CD 3 . In some embodiments, R 10 is hydrogen. In some embodiments, R 10 is a C 1 -C 4 alkyl group. In some embodiments, the C 1 -C 4 alkyl group is methyl. In some embodiments, the C 1 -C 4 alkyl group is ethyl. In some embodiments, R 10 is a —(C═O)—(C 1 -C 4 alkyl) group. In some embodiments, the —(C═O)—(C 1 -C 4 alkyl) group is —(C═O)-methyl. In some embodiments, R 10 is —CD 3 .

In some embodiments, R 15 is hydrogen. In some embodiments, R 15 is R 17 . In some embodiments, R 15 is —C(═O)—R 17 . In some embodiments, R 15 is —C(═O)—O—R 17 .

In some embodiments, R 16 is hydrogen. In some embodiments, R 16 is R 17 . In some embodiments, R 16 is —C(═O)—R 17 . In some embodiments, R 16 is —C(═O)—O—R 17 .

In some embodiments, a is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, a is 1, 2, 3, 4, 5, or 6. In some embodiments, a is 1, 2, 3, 4, or 5. In some embodiments, a is 1, 2, 3, or 4. In some embodiments, a is 1, 2, or 3. In some embodiments, a is 1 or 2. In some embodiments, a is 1. In some embodiments, a is 2. In some embodiments, a is 3. In some embodiments, a is 4. In some embodiments, a is 5. In some embodiments, a is 6. In some embodiments, a is 7. In some embodiments, a is 8. In some embodiments, a is 9. In some embodiments, a is 10.

In some embodiments, the splicing modulator compound of Formula (VI) attaches to the linker L, e.g., in an ADC of Formula (I), as shown in Formula (VI-A):

In various other embodiments, the drug moiety is a splicing modulator compound of Formula (VIII):

or a pharmaceutically acceptable salt thereof, wherein:

R 4 is chosen from hydrogen, hydroxyl groups, —O—(C 1 -C 6 alkyl) groups, —O—C(═O)—(C 1 -C 6 alkyl) groups, and C 1 -C 6 alkyl groups; and

R 15 and R 16 are each independently chosen from hydrogen, R 17 , —C(═O)—R 17 , and —C(═O)—O—R 17 ; and

R 17 is chosen from hydrogen, C 1 -C 6 alkyl groups, C 3 -C 8 cycloalkyl groups, benzyl groups, and C 3 -C 8 heterocyclyl groups;

›Definitions · 31 of 64

wherein each R a is independently substituted with 0 to 3 groups independently chosen from halogens, hydroxyl groups, —NR 15 R 16 , C 1 -C 6 alkyl groups, —(C═O)—(C 1 -C 6 alkyl) groups, —(C═O)—(C 1 -C 6 alkyl)-(C 3 -C 10 heterocyclyl groups), and C 1 -C 6 alkylcarboxylic acid groups, each of which is substituted with 0, 1, or 2 groups independently chosen from halogens, hydroxyl groups, —NR 15 R 16 , and C 1 -C 3 alkyl groups; and

w is 0, 1, or 2.

In some embodiments, R 1 is chosen from absent, hydrogen, C 1 -C 4 alkyl groups, C 1 -C 4 alkylcarboxylic acid groups, and C 3 -C 8 cycloalkyl groups. In some embodiments, R 1 is hydrogen. In some embodiments, R 1 is a C 1 -C 4 alkyl group. In some embodiments, R 1 is methyl. In some embodiments, R 1 is ethyl. In some embodiments, R 1 is a C 1 -C 4 alkylcarboxylic acid group. In some embodiments, R 1 is —CH 2 CH 2 CH 2 CO 2 H. In some embodiments, R 1 is a C 3 -C 8 cycloalkyl group. In some embodiments, R 1 is cycloheptyl.

In some embodiments, R 10 is chosen from 6 to 9 membered carbocycles and 6 to 9 membered heterocycles, each of which is substituted with 0 to 2 R a , wherein each R a is independently substituted with 0 to 3 groups independently chosen from halogens, hydroxyl groups, C 1 -C 6 alkyl groups, —(C═O)—(C 1 -C 6 alkyl) groups, —(C═O)—(C 1 -C 6 alkyl)-(3 to 10 membered heterocycle) groups, and C 1 -C 6 alkylcarboxylic acid groups.

In some embodiments, the carbocycle is a phenyl substituted with 0 to 2 R a , wherein each R a is independently substituted with 0 to 3 groups independently chosen from halogens, hydroxyl groups, C 1 -C 6 alkyl groups, —(C═O)—(C 1 -C 6 alkyl) groups, —(C═O)—(C 1 -C 6 alkyl)-(3 to 10 membered heterocycle) groups, and C 1 -C 6 alkylcarboxylic acid groups. In some embodiments, the phenyl is substituted with 2 R a , wherein each R a is independently substituted with 0 to 3 groups independently chosen from halogens, hydroxyl groups, C 1 -C 6 alkyl groups, —(C═O)—(C 1 -C 6 alkyl) groups, —(C═O)—(C 1 -C 6 alkyl)-(3 to 10 membered heterocycle) groups, and C 1 -C 6 alkylcarboxylic acid groups. In some embodiments, the phenyl is

In some embodiments, the heterocycle is a 9 membered heterocycle substituted with 0 to 2 R a , wherein each R a is independently substituted with 0 to 3 groups independently chosen from halogens, hydroxyl groups, C 1 -C 6 alkyl groups, —(C═O)—(C 1 -C 6 alkyl) groups, —(C═O)—(C 1 -C 6 alkyl)-(3 to 10 membered heterocycle) groups, and C 1 -C 6 alkylcarboxylic acid groups. In some embodiments, the 9 membered heterocycle is

In some embodiments, R a is chosen from halogens, 3 to 10 membered carbocycles, and 3 to 10 membered heterocycles, wherein each R a is independently substituted with 0 to 3 groups independently chosen from halogens, hydroxyl groups, C 1 -C 6 alkyl groups, —(C═O)—(C 1 -C 6 alkyl) groups, —(C═O)—(C 1 -C 6 alkyl)-(3 to 10 membered heterocycle) groups, and C 1 -C 6 alkylcarboxylic acid groups. In some embodiments, R a is chosen from halogens,

In some embodiments, R 15 is hydrogen. In some embodiments, R 15 is R 17 . In some embodiments, R 15 is —C(═O)—R 17 . In some embodiments, R 15 is —C(═O)—O—R 17 .

In some embodiments, R 16 is hydrogen. In some embodiments, R 16 is R 17 . In some embodiments, R 16 is —C(═O)—R 17 . In some embodiments, R 16 is —C(═O)—O—R 17 .

In some embodiments, the splicing modulator compound of Formula (VIII) attaches to the linker L, e.g., in an ADC of Formula (I), as shown in Formula (VIII-A):

In various embodiments, the drug moiety is a splicing modulator selected from D2 and D1.

In various embodiments, the drug moiety is D2. In various embodiments, the structure of the D2 drug mole used in the disclosed ADCs is shown below:

In various embodiments, the linker in the ADCs (e.g., ADCs of Formula (I)) described herein covalently attaches to the D2 drug moiety via an amine on the piperazine group. In various embodiments, the drug moiety is a derivative of D2. In various embodiments, the D2 derivative retains at least one biological function or activity as D2 (e.g., SF3b complex binding, in vitro splicing activity, cytotoxicity) but has an altered chemical structure.

›Definitions · 32 of 64

In various embodiments, the drug moiety is D1 or a pharmaceutically acceptable salt thereof. In various embodiments, the structure of the D1 drug moiety used in the disclosed ADCs is shown below:

In various embodiments, the linker in the ADCs (e.g., ADCs of Formula (I)) described herein covalently attaches to the D1 drug moiety via an amine on the piperazine group. In various embodiments, the drug moiety is a derivative of D1. In various embodiments, the D1 derivative retains at least one biological function or activity as D1 (e.g., SF3b complex binding, in vitro splicing activity, cytotoxicity) but has an altered chemical structure.

In some embodiments, the splicing modulator comprises D1:

In some embodiments, the splicing modulator comprises D2:

In some embodiments, the splicing modulator comprises D3:

In some embodiments, the splicing modulator comprises D4:

In some embodiments, the splicing modulator comprises D4′:

In some embodiments, the splicing modulator comprises D5:

In some embodiments, the splicing modulator comprises D6:

In some embodiments, the splicing modulator comprises D7:

In some embodiments, the splicing modulator comprises D8:

In some embodiments, the splicing modulator comprises D9:

In some embodiments, the splicing modulator comprises D10:

In some embodiments, the splicing modulator comprises D11:

In some embodiments, the splicing modulator comprises D12:

In some embodiments, the splicing modulator comprises D13:

In some embodiments, the splicing modulator comprises D14:

In some embodiments, the splicing modulator comprises D15:

In some embodiments, the splicing modulator comprises D16:

In some embodiments, the splicing modulator comprises D17:

In some embodiments, the splicing modulator comprises D18:

In some embodiments, the splicing modulator comprises D19:

In some embodiments, the splicing modulator comprises D20:

In some embodiments, the splicing modulator comprises D21:

In some embodiments, the splicing modulator comprises D22:

In some embodiments, the splicing modulator comprises D23:

In some embodiments, the splicing modulator comprises D24:

In some embodiments, the splicing modulator comprises D25:

In some embodiments, the splicing modulator comprises D26:

In some embodiments, the splicing modulator comprises D27:

In some embodiments, the splicing modulator comprises D28:

In some embodiments, the splicing modulator comprises D29:

In some embodiments, the splicing modulator comprises D30:

In some embodiments, the splicing modulator comprises D31:

In some embodiments, the splicing modulator comprises D32:

In some embodiments, the splicing modulator comprises D33:

In some embodiments, the splicing modulator comprises D34:

In some embodiments, the splicing modulator comprises D35:

An exemplary ADC has Formula (I):

Ab-(L-D) p (I)

wherein

Ab is an antibody or antigen binding fragment which targets a neoplastic cell;

D is D2;

L is a linker that covalently attaches Ab to D; and

p is an integer from 1 to 15.

In some embodiments, the antibody or antigen binding fragment targets a cell expressing HER2, CD138, EPHA2, MSLN, FOLH1, CDH6, CEACAM5, CFC1B, ENPP3, FOLR1, HAVCR1, KIT, MET, MUC16, SLC39A6, SLC44A4, and/or STEAP1.

In some embodiments, the antibody or antigen binding fragment targets a HER2-expressing cell. In some embodiments, the antibody or antigen binding fragment is an anti-HER2 antibody or antigen binding fragment. In some embodiments, the antibody or antigen binding fragment comprises three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) comprising amino acid sequences of SEQ ID NO:1 (HCDR1), SEQ ID NO:2 (HCDR2), and SEQ ID NO:3 (HCDR3); and three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) comprising amino acid sequences of SEQ ID NO:4 (LCDR1), SEQ ID NO:5 (LCDR2), and SEQ ID NO:6 (LCDR3). In some embodiments, the antibody or antigen binding fragment comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO:19, and a light chain variable region comprising an amino acid sequence of SEQ ID NO:20. In some embodiments, the antibody or antigen binding fragment comprises a human IgG1 heavy chain constant region. In some embodiments, the antibody or antigen binding fragment comprises a human Ig kappa light chain constant region.

In some other embodiments, the antibody or antigen binding fragment targets a CD138-expressing cell. In some embodiments, the antibody or antigen binding fragment is an anti-CD138 antibody or antigen binding fragment. In some embodiments, the antibody or antigen binding fragment comprises three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) comprising amino acid sequences of SEQ ID NO:7 (HCDR1), SEQ ID NO:8 (HCDR2), and SEQ ID NO:9 (HCDR3); and three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) comprising amino acid sequences of SEQ ID NO:10 (LCDR1), SEQ ID NO:11 (LCDR2), and SEQ ID NO:12 (LCDR3). In some embodiments, the antibody or antigen binding fragment comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO:21, and a light chain variable region comprising an amino acid sequence of SEQ ID NO:22. In some embodiments, the antibody or antigen binding fragment comprises a murine IgG2a heavy chain constant region. In some embodiments, the antibody or antigen binding fragment comprises a murine Ig kappa light chain constant region. In some embodiments, the antibody or antigen binding fragment comprises a human IgG2a heavy chain constant region. In some embodiments, the antibody or antigen binding fragment comprises a human Ig kappa light chain constant region.

In some other embodiments, the antibody or antigen binding fragment targets an EPHA2-expressing cell. In some embodiments, the antibody or antigen binding fragment is an anti-EPHA2 antibody or antigen binding fragment. In some embodiments, the antibody or antigen binding fragment comprises three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) comprising amino acid sequences of SEQ ID NO:13 (HCDR1), SEQ ID NO:14 (HCDR2), and SEQ ID NO:15 (HCDR3); and three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) comprising amino acid sequences of SEQ ID NO:16 (LCDR1), SEQ ID NO:17 (LCDR2), and SEQ ID NO:18 (LCDR3). In some embodiments, the antibody or antigen binding fragment comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO:23, and a light chain variable region comprising an amino acid sequence of SEQ ID NO:24. In some embodiments, the antibody or antigen binding fragment comprises a human IgG1 heavy chain constant region. In some embodiments, the antibody or antigen binding fragment comprises a human Ig kappa light chain constant region.

›Definitions · 33 of 64

In some other embodiments, the antibody or antigen binding fragment targets a MSLN-expressing cell. In some embodiments, the antibody or antigen binding fragment is an anti-MSLN antibody or antigen binding fragment.

In some other embodiments, the antibody or antigen binding fragment targets a FOLH1-expressing cell. In some embodiments, the antibody or antigen binding fragment is an anti-FOLH1 antibody or antigen binding fragment.

In some other embodiments, the antibody or antigen binding fragment targets a CDH6-expressing cell. In some embodiments, the antibody or antigen binding fragment is an anti-CDH6 antibody or antigen binding fragment.

In some other embodiments, the antibody or antigen binding fragment targets a CEACAM5-expressing cell. In some embodiments, the antibody or antigen binding fragment is an anti-CEACAM5 antibody or antigen binding fragment.

In some other embodiments, the antibody or antigen binding fragment targets a CFC1B-expressing cell. In some embodiments, the antibody or antigen binding fragment is an anti-CFC1B antibody or antigen binding fragment.

In some other embodiments, the antibody or antigen binding fragment targets an ENPP3-expressing cell. In some embodiments, the antibody or antigen binding fragment is an anti-ENPP3 antibody or antigen binding fragment.

In some other embodiments, the antibody or antigen binding fragment targets a FOLR1-expressing cell. In some embodiments, the antibody or antigen binding fragment is an anti-FOLR1 antibody or antigen binding fragment.

In some other embodiments, the antibody or antigen binding fragment targets a HAVCR1-expressing cell. In some embodiments, the antibody or antigen binding fragment is an anti-HAVCR1 antibody or antigen binding fragment.

In some other embodiments, the antibody or antigen binding fragment targets a KIT-expressing cell. In some embodiments, the antibody or antigen binding fragment is an anti-KIT antibody or antigen binding fragment.

In some other embodiments, the antibody or antigen binding fragment targets a MET-expressing cell. In some embodiments, the antibody or antigen binding fragment is an anti-MET antibody or antigen binding fragment.

In some other embodiments, the antibody or antigen binding fragment targets a MUC16-expressing cell. In some embodiments, the antibody or antigen binding fragment is an anti-MUC16 antibody or antigen binding fragment.

In some other embodiments, the antibody or antigen binding fragment targets a SLC39A6-expressing cell. In some embodiments, the antibody or antigen binding fragment is an anti-SLC39A6 antibody or antigen binding fragment.

In some other embodiments, the antibody or antigen binding fragment targets a SLC44A4-expressing cell. In some embodiments, the antibody or antigen binding fragment is an anti-SLC44A4 antibody or antigen binding fragment.

In some other embodiments, the antibody or antigen binding fragment targets a STEAP1-expressing cell. In some embodiments, the antibody or antigen binding fragment is an anti-STEAP1 antibody or antigen binding fragment.

Another exemplary ADC has Formula (I):

Ab-(L-D) p (I)

wherein

Ab is an antibody or antigen binding fragment which targets a neoplastic cell;

D is D1;

L is a linker that covalently attaches Ab to D; and

p is an integer from 1 to 15.

›Definitions · 34 of 64

Another exemplary ADC has Formula (I):

Ab-(L-D) p (I)

wherein

Ab is an antibody or antigen binding fragment which targets a neoplastic cell;

D is D4;

L is a linker that covalently attaches Ab to D; and

p is an integer from 1 to 15.

›Definitions · 35 of 64

In some other embodiments, the antibody or antigen binding fragment targets a ENPP3-expressing cell. In some embodiments, the antibody or antigen binding fragment is an anti-ENPP3 antibody or antigen binding fragment.

Another exemplary ADC has Formula (I):

Ab-(L-D) p (I)

wherein

Ab is an antibody or antigen binding fragment which targets a neoplastic cell;

D is D12;

L is a linker that covalently attaches Ab to D; and

p is an integer from 1 to 15.

›Definitions · 36 of 64

Another exemplary ADC has Formula (I):

Ab-(L-D) p (I)

wherein

Ab is an antibody or antigen binding fragment which targets a neoplastic cell;

D is D15;

L is a linker that covalently attaches Ab to D; and

p is an integer from 1 to 15.

›Definitions · 37 of 64

In some embodiments, L is selected from any of the linkers disclosed herein, or any combination of linker components disclosed herein. In some embodiments, L is a cleavable linker. In some embodiments, L is a non-cleavable linker. In some embodiments, L is a linker comprising MC-Val-Cit-pABC, MC-Val-Ala-pABC, MC-Val-Ala-pAB, MC-Glu-Val-Cit-pABC, Ala-Ala-Asn-pABC, or @-glucuronide. In some embodiments, L is a linker comprising Mal-Hex, Mal-Et, or Mal-Et-O-Et. In some embodiments, L is a linker comprising MC-Val-Cit-pABC, Mal-(PEG) 2 -CO, MC-Val-Ala-pAB, MC-Val-Ala-pABC, MC-Val-Cit-pAB, Mal-Hex, Mal-Et, or Mal-Et-O-Et. In some embodiments, the linker may also comprise one or more additional spacer units. In some embodiments, L is an ADL1, ADL2, ADL5, ADL6, ADL7, ADL12, ADL13, ADL14, ADL21, ADL23, or ADL15 linker. In some embodiments, L is an ADL12, ADL14, or ADL15 linker. In some embodiments, the ADL1, ADL2, ADL5, ADL6, ADL7, ADL12, ADL13, ADL14, ADL21, ADL23, or ADL15 linker may also comprise one or more additional spacer units.

In some embodiments, p is from 1 to 10. In some embodiments, p is from 2 to 8. In some embodiments, p is from 4 to 8. In some embodiments, p is 4. In some embodiments, p is 8.

Another exemplary ADC has Formula (I):

Ab-(L-D) p (I)

wherein Ab is an anti-HER2 antibody or antigen binding fragment comprising three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) comprising amino acid sequences of SEQ ID NO:1 (HCDR1), SEQ ID NO:2 (HCDR2), and SEQ ID NO:3 (HCDR3); and three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) comprising amino acid sequences of SEQ ID NO:4 (LCDR1), SEQ ID NO:5 (LCDR2), and SEQ ID NO:6 (LCDR3);

D is D2, D1, D4, D12, or D15;

L is a linker that covalently attaches Ab to D; and

p is an integer from 1 to 15.

In some embodiments, the antibody or antigen binding fragment comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO:19, and a light chain variable region comprising an amino acid sequence of SEQ ID NO:20.

›Definitions · 38 of 64

Another exemplary ADC has Formula (I):

Ab-(L-D) p (I)

wherein Ab is an anti-CD138 antibody or antigen binding fragment comprising three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) comprising amino acid sequences of SEQ ID NO:7 (HCDR1), SEQ ID NO:8 (HCDR2), and SEQ ID NO:9 (HCDR3); and three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) comprising amino acid sequences of SEQ ID NO:10 (LCDR1), SEQ ID NO:11 (LCDR2), and SEQ ID NO:12 (LCDR3);

D is D2, D1, D4, D12, or D15;

L is a linker that covalently attaches Ab to D; and

p is an integer from 1 to 15.

In some embodiments, the antibody or antigen binding fragment comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO:21, and a light chain variable region comprising an amino acid sequence of SEQ ID NO:22.

Another exemplary ADC has Formula (I):

Ab-(L-D) p (I)

wherein Ab is an anti-EPHA2 antibody or antigen binding fragment comprising three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) comprising amino acid sequences of SEQ ID NO:13 (HCDR1), SEQ ID NO:14 (HCDR2), and SEQ ID NO:15 (HCDR3); and three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) comprising amino acid sequences of SEQ ID NO:16 (LCDR1), SEQ ID NO:17 (LCDR2), and SEQ ID NO:18 (LCDR3);

D is D2, D1, D4, D12, or D15;

L is a linker that covalently attaches Ab to D; and

p is an integer from 1 to 15.

In some embodiments, the antibody or antigen binding fragment comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO:23, and a light chain variable region comprising an amino acid sequence of SEQ ID NO:24.

In various embodiments, ADCs comprising a D2 or D1 drug moiety can include a cleavable or non-cleavable linker.

In various embodiments, the linker is a cleavable linker. In various embodiments, the cleavable linker comprises MC-Val-Cit-pABC. In various embodiments, the cleavable linker comprises MC-Val-Ala-pABC. In various embodiments, the cleavable linker comprises MC-Val-Ala-pAB. In various embodiments, the cleavable linker comprises MC-Glu-Val-Cit-pABC. In various embodiments, the cleavable linker comprises MC-Ala-Ala-Asn-pABC.

In various other embodiments, the linker is a non-cleavable linker. In various embodiments, the non-cleavable linker comprises MC, alone or in combination with at least one additional spacer unit. In various embodiments, the non-cleavable linker comprises Mal-Hex, alone or in combination with at least one additional spacer unit. In various embodiments, the non-cleavable linker comprises Mal-Et, alone or in combination with at least one additional spacer unit. In various embodiments, the non-cleavable linker comprises Mal-Et-O-Et, alone or in combination with at least one additional spacer unit.

In various embodiments, in ADCs comprising a D2 or D1 drug moiety, p is from 1 to 10. In various embodiments, p is from 2 to 8. In various embodiments, p is from 4 to 8. In various embodiments, p is 4. In various embodiments, p is 8.

In various embodiments, an ADC comprising a D2 or D1 drug moiety as disclosed herein demonstrates improved drug:antibody ratio, lower aggregation levels, increased stability, increased on-target killing of cancer cells, decreased off-target killing of non-cancer cells, and/or increased cytotoxicity and/or potency relative to an ADC comprising an alternate drug moiety (e.g., an alternate splicing modulator drug moiety). In some embodiments, an ADC comprising a D2 or D1 drug moiety as disclosed herein provides good or superior properties in one or more of the categories listed above, or across a spectrum of functional properties for a therapeutic ADC. In some embodiments, an ADC comprising a D2 or D1 drug moiety exhibits surprisingly effective potency and increased inhibition of cell growth and/or proliferation in cells that express the antigen targeted by the ADC, as compared to an ADC comprising an alternate drug moiety (e.g., an alternate splicing modulator drug moiety). In some embodiments, potency can be measured in terms of the concentration of compound to cause 50% reduction in cell proliferation (GI 50 ). In various embodiments, ADCs comprising a D2 or D1 drug moiety exhibit surprisingly increased in vivo stability (e.g., plasma stability), as compared to an ADC with an alternate drug moiety (e.g., an alternate splicing modulator drug moiety, e.g., thailanstatin A). See, e.g., the ADC described in Puthenveetil et al. (Bioconjugate Chem. (2016) 27:1880-8), which shows complete bioconversion of payload by 72 hours (i.e., acetate is completely hydrolyzed).

›Definitions · 39 of 64

In certain embodiments, an intermediate, which is the precursor of the linker moiety, is reacted with the drug moiety (e.g., the splicing modulator) under appropriate conditions. In certain embodiments, reactive groups are used on the drug and/or the intermediate or linker. The product of the reaction between the drug and the intermediate, or the derivatized drug (drug plus linker), is subsequently reacted with the antibody or antigen binding fragment under appropriate conditions. Alternatively, the intermediate or linker may first be reacted with the antibody or antigen binding fragment, or a derivatized antibody or antigen binding fragment, and then reacted with the drug or derivatized drug.

A number of different reactions are available for covalent attachment of the drug moiety and/or linker moiety to the antibody or antigen binding fragment. This is often accomplished by reaction of one or more amino acid residues of the antibody or antigen binding fragment, including the amine groups of lysine, the free carboxylic acid groups of glutamic acid and aspartic acid, the sulfhydryl groups of cysteine, and the various moieties of the aromatic amino acids. For instance, non-specific covalent attachment may be undertaken using a carbodiimide reaction to link a carboxy (or amino) group on a drug moiety to an amino (or carboxy) group on an antibody or antigen binding fragment. Additionally, bifunctional agents such as dialdehydes or imidoesters may also be used to link the amino group on a drug moiety to an amino group on an antibody or antigen binding fragment. Also available for attachment of drugs (e.g., a splicing modulator) to binding agents is the Schiff base reaction. This method involves the periodate oxidation of a drug that contains glycol or hydroxy groups, thus forming an aldehyde which is then reacted with the binding agent. Attachment occurs via formation of a Schiff base with amino groups of the binding agent. Isothiocyanates may also be used as coupling agents for covalently attaching drugs to binding agents. Other techniques are known to the skilled artisan and within the scope of the present disclosure. Examples of drug moieties that can be generated and linked to an antibody or antigen binding fragment using various chemistries known to in the art include splicing modulators, e.g., the splicing modulators described and exemplified herein.

Linker-Drug/Drug Compounds

Further disclosed herein are exemplary linker-drug (L-D) compounds, as well as compositions comprising multiple copies of such compounds. In various embodiments, the linker-drug compounds disclosed herein can be defined by the generic formula: L-D, wherein L=a linker moiety, and D=a drug moiety (e.g., a splicing modulator drug moiety). In certain embodiments, the disclosed L-D compounds are suitable for use in the ADCs described herein, e.g., in ADCs of Formula (I).

or a pharmaceutically acceptable salt thereof, wherein:

R 2 is absent or a linker;

R 4 , R 5 , and R 8 are each independently chosen from hydrogen, hydroxyl groups, —O—(C 1 -C 6 alkyl) groups, —O—C(═O)—(C 1 -C 6 alkyl) groups, and C 1 -C 6 alkyl groups;

R 6 and R 7 are each independently chosen from hydrogen, —O—R 17 , —O—C(═O)—R 17 , —O—C(═O)—NR 15 R 16 , C 1 -C 6 alkyl groups, —NR 15 R 16 , and a linker;

R 15 and R 16 are each independently chosen from hydrogen, R 17 , —C(═O)—R 17 , and —C(═O)—O—R 17 ;

R 17 is chosen from hydrogen, C 1 -C 6 alkyl groups, C 3 -C 8 cycloalkyl groups, benzyl groups, and C 3 -C 8 heterocyclyl groups; and

Z″ is chosen from

wherein at least one of R 6 and R 7 is hydrogen; and

wherein if R 2 is a linker, then neither R 6 or R 7 is a linker, and if R 6 or R 7 is a linker, then R 2 is absent.

In some embodiments, R 1 is chosen from absent, hydrogen, C 1 -C 4 alkyl groups, C 1 -C 4 alkylcarboxylic acid groups, and C 3 -C 8 cycloalkyl groups. In some embodiments, R 1 is absent. In some embodiments, R 1 is hydrogen. In some embodiments, R 1 is a C 1 -C 4 alkyl group. In some embodiments, R 1 is methyl. In some embodiments, R 1 is ethyl. In some embodiments, R 1 is a C 1 -C 4 alkylcarboxylic acid group. In some embodiments, R 1 is —CH 2 CH 2 CH 2 CO 2 H. In some embodiments, R 1 is a C 3 -C 8 cycloalkyl group. In some embodiments, R 1 is cycloheptyl.

›Definitions · 40 of 64

In some embodiments, R 6 is hydrogen and R 7 is —O—R 17 . In some embodiments, R 6 is hydrogen and R 7 is —OR 17 , wherein R 17 is chosen from hydrogen and C 1 -C 4 alkyl groups. In some embodiments, R 6 is hydrogen and R 7 is —O—R 17 , wherein R 17 is hydrogen. In some embodiments, R 6 is —O—R 17 and R 7 is hydrogen. In some embodiments, R 6 is —O—R 17 and R 7 is hydrogen, wherein R 17 is chosen from hydrogen and C 1 -C 4 alkyl groups. In some embodiments, R 6 is —O—R 17 and R 7 is hydrogen, wherein R 17 is hydrogen. In some embodiments, R 6 is hydrogen and R 7 is —NR 15 R 16 . In some embodiments, R 6 is hydrogen and R 7 is —NR 15 R 16 , wherein R 15 is H and R 16 is chosen from hydrogen, R 17 , —C(═O)—R 17 , and —C(═O)—O—R 17 . In some embodiments, Re is hydrogen and R 6 is —NR 15 R 16 , wherein R 15 is H and R 16 is chosen from hydrogen, R 17 , —C(═O)—R 17 , and —C(═O)—O—R 17 , and wherein R 17 is chosen from hydrogen, C 1 -C 6 alkyl groups, C 3 -C 8 cycloalkyl groups, and C 3 -C 8 heterocyclyl groups. In some embodiments, R 6 is —O—R 17 . In some embodiments, R 6 is —O—C(═O)—R 17 . In some embodiments, R 6 is C 1 -C 6 alkyl. In some embodiments, R 6 is C 1 -C 4 alkyl. In some embodiments, R 6 is C 1 alkyl. In some embodiments, R 6 is —NR 15 R 16 .

In some embodiments, R 15 is hydrogen. In some embodiments, R 15 is R 17 . In some embodiments, R 15 is —C(═O)—R 17 . In some embodiments, R 15 is —C(═O)—O—R 17 .

In some embodiments, R 16 is hydrogen. In some embodiments, R 16 is R 17 . In some embodiments, R 16 is —C(═O)—R 17 . In some embodiments, R 16 is —C(═O)—O—R 17 .

In some embodiments, Z′ is

In some embodiments, R 2 is a linker. In some embodiments, the linker comprises at least one cleavable peptide moiety. In some embodiments, the at least one cleavable peptide moiety is cleavable by an enzyme. In some embodiments, the linker or cleavable peptide moiety comprises at least one amino acid unit. In some embodiments, the at least one amino acid unit is chosen from arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, cysteine, selenocysteine, glycine, proline, alanine, valine, isoleucine, methionine, phenylalanine, tyrosine, tryptophan, and citrulline. In some embodiments, the at least one amino acid unit is chosen from alanine, citrulline, and valine. In some embodiments, the linker comprises citrulline and valine. In some embodiments, the linker comprises alanine and valine.

In some embodiments, the linker comprises a moiety chosen from a sulfonamide, a β-glucuronide, a disulfide, and a carbonyl. In some embodiments, the linker comprises a sulfonamide. In some embodiments, the linker comprises a β-glucuronide. In some embodiments, the linker comprises a disulfide. In some embodiments, the linker comprises a carbonyl.

In some embodiments, the linker comprises a spacer unit. In some embodiments, the spacer unit is chosen from alkyl groups and polyethylene glycol (PEG) moieties. In some embodiments, the alkyl group is a C 1 -C 12 alkyl group. In some embodiments, the alkyl group is a C 1 -C 6 alkyl group. In some embodiments, the alkyl group is methylene. In some embodiments, the alkyl group is ethylene. In some embodiments, the alkyl group is n-propylene. In some embodiments, the alkyl group is n-butylene. In some embodiments, the alkyl group is n-pentylene. In some embodiments, the alkyl group is n-hexylene. In some embodiments, the PEG moiety comprises -(PEG) m -, wherein m is an integer from 1 to 10. In some embodiments, m is 1. In some embodiments, m is 2. In some embodiments, m is 3. In some embodiments, m is 4. In some embodiments, m is 5. In some embodiments, m is 6.

In some embodiments, the linker comprises a maleimide (Mal) moiety (“Mal-spacer unit”). In some embodiments, the linker comprises a self-immolative spacer unit. In some embodiments, the self-immolative spacer unit is chosen from p-aminobenzyloxycarbonyl (pABC) and p-aminobenzyl (pAB).

›Definitions · 41 of 64

In some embodiments, the linker comprises a Mal-spacer unit, an alkyl group, at least one amino acid unit, and a self-immolative spacer. In some embodiments, at least one amino acid unit is chosen from alanine, citrulline, and valine. In some embodiments, the at least one amino acid unit comprises alanine and valine. In some embodiments, the at least one amino acid unit comprises citrulline and valine. In some embodiments, the self-immolative spacer is chosen from pAB and pABC. In some embodiments, the self-immolative spacer comprises pAB. In some embodiments, the self-immolative spacer comprises pABC. In some embodiments, the alkyl group comprises a C 1 -C 6 alkyl group.

In some embodiments, the linker comprises a Mal-spacer unit, PEG moiety, at least one amino acid unit, and a self-immolative spacer. In some embodiments, at least one amino acid unit is chosen from alanine, citrulline, and valine. In some embodiments, the at least one amino acid unit comprises alanine and valine. In some embodiments, the at least one amino acid unit comprises citrulline and valine. In some embodiments, the self-immolative spacer is chosen from pAB and pABC. In some embodiments, the self-immolative spacer comprises pAB. In some embodiments, the self-immolative spacer comprises pABC. In some embodiments, the PEG moiety comprises -(PEG) m -, wherein m is an integer from 1 to 6.

or a pharmaceutically acceptable salt thereof, wherein:

R 2 is absent or a linker;

R 4 , R 5 , and R 8 are each independently chosen from hydrogen, hydroxyl groups, —O—(C 1 -C 6 alkyl) groups, —O—C(═O)—(C 1 -C 6 alkyl) groups, and C 1 -C 6 alkyl groups;

R 6 and R 7 are each independently chosen from hydrogen, —O—R 17 , —O—C(═O)—R 17 , —O—C(═O)—NR 15 R 16 , C 1 -C 6 alkyl groups, —NR 15 R 16 , and a linker;

R 15 and R 16 are each independently chosen from hydrogen, R 17 , —C(═O)—R 17 , and —C(═O)—O—R 17 ; and

R 17 is chosen from hydrogen, C 1 -C 6 alkyl groups, C 3 -C 8 cycloalkyl groups, benzyl groups, and C 3 -C 8 heterocyclyl groups;

wherein at least one of R 6 and R 7 is hydrogen; and

wherein if R 2 is a linker, then neither R 6 or R 7 is a linker, and if R 6 or R 7 is a linker, then R 2 is absent.

In some embodiments, R 6 is hydrogen and R 7 is —O—R 17 . In some embodiments, R 6 is hydrogen and R 7 is —OR 17 , wherein R 17 is chosen from hydrogen and C 1 -C 4 alkyl groups. In some embodiments, R 6 is hydrogen and R 7 is —O—R 17 , wherein R 17 is hydrogen. In some embodiments, R 6 is —O—R 17 and R 7 is hydrogen. In some embodiments, R 6 is —O—R 17 and R 7 is hydrogen, wherein R 17 is chosen from hydrogen and C 1 -C 4 alkyl groups. In some embodiments, R 6 is —O—R 17 and R 7 is hydrogen, wherein R 17 is hydrogen. In some embodiments, R 6 is hydrogen and R 7 is —NR 15 R 16 . In some embodiments, R 6 is hydrogen and R 7 is —NR 15 R 16 , wherein R 15 is H and R 16 is chosen from hydrogen, R 17 , —C(═O)—R 17 , and —C(═O)—O—R 17 . In some embodiments, Re is hydrogen and R 7 is —NR 15 R 16 , wherein R 15 is H and R 16 is chosen from hydrogen, R 17 , —C(═O)—R 17 , and —C(═O)—O—R 17 , and wherein R 17 is chosen from hydrogen, C 1 -C 6 alkyl groups, C 3 -C 8 cycloalkyl groups, and C 3 -C 8 heterocyclyl groups. In some embodiments, R 6 is —O—R 17 . In some embodiments, R 6 is —O—C(═O)—R 17 . In some embodiments, R 6 is C 1 -C 6 alkyl. In some embodiments, R 6 is C 1 -C 4 alkyl. In some embodiments, R 6 is C 1 alkyl. In some embodiments, R 6 is —NR 15 R 16 .

›Definitions · 42 of 64

In some embodiments, R 15 is hydrogen. In some embodiments, R 15 is R 17 . In some embodiments, R 15 is —C(═O)—R 17 . In some embodiments, R 15 is —C(═O)—O—R 17 .

In some embodiments, R 16 is hydrogen. In some embodiments, R 16 is R 17 . In some embodiments, R 16 is —C(═O)—R 17 . In some embodiments, R 16 is —C(═O)—O—R 17 .

or a pharmaceutically acceptable salt thereof, wherein:

›Definitions · 43 of 64

R 2 is absent or a linker;

R 4 , R 5 , and R 8 are each independently chosen from hydrogen, hydroxyl groups, —O—(C 1 -C 6 alkyl) groups, —O—C(═O)—(C 1 -C 6 alkyl) groups, and C 1 -C 6 alkyl groups;

R 6 and R 7 are each independently chosen from hydrogen, —O—R 17 , —O—C(═O)—R 17 , —O—C(═O)—NR 15 R 16 , C 1 -C 6 alkyl groups, —NR 15 R 16 , and a linker;

R 10 is chosen from hydrogen, C 1 -C 6 alkyl groups, —C(═O)—(C 1 -C 6 alkyl) groups, and —CD 3 ;

R 15 and R 16 are each independently chosen from hydrogen, R 17 , —C(═O)—R 17 , and —C(═O)—O—R 17 ;

R 17 is chosen from hydrogen, C 1 -C 6 alkyl groups, C 3 -C 8 cycloalkyl groups, benzyl groups, and C 3 -C 8 heterocyclyl groups; and

a is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

wherein at least one of R 6 and R 7 is hydrogen;

wherein if R 2 is a linker, then neither R 6 or R 7 is a linker, and if R 6 or R 7 is a linker, then R 2 is absent; and

wherein R 1 and R 9 cannot both be absent.

In some embodiments, R 2 is absent and R 6 is a linker. In some embodiments, R 2 is absent and R 7 is a linker. In some embodiments, R 2 is a linker. In some embodiments, R 6 is hydrogen. In some embodiments, R 7 is hydrogen. In some embodiments, R 6 is hydrogen and R 7 is —O—R 17 . In some embodiments, R 6 is hydrogen and R 7 is —OR 17 , wherein R 17 is chosen from hydrogen and C 1 -C 4 alkyl groups. In some embodiments, R 6 is hydrogen and R 7 is —O—R 17 , wherein R 17 is hydrogen. In some embodiments, R 6 is —O—R 17 and R 7 is hydrogen. In some embodiments, R 6 is —O—R 17 and R 7 is hydrogen, wherein R 17 is chosen from hydrogen and C 1 -C 4 alkyl groups. In some embodiments, R 6 is —O—R 17 and R 7 is hydrogen, wherein R 17 is hydrogen. In some embodiments, R 6 is hydrogen and R 7 is —NR 15 R 16 . In some embodiments, R 6 is hydrogen and R 7 is —NR 15 R 16 , wherein R 15 is H and R 16 is chosen from hydrogen, R 17 , —C(═O)—R 17 , and —C(═O)—O—R 17 . In some embodiments, R 6 is hydrogen and R 7 is —NR 15 R 16 , wherein R 15 is H and R 16 is chosen from hydrogen, R 17 , —C(═O)—R 17 , and —C(═O)—O—R 17 , and wherein R 17 is chosen from hydrogen, C 1 -C 6 alkyl groups, C 3 -C 8 cycloalkyl groups, and C 3 -C 8 heterocyclyl groups. In some embodiments, R 6 is —O—R 17 . In some embodiments, R 6 is —O—C(═O)—R 17 . In some embodiments, R 6 is C 1 -C 6 alkyl. In some embodiments, R 6 is C 1 -C 4 alkyl. In some embodiments, R 6 is C 1 alkyl. In some embodiments, R 6 is —NR 15 R 16 .

›Definitions · 44 of 64

In some embodiments, R 15 is hydrogen. In some embodiments, R 15 is R 17 . In some embodiments, R 15 is —C(═O)—R 17 . In some embodiments, R 15 is —C(═O)—O—R 17 .

In some embodiments, R 16 is hydrogen. In some embodiments, R 16 is R 17 . In some embodiments, R 16 is —C(═O)—R 17 . In some embodiments, R 16 is —C(═O)—O—R 17 .

›Definitions · 45 of 64

or a pharmaceutically acceptable salt thereof, wherein:

R 2 is a linker;

R 4 is chosen from hydrogen, hydroxyl groups, —O—(C 1 -C 6 alkyl) groups, —O—C(═O)—(C 1 -C 6 alkyl) groups, and C 1 -C 6 alkyl groups;

R 15 and R 16 are each independently chosen from hydrogen, R 17 , —C(═O)—R 17 , and —C(═O)—O—R 17 ; and

R 17 is chosen from hydrogen, C 1 -C 6 alkyl groups, C 3 -C 8 cycloalkyl groups, benzyl groups, and C 3 -C 8 heterocyclyl groups;

wherein each R a is independently substituted with 0 to 3 groups independently chosen from halogens, hydroxyl groups, —NR 15 R 16 , C 1 -C 6 alkyl groups, —(C═O)—(C 1 -C 6 alkyl) groups, —(C═O)—(C 1 -C 6 alkyl)-(C 3 -C 10 heterocyclyl groups), and C 1 -C 6 alkylcarboxylic acid groups, each of which is substituted with 0, 1, or 2 groups independently chosen from halogens, hydroxyl groups, —NR 15 R 16 , and C 1 -C 3 alkyl groups; and

w is 0, 1, or 2.

›Definitions · 46 of 64

is chosen from halogens,

In some embodiments, R 15 is hydrogen. In some embodiments, R 15 is R 17 . In some embodiments, R 15 is —C(═O)—R 17 . In some embodiments, R 15 is —C(═O)—O—R 17 .

In some embodiments, R 16 is hydrogen. In some embodiments, R 16 is R 17 . In some embodiments, R 16 is —C(═O)—R 17 . In some embodiments, R 16 is —C(═O)—O—R 17 .

In some embodiments, the drug moiety is a splicing modulator chosen from D1, D2, D3, D4, D4′, D5, D6, D7, D8, D9, D10, D11, D12, D13, D14, D15, D16, D17, D18, D19, D20, D21, D22, D23, D24, D25, D26, D27, D28, D29, D30, D31, D32, D33, D34, and D35.

In various embodiments, the drug moiety is a splicing modulator selected from D2 and D1.

In various embodiments, the drug moiety is D2. In various embodiments, the structure of the D2 drug moiety used in the disclosed linker-drug (L-D) compounds is shown below:

In various embodiments, the linker in the linker-drug (L-D) compounds described herein covalently attaches to the D2 drug moiety via an amine on the piperazine group. In various embodiments, the drug moiety is a derivative of D2. In various embodiments, the D2 derivative retains at least one biological function or activity as D2 (e.g., SF3b complex binding, in vitro splicing activity, cytotoxicity) but has an altered chemical structure.

In various embodiments, the drug moiety is D1. In various embodiments, the structure of the D1 drug moiety used in the disclosed linker-drug (L-D) compounds is shown below:

›Definitions · 47 of 64

In various embodiments, the linker in the linker-drug (L-D) compounds described herein covalently attaches to the D1 drug moiety via an amine on the piperazine group. In various embodiments, the drug moiety is a derivative of D1. In various embodiments, the D1 derivative retains at least one biological function or activity as D1 (e.g., SF3b complex binding, in vitro splicing activity, cytotoxicity) but has an altered chemical structure

Also disclosed herein are exemplary splicing modulator drug compounds, for use on their own or as drug moieties in the ADCs disclosed herein. The present disclosure, in various embodiments, further provides compositions comprising multiple copies of such compounds, e.g., pharmaceutical compositions comprising a splicing modulator drug compound and a pharmaceutically acceptable carrier.

In various embodiments, the present disclosure provides a compound chosen from a compound of formula

and pharmaceutically acceptable salts thereof. In various embodiments, the present disclosure further provides a pharmaceutical composition comprising the compound, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

In various other embodiments, the present disclosure provides a compound chosen from a compound of formula

and a pharmaceutically acceptable sals thereof. In various embodiments, the present disclosure further provides a pharmaceutical composition comprising the compound, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier

In various other embodiments, the present disclosure provides a compound chosen from a compound of formula

Bioconjugation

In some embodiments, a linker-drug compound may be conjugated to an antibody or antigen binding fragment according to the exemplary scheme depicted in FIG. 26 . Briefly, in some embodiments, an antibody or antigen binding fragment may be treated with a reagent, e.g., a reducing agent, e.g., tris(2-carboxyethyl)phosphine, to activate the antibody or antigen binding fragment by reducing one or more disulfide bonds. In some embodiments, the activated antibody or antigen binding fragment is then treated with a linker-drug compound at a predetermined stoichiometry. Subsequently, in some embodiments, the mixture is then subjected to a purification technique, e.g., size exclusion resin or ultrafiltration to afford the desired antibody-drug conjugate.

In some embodiments, provided herein is an antibody-drug conjugate having the structure

wherein Ab is an antibody or antigen binding fragment covalently bound to a maleimide group of the linker-drug compound (ADL1-D1) through the sulfur atom of a thiol group on the antibody or antigen binding fragment.

›Definitions · 48 of 64

In some embodiments, provided herein is an antibody-drug conjugate having the structure

wherein Ab is an antibody or antigen binding fragment covalently bound to a maleimide group of the linker-drug compound (ADL1-D4) through the sulfur atom of a thiol group on the antibody or antigen binding fragment.

In some embodiments, provided herein is an antibody-drug conjugate having the structure

wherein Ab is an antibody or antigen binding fragment covalently bound to a maleimide group of the linker-drug compound (ADL1-D12) through the sulfur atom of a thiol group on the antibody or antigen binding fragment.

In some embodiments, provided herein is an antibody-drug conjugate having the structure

wherein Ab is an antibody or antigen binding fragment covalently bound to a maleimide group of the linker-drug compound (ADL5-D2) through the sulfur atom of a thiol group on the antibody or antigen binding fragment.

In some embodiments, provided herein is an antibody-drug conjugate having the structure

wherein Ab is an antibody or antigen binding fragment covalently bound to a maleimide group of the linker-drug compound (ADL5-D15) through the sulfur atom of a thiol group on the antibody or antigen binding fragment.

In some embodiments, provided herein is an antibody-drug conjugate having the structure

wherein Ab is an antibody or antigen binding fragment covalently bound to a maleimide group of the linker-drug compound (ADL13-D4) through the sulfur atom of a thiol group on the antibody or antigen binding fragment.

In some embodiments, the Ab is an anti-HER2 antibody or an antigen binding fragment thereof. In some embodiments, the Ab binds to HER2 and targets HER2-expressing neoplastic cells (i.e., the ADC targets HER2-expressing neoplastic cells). In some embodiments, the Ab is an internalizing anti-HER2 antibody or internalizing antigen binding fragment thereof.

In some embodiments, the anti-HER2 antibody or antigen binding fragment comprises three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) comprising amino acid sequences of SEQ ID NO:1 (HCDR1), SEQ ID NO:2 (HCDR2), and SEQ ID NO:3 (HCDR3); and three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3) comprising amino acid sequences of SEQ ID NO:4 (LCDR1), SEQ ID NO:5 (LCDR2), and SEQ ID NO:6 (LCDR3). In some embodiments, the anti-HER2 antibody or antigen binding fragment is an internalizing antibody or internalizing antigen binding fragment. In some embodiments, the anti-HER2 antibody or antigen binding fragment comprises human framework sequences. In some embodiments, the anti-HER2 antibody or antigen binding fragment comprises a heavy chain variable region comprising an amino acid sequence of SEQ ID NO:19, and a light chain variable region comprising an amino acid sequence of SEQ ID NO:20. In some embodiments, the anti-HER2 antibody or antigen binding fragment comprises a human IgG1 heavy chain constant region. In some embodiments, the anti-HER2 antibody or antigen binding fragment comprises a human Ig kappa light chain constant region.

Drug Loading

Drug loading is represented by p, and is also referred to herein as the drug-to-antibody ratio (DAR). Drug loading may range from 1 to 10 drug moieties per antibody or antigen binding fragment. In some embodiments, p is an integer from 1 to 10. In some embodiments, p is an integer from 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2. In some embodiments, p is an integer from 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, or 2 to 3. In some embodiments, p is an integer from 1 to 8. In some embodiments, p is an integer from 2 to 4. In other embodiments, p is an integer from 4 to 8. In other embodiments, p is 1, 2, 3, 4, 5, 6, 7, or 8, preferably 4 or 8.

Drug loading may be limited by the number of attachment sites on the antibody or antigen binding fragment. In some embodiments, the linker moiety (L) of the ADC attaches to the antibody or antigen binding fragment through a chemically active group on one or more amino acid residues on the antibody or antigen binding fragment. For example, the linker may be attached to the antibody or antigen binding fragment via a free amino, imino, hydroxyl, thiol, or carboxyl group (e.g., to the N- or C-terminus, to the epsilon amino group of one or more lysine residues, to the free carboxylic acid group of one or more glutamic acid or aspartic acid residues, or to the sulfhydryl group of one or more cysteine residues). The site to which the linker is attached can be a natural residue in the amino acid sequence of the antibody or antigen binding fragment, or it can be introduced into the antibody or antigen binding fragment, e.g., by DNA recombinant technology (e.g., by introducing a cysteine residue into the amino acid sequence) or by protein biochemistry (e.g., by reduction, pH adjustment, or hydrolysis).

In some embodiments, the number of drug moieties that can be conjugated to an antibody or antigen binding fragment is limited by the number of free cysteine residues. For example, where the attachment is a cysteine thiol group, an antibody may have only one or a few cysteine thiol groups, or may have only one or a few sufficiently reactive thiol groups through which a linker may be attached. Generally, antibodies do not contain many free and reactive cysteine thiol groups that may be linked to a drug moiety. Indeed, most cysteine thiol residues in antibodies are involved in either interchain or intrachain disulfide bonds. Conjugation to cysteines can, in some embodiments, therefore require at least partial reduction of the antibody. Over-attachment of linker-toxin to an antibody may destabilize the antibody by reducing the cysteine residues available to form disulfide bonds. Therefore, an optimal drug:antibody ratio should increase potency of the ADC (by increasing the number of attached drug moieties per antibody) without destabilizing the antibody or antigen binding fragment. In some embodiments, an optimal ratio may be 2, 4, 6, or 8.

›Definitions · 49 of 64

In some embodiments, an antibody or antigen binding fragment is exposed to reducing conditions prior to conjugation in order to generate one or more free cysteine residues. An antibody, in some embodiments, may be reduced with a reducing agent such as dithiothreitol (DTT) or tris(2-carboxyethyl)phosphine (TCEP), under partial or total reducing conditions, to generate reactive cysteine thiol groups. Unpaired cysteines may be generated through partial reduction with limited molar equivalents of TCEP, which can reduce the interchain disulfide bonds which link the light chain and heavy chain (one pair per H-L pairing) and the two heavy chains in the hinge region (two pairs per H—H pairing in the case of human IgG1) while leaving the intrachain disulfide bonds intact (Stefano et al. (2013) Methods Mol Biol. 1045:145-71). In embodiments, disulfide bonds within the antibodies are reduced electrochemically, e.g., by employing a working electrode that applies an alternating reducing and oxidizing voltage. This approach can allow for on-line coupling of disulfide bond reduction to an analytical device (e.g., an electrochemical detection device, an NMR spectrometer, or a mass spectrometer) or a chemical separation device (e.g., a liquid chromatograph (e.g., an HPLC) or an electrophoresis device (see, e.g., U.S. Publ. No. 20140069822)). In certain embodiments, an antibody is subjected to denaturing conditions to reveal reactive nucleophilic groups on amino acid residues, such as cysteine.

The drug loading of an ADC may be controlled in different ways, e.g., by: (i) limiting the molar excess of drug-linker intermediate or linker reagent relative to antibody; (ii) limiting the conjugation reaction time or temperature; (iii) partial or limiting reductive conditions for cysteine thiol modification; and/or (iv) engineering by recombinant techniques the amino acid sequence of the antibody such that the number and position of cysteine residues is modified for control of the number and/or position of linker-drug attachments.

In some embodiments, free cysteine residues are introduced into the amino acid sequence of the antibody or antigen binding fragment. For example, cysteine engineered antibodies can be prepared wherein one or more amino acids of a parent antibody are replaced with a cysteine amino acid. Any form of antibody may be so engineered, i.e. mutated. For example, a parent Fab antibody fragment may be engineered to form a cysteine engineered Fab referred to as a “ThioFab.” Similarly, a parent monoclonal antibody may be engineered to form a “ThioMab.” A single site mutation yields a single engineered cysteine residue in a ThioFab, whereas a single site mutation yields two engineered cysteine residues in a ThioMab, due to the dimeric nature of the IgG antibody. DNA encoding an amino acid sequence variant of the parent polypeptide can be prepared by a variety of methods known in the art (see, e.g., the methods described in Intl. Pub. No. WO 2006/034488). These methods include, but are not limited to, preparation by site-directed (or oligonucleotide-mediated) mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlier prepared DNA encoding the polypeptide. Variants of recombinant antibodies may also be constructed by restriction fragment manipulation or by overlap extension PCR with synthetic oligonucleotides. ADCs of Formula (I) include, but are not limited to, antibodies that have 1, 2, 3, or 4 engineered cysteine amino acids (Lyon et al. (2012) Methods Enzymol. 502:123-38). In some embodiments, one or more free cysteine residues are already present in an antibody or antigen binding fragment, without the use of engineering, in which case the existing free cysteine residues may be used to conjugate the antibody or antigen binding fragment to a drug moiety.

Where more than one nucleophilic group reacts with a drug-linker intermediate or a linker moiety reagent followed by drug moiety reagent, in a reaction mixture comprising multiple copies of the antibody or antigen binding fragment and linker moiety, then the resulting product can be a mixture of ADC compounds with a distribution of one or more drug moieties attached to each copy of the antibody or antigen binding fragment in the mixture. In some embodiments, the drug loading in a mixture of ADCs resulting from a conjugation reaction ranges from 1 to 10 drug moieties attached per antibody or antigen binding fragment. The average number of drug moieties per antibody or antigen binding fragment (i.e., the average drug loading, or average p) may be calculated by any conventional method known in the art, e.g., by mass spectrometry (e.g., reverse-phase LC-MS), and/or high-performance liquid chromatography (e.g., HIC-HPLC). In some embodiments, the average number of drug moieties per antibody or antigen binding fragment is determined by hydrophobic interaction chromatography-high performance liquid chromatography (HIC-HPLC). In some embodiments, the average number of drug moieties per antibody or antigen binding fragment is determined by reverse-phase liquid chromatography-mass spectrometry (LC-MS). In some embodiments, the average number of drug moieties per antibody or antigen binding fragment is from about 1.5 to about 3.5, about 2.5 to about 4.5, about 3.5 to about 5.5, about 4.5 to about 6.5, about 5.5 to about 7.5, about 6.5 to about 8.5, or about 7.5 to about 9.5. In some embodiments, the average number of drug moieties per antibody or antigen binding fragment is from about 2 to about 4, about 3 to about 5, about 4 to about 6, about 5 to about 7, about 6 to about 8, about 7 to about 9, about 2 to about 8, or about 4 to about 8.

In some embodiments, the average number of drug moieties per antibody or antigen binding fragment is about 2. In some embodiments, the average number of drug moieties per antibody or antigen binding fragment is about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2, about 2.1, about 2.2, about 2.3, about 2.4, or about 2.5. In some embodiments, the average number of drug moieties per antibody or antigen binding fragment is 2.

›Definitions · 50 of 64

In some embodiments, the average number of drug moieties per antibody or antigen binding fragment is about 4. In some embodiments, the average number of drug moieties per antibody or antigen binding fragment is about 3.5, about 3.6, about 3.7, about 3.8, about 3.9, about 4, about 4.1, about 4.2, about 4.3, about 4.4, or about 4.5. In some embodiments, the average number of drug moieties per antibody or antigen binding fragment is 4.

In some embodiments, the average number of drug moieties per antibody or antigen binding fragment is about 8. In some embodiments, the average number of drug moieties per antibody or antigen binding fragment is about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8, about 8.1, about 8.2, about 8.3, about 8.4, or about 8.5. In some embodiments, the average number of drug moieties per antibody or antigen binding fragment is 8.

In various embodiments, the term “about,” as used with respect to the average number of drug moieties per antibody or antigen binding fragment, means plus or minus 10%.

Individual ADC compounds, or “species,” may be identified in the mixture by mass spectroscopy and separated by UPLC or HPLC, e.g. hydrophobic interaction chromatography (HIC-HPLC). In certain embodiments, a homogeneous or nearly homogenous ADC product with a single loading value may be isolated from the conjugation mixture, e.g., by electrophoresis or chromatography.

In some embodiments, higher drug loading (e.g., p>8) may cause aggregation, insolubility, toxicity, or loss of cellular permeability of certain antibody-drug conjugates. Higher drug loading may also negatively affect the pharmacokinetics (e.g., clearance) of certain ADCs. In some embodiments, lower drug loading (e.g., p<2) may reduce the potency of certain ADCs against target-expressing cells and/or bystander cells. In some embodiments, the drug loading for an ADC of the present disclosure ranges from about 2 to about 8; from about 2 to about 6; from about 2 to about 5; from about 3 to about 5; from about 2 to about 4; or from about 4 to about 8.

In some embodiments, a drug loading and/or an average drug loading of about 2 is achieved, e.g., using partial reduction of intrachain disulfides on the antibody or antigen binding fragment, and provides beneficial properties. In some embodiments, a drug loading and/or an average drug loading of about 4 is achieved, e.g., using partial reduction of intrachain disulfides on the antibody or antigen binding fragment, and provides beneficial properties. In some embodiments, a drug loading and/or an average drug loading of about 8 is achieved, e.g., using partial reduction of intrachain disulfides on the antibody or antigen binding fragment, and provides beneficial properties. In some embodiments, a drug loading and/or an average drug loading of less than about 2 may result in an unacceptably high level of unconjugated antibody species, which can compete with the ADC for binding to a target antigen and/or provide for reduced treatment efficacy. In some embodiments, a drug loading and/or average drug loading of more than about 8 may result in an unacceptably high level of product heterogeneity and/or ADC aggregation. A drug loading and/or an average drug loading of more than about 8 may also affect stability of the ADC, due to loss of one or more chemical bonds required to stabilize the antibody or antigen binding fragment.

The present disclosure includes methods of producing the described ADCs. Briefly, the ADCs comprise an antibody or antigen binding fragment as the antibody or antigen binding fragment, a drug moiety (e.g., a splicing modulator), and a linker that joins the drug moiety and the antibody or antigen binding fragment. In some embodiments, the ADCs can be prepared using a linker having reactive functionalities for covalently attaching to the drug moiety and to the antibody or antigen binding fragment. For example, in some embodiments, a cysteine thiol of an antibody or antigen binding fragment can form a bond with a reactive functional group of a linker or a drug-linker intermediate (e.g., a maleimide moiety) to make an ADC. The generation of the ADCs can be accomplished by any technique known to the skilled artisan.

In some embodiments, an ADC is produced by contacting an antibody or antigen binding fragment with a linker and a drug moiety (e.g., a splicing modulator) in a sequential manner, such that the antibody or antigen binding fragment is covalently linked to the linker first, and then the pre-formed antibody-linker intermediate reacts with the drug moiety. The antibody-linker intermediate may or may not be subjected to a purification step prior to contacting the drug moiety. In other embodiments, an ADC is produced by contacting an antibody or antigen binding fragment with a linker-drug compound pre-formed by reacting a linker with a drug moiety. The pre-formed linker-drug compound may or may not be subjected to a purification step prior to contacting the antibody or antigen binding fragment. In other embodiments, the antibody or antigen binding fragment contacts the linker and the drug moiety in one reaction mixture, allowing simultaneous formation of the covalent bonds between the antibody or antigen binding fragment and the linker, and between the linker and the drug moiety. This method of producing ADCs may include a reaction, wherein the antibody or antigen binding fragment contacts the antibody or antigen binding fragment prior to the addition of the linker to the reaction mixture, and vice versa. In certain embodiments, an ADC is produced by reacting an antibody or antigen binding fragment with a linker joined to a drug moiety, such as ADL1-splicing modulator (e.g., ADL1-D1) or ADL5-splicing modulator (e.g., ADL5-D2), under conditions that allow conjugation.

The ADCs prepared according to the methods described above may be subjected to a purification step. The purification step may involve any biochemical methods known in the art for purifying proteins, or any combination of methods thereof. These include, but are not limited to, tangential flow filtration (TFF), affinity chromatography, ion exchange chromatography, any charge or isoelectric point-based chromatography, mixed mode chromatography, e.g., CHT (ceramic hydroxyapatite), hydrophobic interaction chromatography, size exclusion chromatography, dialysis, filtration, selective precipitation, or any combination thereof.

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Therapeutic Uses and Compositions

Disclosed herein are methods of using the disclosed ADCs and compositions in treating a subject for a disorder, e.g., a neoplastic disorder. ADCs may be administered alone or in combination with a second therapeutic agent, and may be administered in any pharmaceutically acceptable formulation, dosage, and dosing regimen. ADC treatment efficacy may be evaluated for toxicity as well as indicators of efficacy and adjusted accordingly. Efficacy measures include, but are not limited to, a cytostatic and/or cytotoxic effect observed in vitro or in vivo, reduced tumor volume, tumor growth inhibition, and/or prolonged survival.

Methods of determining whether an ADC exerts a cytostatic and/or cytotoxic effect on a cell are known. For example, the cytotoxic or cytostatic activity of an ADC can be measured by: exposing mammalian cells expressing a target protein of the ADC in a cell culture medium; culturing the cells for a period from about 6 hours to about 6 days; and measuring cell viability. Cell-based in vitro assays may also be used to measure viability (proliferation), cytotoxicity, and induction of apoptosis (caspase activation) of the ADC.

For determining whether an ADC exerts a cytostatic effect, a thymidine incorporation assay may be used. For example, cancer cells expressing a target antigen at a density of 5,000 cells/well of a 96-well plated can be cultured for a 72-hour period and exposed to 0.5 μCi of 3 H-thymidine during the final 8 hours of the 72-hour period. The incorporation of 3 H-thymidine into cells of the culture is measured in the presence and absence of the ADC.

For determining cytotoxicity, necrosis or apoptosis (programmed cell death) may be measured. Necrosis is typically accompanied by increased permeability of the plasma membrane; swelling of the cell, and rupture of the plasma membrane. Apoptosis can be quantitated, for example, by measuring DNA fragmentation. Commercial photometric methods for the quantitative in vitro determination of DNA fragmentation are available. Examples of such assays, including TUNEL (which detects incorporation of labeled nucleotides in fragmented DNA) and ELISA-based assays, are described in Biochemica (1999) No. 2, pp. 34-37 (Roche Molecular Biochemicals).

Apoptosis may also be determined by measuring morphological changes in a cell. For example, as with necrosis, loss of plasma membrane integrity can be determined by measuring uptake of certain dyes (e.g., a fluorescent dye such as, for example, acridine orange or ethidium bromide). A method for measuring apoptotic cell number has been described by Duke and Cohen, Current Protocols in Immunology (Coligan et al., eds. (1992) pp. 3.17.1-3.17.16). Cells also can be labeled with a DNA dye (e.g., acridine orange, ethidium bromide, or propidium iodide) and the cells observed for chromatin condensation and margination along the inner nuclear membrane. Apoptosis may also be determined, in some embodiments, by screening for caspase activity. In some embodiments, a Caspase-Glo® Assay can be used to measure activity of caspase-3 and caspase-7. In some embodiments, the assay provides a luminogenic caspase-3/7 substrate in a reagent optimized for caspase activity, luciferase activity, and cell lysis. In some embodiments, adding Caspase-Glo® 3/7 Reagent in an “add-mix-measure” format may result in cell lysis, followed by caspase cleavage of the substrate and generation of a “glow-type” luminescent signal, produced by luciferase. In some embodiments, luminescence may be proportional to the amount of caspase activity present, and can serve as an indicator of apoptosis. Other morphological changes that can be measured to determine apoptosis include, e.g., cytoplasmic condensation, increased membrane blebbing, and cellular shrinkage. Determination of any of these effects on cancer cells indicates that an ADC is useful in the treatment of cancers.

Cell viability may be measured, e.g., by determining in a cell the uptake of a dye such as neutral red, trypan blue, Crystal Violet, or ALAMAR™ blue (see, e.g., Page et al. (1993) Intl J Oncology 3:473-6). In such an assay, the cells are incubated in media containing the dye, the cells are washed, and the remaining dye, reflecting cellular uptake of the dye, is measured spectrophotometrically. Cell viability may also be measured, e.g., by quantifying ATP, an indicator of metabolically active cells. In certain embodiments, in vitro potency and/or cell viability of prepared ADCs or splicing modulator compounds may be assessed using a CellTiter-Glo® Luminescent Cell Viability Assay, as described in the examples provided herein. In this assay, in certain embodiments, the single reagent (CellTiter-Glo® Reagent) is added directly to cells cultured in serum-supplemented medium. The addition of reagent results in cell lysis and generation of a luminescent signal proportional to the amount of ATP present. The amount of ATP is directly proportional to the number of cells present in culture. The protein-binding dye sulforhodamine B (SRB) can also be used to measure cytotoxicity (Skehan et al. (1990) J Natl Cancer Inst. 82:1107-12).

The disclosed ADCs may also be evaluated for bystander killing activity. Bystander killing activity may be determined, e.g., by an assay employing two cell lines, one positive for a target antigen and one negative for a target antigen. In certain embodiments, the design of the assay allows tracking of only target negative cells. In certain embodiments, cells are plated under three conditions: (i) target negative cells alone (tagged or labeled); (ii) target positive cells alone; and (iii) co-culture of target negative cells and target positive cells. Cells are then treated with an ADC followed by monitoring of cytotoxicity. When plates are read with CellTiter-Glo® Reagent, viability of all cell populations can be monitored. Men plates are read with OneGlo® Reagent, only the tagged or labeled target negative cells produce a signal. Killing of the target-negative cells when mixed with target-positive cells is indicative of bystander killing, whereas killing of the target-negative cells in the absence of the target-positive cells is indicative of off-target killing.

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In certain aspects, the present disclosure features a method of killing, inhibiting or modulating the growth of, or interfering with the metabolism of, a cancer cell or tissue by disrupting RNA splicing. The method may be used with any subject where disruption of RNA splicing provides a therapeutic benefit. Subjects that may benefit from disrupting RNA splicing include, but are not limited to, those having or at risk of having a neoplastic disorder such as a hematological malignancy or a solid tumor. In certain embodiments, the hematological malignancy is a B-cell malignancy, a cancer of the blood (leukemia), a cancer of plasma cells (myeloma, e.g., multiple myeloma), or a cancer of the lymph nodes (lymphoma). In certain embodiments, the hematological malignancy is acute myelogenous leukemia or multiple myeloma. In certain embodiments, the leukemia is acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), chronic myelomonocytic leukemia (CMML), or acute monocytic leukemia (AMoL). In certain embodiments, the lymphoma is Hodgkin's lymphoma or non-Hodgkin's lymphoma. In certain embodiments, the hematological malignancy is myelodysplasia syndrome (MDS). In certain embodiments, the solid tumor is a carcinoma such as breast cancer, pancreatic cancer, prostate cancer, colon or colorectal cancer, lung cancer, gastric cancer, cervical cancer, endometrial cancer, ovarian cancer, cholangiocarcinoma, glioma, or melanoma. In certain embodiments, the solid tumor is breast cancer, gastric cancer, prostate cancer, ovarian cancer, lung cancer (e.g., lung adenocarcinoma), uterine cancer (e.g., uterine serous endometrial carcinoma), salivary duct carcinoma, melanoma, colon cancer, cervical cancer, pancreatic cancer, kidney cancer, colorectal cancer, and esophageal cancer. In certain embodiments, the lung cancer is lung adenocarcinoma. In certain embodiments, the uterine cancer is uterine serous endometrial carcinoma.

In various embodiments, the disclosed ADCs may be administered in any cell or tissue that expresses HER2, such as a HER2-expressing neoplastic cell or tissue. An exemplary embodiment includes a method of inhibiting HER2-mediated cell signaling or a method of killing a cell. The method may be used with any cell or tissue that expresses HER2, such as a cancerous cell or a metastatic lesion. Non-limiting examples of HER2-expressing cancers include breast cancer, gastric cancer, bladder cancer, urothelial cell carcinoma, esophageal cancer, lung cancer (e.g., lung adenocarcinoma), uterine cancer (e.g., uterine serous endometrial carcinoma), salivary duct carcinoma, cervical cancer, endometrial cancer, and ovarian cancer (English et al. (2013) Mol Diagn Ther. 17:85-99). Non-limiting examples of HER2-expressing cells include HCC1954 and SKBR3 human breast ductal carcinoma cells, N87 human gastric carcinoma cells, and cells comprising a recombinant nucleic acid encoding HER2 or a portion thereof.

In various embodiments, the disclosed ADCs may be administered in any cell or tissue that expresses CD138, such as a CD138-expressing neoplastic cell or tissue. An exemplary embodiment includes a method of inhibiting CD138-mediated cell signaling or a method of killing a cell. The method may be used with any cell or tissue that expresses CD138, such as a cancerous cell or a metastatic lesion. Non-limiting examples of CD138-expressing cancers include intrathoracic cancer (e.g., lung cancer, mesothelioma), skin cancer (e.g., basal cell carcinoma, squamous cell carcinoma), head and neck cancer (e.g., laryngeal, hypopharynx, nasopharyngeal), breast cancer, urogenital cancer (e.g., cervical cancer, ovarian cancer, endometrial cancer, prostate cancer, bladder cancer, urothelial cancer), hematological malignancies (e.g., myeloma such as multiple myeloma, Hodgkin's lymphoma), and thyroid cancer (Szatmdri et al. (2015) Dis Markers 2015:796052). Non-limiting examples of CD138-expressing cells include MOLP8 human multiple myeloma cells, and cells comprising a recombinant nucleic acid encoding CD138 or a portion thereof.

In various embodiments, the disclosed ADCs may be administered in any cell or tissue that expresses EPHA2, such as an EPHA2-expressing neoplastic cell or tissue. An exemplary embodiment includes a method of inhibiting EPHA2-mediated cell signaling or a method of killing a cell. The method may be used with any cell or tissue that expresses EPHA2, such as a cancerous cell or a metastatic lesion. Non-limiting examples of EPHA2-expressing cancers include breast cancer, brain cancer, ovarian cancer, bladder cancer, pancreatic cancer, esophageal cancer, lung cancer, prostate cancer, melanoma, esophageal cancer, and gastric cancer (Tandon et al. (2011) Expert Opin Ther Targets 15(1):31-51. Non-limiting examples of EPHA2-expressing cells include PC3 human prostate cancer cells, and cells comprising a recombinant nucleic acid encoding EPHA2 or a portion thereof.

Exemplary methods include the steps of contacting a cell with an ADC, as described herein, in an effective amount, i.e., amount sufficient to kill the cell. The method can be used on cells in culture, e.g. in vitro, in vivo, ex vivo, or in situ. For example, cells that express HER2 (e.g., cells collected by biopsy of a tumor or metastatic lesion; cells from an established cancer cell line; or recombinant cells), can be cultured in vitro in culture medium and the contacting step can be affected by adding the ADC to the culture medium. The method will result in killing of cells expressing HER2, including in particular tumor cells expressing HER2. Alternatively, the ADC can be administered to a subject by any suitable administration route (e.g., intravenous, subcutaneous, or direct contact with a tumor tissue) to have an effect in vivo. This approach can be used for antibodies targeting other cell surface antigens (e.g., CD138, EPHA2).

The in vivo effect of a disclosed ADC therapeutic composition can be evaluated in a suitable animal model. For example, xenogeneic cancer models can be used, wherein cancer explants or passaged xenograft tissues are introduced into immune compromised animals, such as nude or SCID mice (Klein et al. (1997) Nature Med. 3:402-8). Efficacy may be predicted using assays that measure inhibition of tumor formation, tumor regression or metastasis, and the like.

›Definitions · 53 of 64

In vivo assays that evaluate the promotion of tumor death by mechanisms such as apoptosis may also be used. In one embodiment, xenografts from tumor bearing mice treated with the therapeutic composition can be examined for the presence of apoptotic foci and compared to untreated control xenograft-bearing mice. The extent to which apoptotic foci are found in the tumors of the treated mice provides an indication of the therapeutic efficacy of the composition.

Further provided herein are methods of treating a neoplastic disorder, e.g., a cancer. The ADCs disclosed herein can be administered to a non-human mammal or human subject for therapeutic purposes. The therapeutic methods entail administering to a subject having or suspected of having a neoplastic disorder a therapeutically effective amount of an ADC or composition comprising a splicing modulator linked to a targeting antibody that binds to an antigen expressed, is accessible to binding, or is localized on a cancer cell surface. In some embodiments, treatment with the antibody-drug conjugate or composition induces bystander killing of neoplastic cells which do not express a target antigen but are adjacent to neoplastic cells which express a target antigen.

An exemplary embodiment is a method of delivering a splicing modulator to a cell expressing HER2, comprising conjugating the splicing modulator to an antibody that immunospecifically binds to a HER2 epitope and exposing the cell to the ADC. Exemplary tumor cells that express HER2 for which the ADCs of the present disclosure are indicated include gastric carcinoma cells and breast ductal carcinoma cells.

Another exemplary embodiment is a method of delivering a splicing modulator to a cell expressing CD138, comprising conjugating the splicing modulator to an antibody that immunospecifically binds to a CD138 epitope and exposing the cell to the ADC. Exemplary tumor cells that express CD138 for which the ADCs of the present disclosure are indicated include multiple myeloma cells.

Another exemplary embodiment is a method of delivering a splicing modulator to a cell expressing EPHA2, comprising conjugating the splicing modulator to an antibody that immunospecifically binds to an EPHA2 epitope and exposing the cell to the ADC. Exemplary tumor cells that express EPHA2 for which the ADCs of the present disclosure are indicated include prostate cancer cells.

Another exemplary embodiment is a method of reducing or inhibiting growth of a tumor (e.g., a HER2-expressing tumor, a CD138-expressing tumor, an EPHA2-expressing tumor), comprising administering a therapeutically effective amount of an ADC or composition comprising an ADC. In some embodiments, the treatment is sufficient to reduce or inhibit the growth of the patient's tumor, reduce the number or size of metastatic lesions, reduce tumor load, reduce primary tumor load, reduce invasiveness, prolong survival time, and/or maintain or improve the quality of life. In some embodiments, the tumor is resistant or refractory to treatment with the antibody or antigen binding fragment of the ADC (e.g., an anti-HER2 antibody, an anti-CD138 antibody, an anti-EPHA2 antibody) when administered alone, and/or the tumor is resistant or refractory to treatment with the splicing modulator drug moiety when administered alone

In certain aspects, the present disclosure provides a method of reducing or inhibiting growth of a HER2-expressing tumor. In certain embodiments, treatment with the antibody-drug conjugate or composition induces bystander killing of tumor cells which do not express HER2 but that are adjacent to neoplastic tumor cells which do express HER2. Exemplary HER2-expressing tumor types include but are not limited to tumors derived from a HER2-expressing breast cancer, gastric cancer, bladder cancer, urothelial cell carcinoma, esophageal cancer, lung cancer (e.g., lung adenocarcinoma), uterine cancer (e.g., uterine serous endometrial carcinoma), salivary duct carcinoma, cervical cancer, endometrial cancer, and ovarian cancer. In certain embodiments, the HER2-expressing tumor is a tumor derived from a HER2-expressing breast cancer, ovarian cancer, gastric cancer, lung cancer (e.g., lung adenocarcinoma), uterine cancer (e.g., uterine serous endometrial carcinoma), osteosarcoma, or salivary duct carcinoma. In certain embodiments, the HER2-expressing tumor is a lung adenocarcinoma or uterine serous endometrial carcinoma.

In certain aspects, the present disclosure provides a method of reducing or inhibiting growth of a CD138-expressing tumor. In certain embodiments, treatment with the antibody-drug conjugate or composition induces bystander killing of tumor cells which do not express CD138 but that are adjacent to neoplastic tumor cells which do express CD138. Exemplary CD138-expressing tumor types include but are not limited to tumors derived from a CD138-expressing intrathoracic cancer (e.g., lung cancer, mesothelioma), skin cancer (e.g., basal cell carcinoma, squamous cell carcinoma), head and neck cancer (e.g., laryngeal, hypopharynx, nasopharyngeal), breast cancer, urogenital cancer (e.g., cervical cancer, ovarian cancer, endometrial cancer, prostate cancer, bladder cancer, urothelial cancer), and thyroid cancer.

In certain aspects, the present disclosure provides a method of reducing or inhibiting growth of an EPHA2-expressing tumor. In certain embodiments, treatment with the antibody-drug conjugate or composition induces bystander killing of tumor cells which do not express EPHA2 but that are adjacent to neoplastic tumor cells which do express EPHA2. Exemplary EPHA2-expressing tumor types include but are not limited to tumors derived from an EPHA2-expressing breast cancer, brain cancer, ovarian cancer, bladder cancer, pancreatic cancer, esophageal cancer, lung cancer, prostate cancer, melanoma, esophageal cancer, and gastric cancer. In certain embodiments, the EPHA2-expressing tumor is a tumor derived from an EPHA2-expressing breast cancer, prostate cancer, ovarian cancer, lung cancer, melanoma, colon cancer, or esophageal cancer.

›Definitions · 54 of 64

Moreover, antibodies of the present disclosure may be administered to a non-human mammal expressing an antigen with which the ADC is capable of binding for veterinary purposes or as an animal model of human disease. Regarding the latter, such animal models may be useful for evaluating the therapeutic efficacy of the disclosed ADCs (e.g., testing of dosages and time courses of administration).

Further provided herein are therapeutic uses of the disclosed ADCs and compositions. An exemplary embodiment is the use of an ADC in the treatment of a neoplastic disorder (e.g., a HER2-expressing cancer, a CD138-expressing cancer, an EPHA2-expressing cancer). Another exemplary embodiment is an ADC for use in the treatment of a neoplastic disorder (e.g., a HER2-expressing cancer, a CD138-expressing cancer, an EPHA2-expressing cancer). Methods for identifying subjects having cancers that express a target antigen (e.g., HER2, CD138, EPHA2, MSLN, FOLH1, CDH6, CEACAM5, CFC1B, ENPP3, FOLR1, HAVCR1, KIT, MET, MUC16, SLC39A6, SLC44A4, STEAP1) are known in the art and may be used to identify suitable patients for treatment with a disclosed ADC.

Another exemplary embodiment is the use of an ADC in a method of manufacturing a medicament for the treatment of a neoplastic disorder (e.g., a HER2-expressing cancer, a CD138-expressing cancer, an EPHA2-expressing cancer).

The therapeutic compositions used in the practice of the foregoing methods may be formulated into pharmaceutical compositions comprising a pharmaceutically acceptable carrier suitable for the desired delivery method. An exemplary embodiment is a pharmaceutical composition comprising an ADC of the present disclosure and a pharmaceutically acceptable carrier. Suitable carriers include any material that, when combined with the therapeutic composition, retains the anti-tumor function of the therapeutic composition and is generally non-reactive with the patient's immune system. Pharmaceutically acceptable carriers include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Examples of pharmaceutically acceptable carriers include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, mesylate salt, and the like, as well as combinations thereof. In many cases, isotonic agents are included, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Pharmaceutically acceptable carriers may further comprise minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the ADC.

Therapeutic formulations may be solubilized and administered via any route capable of delivering the therapeutic composition to the tumor site. Potentially effective routes of administration include, but are not limited to, intravenous, parenteral, intraperitoneal, intramuscular, intratumor, intradermal, intraorgan, orthotopic, and the like. Therapeutic protein preparations can be lyophilized and stored as sterile powders, e.g., under vacuum, and then reconstituted in bacteriostatic water (containing for example, benzyl alcohol preservative) or in sterile water prior to injection. Therapeutic formulations may comprise an ADC or a pharmaceutically acceptable salt thereof, e.g., a mesylate salt.

In some embodiments, the ADC is administered to the patient daily, bimonthly, or any time period in between. Dosages and administration protocols for the treatment of cancers using the foregoing methods will vary with the method and the target cancer, and will generally depend on a number of other factors appreciated in the art.

Various delivery systems are known and may be used to administer one or more ADCs of the present disclosure. Methods of administering the ADCs include, but are not limited to, parenteral administration (e.g., intradermal, intramuscular, intraperitoneal, intravenous and subcutaneous), epidural administration, intratumoral administration, and mucosal administration (e.g., intranasal and oral routes). In addition, pulmonary administration may be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent. See, e.g., the compositions and methods for pulmonary administration described in U.S. Pat. Nos. 6,019,968, 5,985,320, 5,985,309, 5,934,272, 5,874,064, 5,855,913, 5,290,540, and 4,880,078; and Intl. Publ. Nos. WO 1992/019244, WO 1997/032572, WO 1997/044013, WO 1998/031346, and WO 1999/066903. The ADCs may be administered by any convenient route, for example, by infusion or bolus injection, or by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.). Administration can be either systemic or local.

Therapeutic compositions disclosed herein may be sterile and stable under the conditions of manufacture and storage. In some embodiments, one or more of the ADCs, or pharmaceutical compositions, is supplied as a dry sterilized lyophilized powder or water free concentrate in a hermetically sealed container and can be reconstituted (e.g., with water or saline) to the appropriate concentration for administration to a subject. In some embodiments, one or more of the prophylactic or therapeutic agents or pharmaceutical compositions is supplied as a dry sterile lyophilized powder in a hermetically sealed container at a unit dosage of at least 5 mg, at least 10 mg, at least 15 mg, at least 25 mg, at least 35 mg, at least 45 mg, at least 50 mg, at least 75 mg, or at least 100 mg, or any amount in between. In some embodiments, the lyophilized ADCs or pharmaceutical compositions is stored at between 2° C. and 8° C. in the original container. In some embodiments, one or more of the ADCs or pharmaceutical compositions described herein is supplied in liquid form in a hermetically sealed container, e.g., a container indicating the quantity and concentration of the agent. In some embodiments, the liquid form of the administered composition is supplied in a hermetically sealed container of at least 0.25 mg/mL, at least 0.5 mg/mL, at least 1 mg/mL, at least 2.5 mg/mL, at least 5 mg/mL, at least 8 mg/mL, at least 10 mg/mL, at least 15 mg/mL, at least 25 mg/mL, at least 50 mg/mL, at least 75 mg/mL, or at least 100 mg/mL ADC. The liquid form may be stored at between 2° C. and 8° C. in the original container.

›Definitions · 55 of 64

In some embodiments, the disclosed ADCs can be incorporated into a pharmaceutical composition suitable for parenteral administration. The injectable solution may be composed of either a liquid or lyophilized dosage form in a flint or amber vial, ampule, or pre-filled syringe, or other known delivery or storage device.

The compositions described herein may be in a variety of forms. These include, for example, liquid, semi-solid, and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes, and suppositories. The preferred form depends on the intended mode of administration and therapeutic application.

In various embodiments, treatment involves single bolus or repeated administration of the ADC preparation via an acceptable route of administration.

Patients may be evaluated for the levels of target antigen in a given sample (e.g. the levels of target antigen expressing cells) in order to assist in determining the most effective dosing regimen, etc. An exemplary embodiment is a method of determining whether a patient will be responsive to treatment with an ADC of the present disclosure, comprising providing a biological sample from the patient and contacting the biological sample with the ADC. Exemplary biological samples include tissue or body fluid, such as an inflammatory exudate, blood, serum, bowel fluid, stool sample, or tumor biopsy (e.g., a tumor biopsy derived from a patient having or at risk of a target antigen-expressing cancer, e.g., a HER2-expressing cancer, a CD138-expressing cancer, an EPHA2-expressing cancer). In some embodiments, a sample (e.g., a tissue and/or body fluid) can be obtained from a subject, and a suitable immunological method can be used to detect and/or measure protein expression of the target antigen (e.g., HER2, CD138, EPHA2, MSLN, FOLH1, CDH6, CEACAM5, CFC1B, ENPP3, FOLR1, HAVCR1, KIT, MET, MUC16, SLC39A6, SLC44A4, STEAP1). Such evaluations are also used for monitoring purposes throughout therapy, and are useful to gauge therapeutic success in combination with the evaluation of other parameters.

In some embodiments, the efficacy of an ADC may be evaluated by contacting a tumor sample from a subject with the ADC and evaluating tumor growth rate or volume. In some embodiments, when an ADC has been determined to be effective, it may be administered to the subject.

The above therapeutic approaches can be combined with any one of a wide variety of additional surgical, chemotherapy, or radiation therapy regimens. In some embodiments, the ADCs or compositions disclosed herein are co-formulated and/or co-administered with one or more additional therapeutic agents, e.g., one or more chemotherapeutic agents. Non-limiting examples of chemotherapeutic agents include alkylating agents, for example, nitrogen mustards, ethyleneimine compounds, and alkyl sulphonates; antimetabolites, for example, folic acid, purine or pyrimidine antagonists; anti-mitotic agents, for example, anti-tubulin agents such as eribulin or eribulin mesylate (Halaven™), vinca alkaloids, and auristatins; cytotoxic antibiotics; compounds that damage or interfere with DNA expression or replication, for example, DNA minor groove binders; and growth factor receptor antagonists. In some embodiments, a chemotherapeutic agent may be a cytotoxic or cytostatic agent. Examples of cytotoxic agents include, but are not limited to, anti-mitotic agents, such as eribulin or eribulin mesylate (Halaven™), auristatins (e.g., monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF)), maytansinoids (e.g., maytansine), dolastatins, duostatins, cryptophycins, vinca alkaloids (e.g., vincristine, vinblastine), taxanes, taxols, and colchicines; anthracyclines (e.g., daunorubicin, doxorubicin, dihydroxyanthracindione); cytotoxic antibiotics (e.g., mitomycins, actinomycins, duocarmycins (e.g., CC-1065), auromycins, duomycins, calicheamicins, endomycins, phenomycins); alkylating agents (e.g., cisplatin); intercalating agents (e.g., ethidium bromide); topoisomerase inhibitors (e.g., etoposide, tenoposide); radioisotopes, such as At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 or 213 , P 32 , and radioactive isotopes of lutetium (e.g., Lu 177 ); and toxins of bacterial, fungal, plant or animal origin (e.g., ricin (e.g., ricin A-chain), diphtheria toxin, Pseudomonas exotoxin A (e.g., PE40), endotoxin, mitogellin, combrestatin, restrictocin, gelonin, alpha-sarcin, abrin (e.g., abrin A-chain), modeccin (e.g., modeccin A-chain), curicin, crotin, Sapaonaria officinalis inhibitor, glucocorticoid).

Also disclosed herein are uses of one or more of the disclosed ADCs in the manufacture of a medicament for treating cancer, e.g., according to the methods described above. In some embodiments, the ADCs disclosed herein are used for treating cancer, e.g., according to the methods described above.

In various embodiments, kits for use in the laboratory and therapeutic applications described herein are within the scope of the present disclosure. Such kits may comprise a carrier, package, or container that is compartmentalized to receive one or more containers such as vials, tubes, and the like, each of the container(s) comprising one of the separate elements to be used in a method disclosed herein, along with a label or insert comprising instructions for use, such as a use described herein.

Kits may comprise a container comprising a drug moiety. The present disclosure also provides one or more of the ADCs, or pharmaceutical compositions thereof, packaged in a hermetically sealed container, such as an ampoule or sachette, indicating the quantity of the agent.

Kits may comprise the container described above, and one or more other containers associated therewith that comprise materials desirable from a commercial and user standpoint, including buffers, diluents, filters, needles, syringes; carrier, package, container, vial and/or tube labels listing contents and/or instructions for use, and package inserts with instructions for use.

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A label may be present on or with the container to indicate that the composition is used for a specific therapy or non-therapeutic application, such as a prognostic, prophylactic, diagnostic, or laboratory application. A label may also indicate directions for either in vivo or in vitro use, such as those described herein. Directions and or other information may also be included on an insert(s) or label(s), which is included with or on the kit. The label may be on or associated with the container. A label may be on a container when letters, numbers, or other characters forming the label are molded or etched into the container itself. A label may be associated with a container when it is present within a receptacle or carrier that also holds the container, e.g., as a package insert. The label may indicate that the composition is used for diagnosing or treating a condition, such as a cancer a described herein.

Neoantigens and Methods of Use

Also disclosed herein, in various embodiments, are methods of treating a patient by inducing neoantigens in tumor cells that can be targeted by the patient's immune system for clearance. Without being bound by theory, in various embodiments, administering a splicing modulator, alone and/or as part of an ADC or composition, may produce neoantigens that induce an immune response, induce a double-stranded RNA immune response, e.g., as a result of re-expressed intron-resident endogenous retroviruses, and/or produce neoantigens that induce immunogenic cell death.

As used herein, the term “neoantigen” refers to any antigen to which the immune system has not previously been exposed that arises from one or more tumor-specific mutations and/or from exposing a tumor to a drug (e.g., any one or more of the splicing modulators disclosed herein, alone and/or as part of an ADC or composition). Tumor-specific mutations can include missense mutations, frameshifts, translocations, and mRNA splicing variants, as well as mutations that influence posttranslational processing, such as phosphorylation and glycosylation. These exemplary mutations, in various embodiments, can be derived from non-synonymous coding changes and/or mutations that alter mRNA processing (e.g., splicing). All of these exemplary mutations, in various embodiments, can result in molecular changes that can be discriminated by an appropriate T-cell receptor. In various preferred embodiments, an exemplary neoantigen is a neoantigen induced by delivery of a splicing modulator, alone and/or as part of an ADC or composition. In various embodiments, delivery of a splicing modulator (e.g., any one or more of the splicing modulators disclosed herein) can induce novel mRNA splicing that results in the translation of proteins containing one or more novel peptide domains to which the immune system has not previously been exposed. In various embodiments, tumor-specific mutations may be mRNA splicing variants resulting from delivery or administration of a splicing modulator, ADC, or composition comprising a splicing modulator or ADC.

Without being bound by theory, in various embodiments, the delivery of splicing modulators, alone and as part of an ADC or composition, may induce novel mRNA splicing (e.g., exon skipping, intron retention) that results in the alteration of the open reading frames and/or coding sequences of various genes. In various embodiments, these altered genes are translated into proteins containing one or more novel peptide domains recognized by the immune system as foreign. In various embodiments, the one or more novel peptide domains do not exist in the proteins or in any other part of the human proteome in the absence of splicing modulator treatment. In various embodiments, the proteins containing the one or more novel peptide domains can be degraded by the proteasome to create novel peptide fragments that act as substrates for the immunopeptide presentation machinery, e.g., via MHC presentation. In various embodiments, the novel peptide fragments representing neoantigens can be presented in the MHC1-bound peptidome, e.g., on tumor cells.

In various embodiments, the delivery of splicing modulators, alone and as part of an ADC or composition, may lead to one or more tumor cell-intrinsic events (e.g., cell growth arrest). In various embodiments, the tumor cell-intrinsic event(s) may lead to (1) enhanced engagement by phagocytic cells (Bracci et al. (2014) Cell Death Differ. 21(1):15-25); (2) the transport of novel peptide fragments to a tumor draining lymph node to engage with antigen-presenting cells; (3) antigen-presenting cells processing novel peptide fragments from a phagocytosed tumor cell and presenting the fragments as neoantigens to circulating naïve T-cell populations; (4) novel peptide fragments interacting with T-cells expressing receptors that recognize the fragments as neoantigens; (5) maturation and activation of effector T-cell responses (e.g., CD4+ and/or CD8+ T-cells; and/or (6) engagement of T-cells with additional tumor cells exposed to the splicing modulator treatment and presenting novel peptide fragments representing neoantigens on their surface MHC1 complexes. In various embodiments, the tumor cell-intrinsic event(s) may result, either directly or indirectly, in T-cell engagement of effector function and/or killing of neoantigen-presenting tumor cells.

Also, without being bound by theory, in various embodiments, the delivery of splicing modulators, alone and as part of an ADC or composition, may cause the re-expression of intron-resident endogenous retroviruses, leading to a double-stranded RNA immune response.

Further, without being bound by theory, in various embodiments, the delivery of splicing modulators, alone and as part of an ADC or composition, may lead to immunogenic cell death triggered by splice modulator-induced release of mutationally-derived neoantigens. In various embodiments, the delivery of splicing modulators, alone and as part of an ADC or composition, may induce a double-stranded RNA immune response. In various embodiments, the double-stranded RNA immune response can result from the re-expression of intron-resident endogenous retroviruses. In various embodiments, the double-stranded RNA immune response can result in tumor cell death. In various embodiments, the delivery of splicing modulators, alone and as part of an ADC or composition, may induce immunogenic cell death. In various embodiments, the immunogenic cell death can result from release of mutational-derived neoantigens and/or a host immune response against tumor cells.

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Accordingly, in various embodiments, methods of treatment are disclosed comprising inducing neoantigens by administering one or more splicing modulators and/or ADCs and/or compositions comprising a splicing modulator or ADC, e.g., any splicing modulator, ADC, or composition disclosed herein. In various embodiments, the method comprises administering a reduced dosage of the splicing modulator, ADC, or composition than would be needed absent the induction of neoantigens. In some embodiments, the method comprises administering one or more initial induction doses to produce neoantigens and induce an immune response (e.g., converting naïve T-cells to memory cells), followed by a reduced dosage or administration frequency (i.e., because of the combinatorial effect of the splicing modulator, ADC, or composition and of immune targeting of the neoantigens). In some embodiments, treatment can comprise a combination of administering the splicing modulator, ADC, or composition to induce a neoantigen-based immune response and at least one additional therapy (e.g., a second anti-cancer therapy). For example, in some embodiments, treatment can comprise a combination of administering the splicing modulator, ADC, or composition to induce a neoantigen-based immune response and one or more checkpoint inhibitors. In some embodiments, treatment can comprise a combination of administering the splicing modulator, ADC, or composition to induce a neoantigen-based immune response and one or more cytokines or cytokine analogs. In some embodiments, treatment can comprise a combination of administering the splicing modulator, ADC, or composition to induce a neoantigen-based immune response and one or more neoantigen vaccines. In some other embodiments, treatment can comprise a combination of administering the splicing modulator, ADC, or composition to induce a neoantigen-based immune response and one or more engineered tumor-targeting T-cells (e.g., CAR-T).

In some embodiments, neoantigens can be used to monitor the effectiveness of treatment with a splicing modulator, ADC, or composition. For instance, after administration of a splicing modulator, ADC, or composition, a patient sample (e.g., a tumor biopsy) can be obtained and screened for neoantigens or for identifiers of an immune or inflammatory response. Further treatment can be provided, e.g., at reduced dosage, if a neoantigen and/or immune response is detected.

In various embodiments, methods of treatment are disclosed comprising inducing a double-stranded RNA immune response by administering one or more splicing modulators and/or ADCs and/or compositions comprising a splicing modulator or ADC, e.g., any splicing modulator, ADC, or composition disclosed herein.

In various embodiments, methods of treatment are disclosed comprising inducing immunogenic cell death by administering one or more splicing modulators and/or ADCs and/or compositions comprising a splicing modulator or ADC, e.g., any splicing modulator, ADC, or composition disclosed herein.

In various embodiments, administration of a splicing modulator, ADC, or composition comprising a splicing modulator can be combined with any known anti-cancer therapy. Examples of current immune activating strategies available for oncology treatment include, but are not limited to, treatment with immune checkpoint inhibitor (ICI) molecules, treatment with cytokines or cytokine analogs, vaccination with tumor-associated vaccines, and engineering tumor-targeting T-cells (e.g., expansion of tumor-infiltrating lymphocytes or CAR-T). These technologies are predominantly focused on enhancing or inducing an immune response to already existing tumor antigens (either mutations or aberrant expression of cell-surface proteins). One or more of these strategies may involve one or more mutations that are capable of inducing an antigenic T-cell response. For example, patient responses to checkpoint inhibition may correlate with non-synonymous mutational burden. In addition, cancer vaccine approaches may be used that rely on pre-existing mutations and the antigenicity of these mutations.

Splicing modulators and/or ADCs comprising such modulators may induce broad-ranging changes in the transcriptome that occur in multiple lineages. Translation of these mRNA changes may produce robust and reproducible protein changes that produce MHC1-bound neopeptides with high affinity across multiple HLA isotypes. Without being bound by theory, due to the large number of changes to the transcriptome and proteome, treatment with splicing modulators and/or ADCs may enrich the number of potentially reactive neoantigens for enhanced engagement of the adaptive immune response.

As described herein, the terms “splicing modulator,” “spliceosome modulator,” or “splice modulator” refer to compounds that have anti-cancer activity by interacting with components of the spliceosome. In some embodiments, a splicing modulator alters the rate or form of splicing in a target cell. Splicing modulators that function as inhibitory agents, for example, are capable of decreasing uncontrolled cellular proliferation. In particular, in some embodiments, the splicing modulators may act by inhibiting the SF3b spliceosome complex. In some embodiments, a splicing modulator is chosen from any one or more of the splicing modulators disclosed herein. In some embodiments, a splicing modulator is used, delivered to a cell, and/or administered to a subject as a stand-alone agent. In some other embodiments, a splicing modulator is used, delivered to a cell, and/or administered to a subject as part of an ADC (e.g., an ADC chosen from any of the exemplary ADCs disclosed herein). In some other embodiments, a splicing modulator is used, delivered to a cell, and/or administered to a subject as part of a composition comprising multiple copies of the splicing modulator or multiple copies of an ADC carrying the splicing modulator. Such compositions are disclosed herein.

In some embodiments, a splicing modulator used, delivered to a cell, and/or administered to a subject as part of an ADC (e.g., an ADC chosen from any of the exemplary ADCs disclosed herein) provides added therapeutic benefits over a splicing modulator used, delivered to a cell, and/or administered to a subject as a stand-alone agent. For example, in some embodiments, a splicing modulator used, delivered to a cell, and/or administered to a subject as part of an ADC provides targeted delivery of the splicing modulator to a neoplastic cell expressing the target antigen (i.e., the antigen targeted by the antibody moiety of the ADC). In some embodiments, such targeted delivery of the splicing modulator reduces off-target treatment and/or off-target cytotoxicity. In some embodiments, such targeted delivery promotes tumor-selective neoantigen presentation on neoplastic cells, but not on healthy cells that do not express the target antigen. In some embodiments, such targeted delivery leads to, e.g., at least 75%, 80%, 85%, 90%, 95%, or 99%, of the alternative splicing and induction of novel mRNAs and MHC-associated peptides representing neoantigens in targeted neoplastic cells rather than off-target cells. Thus, without being bound by theory, in some embodiments, following effector T-cell priming and/or expansion (e.g., using a neoantigen vaccine), the immune system may preferentially attack neoantigen-presenting neoplastic cells rather than healthy cells due to the preferential expression of neoantigens on tumor cells after treatment with an ADC as disclosed herein.

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Immune Induction and Treatment Regimen:

In various embodiments, the present disclosure provides a method of inducing at least one neoantigen by contacting a neoplastic cell with an effective amount of a splicing modulator, a splicing modulator-based antibody-drug conjugate (ADC), or a composition comprising a splicing modulator or ADC. In various embodiments, the present disclosure provides a method of inducing a double-stranded RNA immune response by contacting a neoplastic cell with an effective amount of a splicing modulator, a splicing modulator-based antibody-drug conjugate (ADC), or a composition comprising a splicing modulator or ADC. In various embodiments, the present disclosure provides a method of inducing immunogenic cell death by contacting a neoplastic cell with an effective amount of a splicing modulator, a splicing modulator-based antibody-drug conjugate (ADC), or a composition comprising a splicing modulator or ADC.

In some embodiments, the at least one neoantigen comprises an amino acid sequence of any one of SEQ ID NOs: 37-65. In some embodiments, the at least one neoantigen comprises an amino acid sequence of SEQ ID NO: 37. In some embodiments, the at least one neoantigen comprises an amino acid sequence of SEQ ID NO: 39. In some embodiments, the at least one neoantigen comprises an amino acid sequence of any one of SEQ ID NOs: 46-49.

In some embodiments, the neoplastic cell is present in an in vitro cell culture. In some embodiments, the neoplastic cell is obtained from a subject. In some embodiments, the neoplastic cell is present in a subject. In some embodiments, the neoplastic cell is derived from a hematological malignancy or a solid tumor. In some embodiments, the hematological malignancy is selected from a B-cell malignancy, a leukemia, a lymphoma, and a myeloma. In some embodiments, the hematological malignancy is selected from acute myeloid leukemia and multiple myeloma. In some embodiments, the solid tumor is selected from breast cancer (e.g., HER2-positive breast cancer), gastric cancer (e.g., gastric adenocarcinoma), prostate cancer, ovarian cancer, lung cancer (e.g., lung adenocarcinoma), uterine cancer (e.g., uterine serous endometrial carcinoma), salivary duct carcinoma, melanoma, colon cancer, cervical cancer, pancreatic cancer, kidney cancer, colorectal cancer, and esophageal cancer. In some embodiments, the solid tumor is selected from HER2-positive breast cancer, gastric adenocarcinoma, prostate cancer, and osteosarcoma.

In various embodiments, the present disclosure further provides a method of inducing at least one neoantigen and/or a T-cell response in a subject having or suspected of having a neoplastic disorder by administering to the subject an effective amount of a splicing modulator, an ADC, or a composition comprising a splicing modulator or ADC. Also provided herein, in various embodiments, is a method of treating a subject having or suspected of having a neoplastic disorder by administering to the subject an effective amount of a splicing modulator, an ADC, or a composition comprising a splicing modulator or ADC, wherein administration of the splicing modulator, antibody-drug conjugate, or composition induces at least one neoantigen and/or a T-cell response.

In various other embodiments, the present disclosure provides a method of inducing a double-stranded RNA immune response in a subject having or suspected of having a neoplastic disorder by administering to the subject an effective amount of a splicing modulator, an ADC, or a composition comprising a splicing modulator or ADC. Also provided herein, in various embodiments, is a method of treating a subject having or suspected of having a neoplastic disorder by administering to the subject an effective amount of a splicing modulator, an ADC, or a composition comprising a splicing modulator or ADC, wherein administration of the splicing modulator, antibody-drug conjugate, or composition induces a double-stranded RNA immune response.

In still other embodiments, the present disclosure provides a method of inducing immunogenic cell death in a subject having or suspected of having a neoplastic disorder by administering to the subject an effective amount of a splicing modulator, an ADC, or a composition comprising a splicing modulator or ADC. Further provided herein, in various embodiments, is a method of treating a subject having or suspected of having a neoplastic disorder by administering to the subject an effective amount of a splicing modulator, an ADC, or a composition comprising a splicing modulator or ADC, wherein administration of the splicing modulator, antibody-drug conjugate, or composition induces immunogenic cell death.

In some embodiments of the therapeutic methods described herein, the at least one neoantigen comprises an amino acid sequence of any one of SEQ ID NOs: 37-65. In some embodiments, the at least one neoantigen comprises an amino acid sequence of SEQ ID NO: 37. In some embodiments, the at least one neoantigen comprises an amino acid sequence of SEQ ID NO: 39. In some embodiments, the at least one neoantigen comprises an amino acid sequence of any one of SEQ ID NOs: 46-49.

In some embodiments, the present disclosure further provides a method of treating a subject having or suspected of having a neoplastic disorder by administering to the subject an effective amount of a splicing modulator, an ADC, or a composition comprising a splicing modulator or ADC, wherein administration of the splicing modulator, antibody-drug conjugate, or composition induces immunogenic cell death, in combination with one or more additional therapies comprising a second agent.

In some embodiments of the therapeutic methods described herein, the amount of the splicing modulator, antibody-drug conjugate, composition, or second agent administered is reduced due to induction of at least one neoantigen and/or a T-cell response, as compared to a standard dosage of the splicing modulator, antibody-drug conjugate, composition, or second agent. In some embodiments, the administered amount of the splicing modulator, antibody-drug conjugate, composition, or second agent is reduced by 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 75%, or 90%, as compared to a standard dosage of the splicing modulator, antibody-drug conjugate, composition, or second agent. In some embodiments, the splicing modulator, antibody-drug conjugate, composition, or second agent is administered at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 75%, or 90% less frequently, as compared to a standard dosing regimen of the splicing modulator, antibody-drug conjugate, composition, or second agent. In some embodiments, the administered amount and/or dosage of the splicing modulator, antibody-drug conjugate, composition, or second agent results in lower systemic toxicity and/or improved tolerance.

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As used herein, the term “standard dosage” or “standard dosing regimen” refers to any usual or routine dosing regimen for a therapeutic agent, e.g., a regimen proposed by the manufacturer, approved by regulatory authorities, or otherwise tested in human subjects to meet the average patient's needs. In some embodiments, the therapeutic agent is a splicing modulator, an antibody, or an antibody-drug conjugate with anti-cancer activity.

For instance, a standard dosing regimen for trastuzumab, an exemplary anti-HER2 antibody disclosed herein, may be 8 mg/kg administered intravenously over 90 min (week 1) followed by 6 mg/kg administered intravenously over 30-90 min every 3 weeks (week 4 through the end of the therapy cycle) (Herceptin® (trastuzumab) FDA Label Supplement, 2017).

As another example, a standard dosing regimen for ipilimumab, an exemplary anti-CTLA4 checkpoint inhibitor antibody, may be 3 mg/kg administered intravenously over 90 min every 3 weeks for 4 doses (Yervoy® (ipilimumab) FDA Label Supplement, 2018). Another standard dosing regimen for ipilimumab may be 10 mg/kg administered intravenously over 90 min every 3 weeks for 4 doses, followed by 10 mg/kg every 12 weeks for up to 3 years (Yervoy® (ipilimumab) FDA Label Supplement, 2018).

As another example, a standard dosing regimen for nivolumab, an exemplary anti-PD1 checkpoint inhibitor antibody, may be 3 mg/kg administered intravenously over 60 min every 2 weeks (Opdivo® (nivolumab) FDA Label, 2015).

As another example, a standard dosing regimen for atezolizumab, an exemplary anti-PDL1 checkpoint inhibitor antibody, may be 1200 mg administered intravenously over 60 min every 3 weeks (Tecentriq® (atezolizumab) FDA Label Supplement, 2018).

As yet another example, a standard dosing regimen for T-DM1, an exemplary anti-HER2 antibody-drug conjugate, may be 3.6 mg/kg administered intravenously over 90 min every 3 weeks (Kadcyla® (T-DM1) FDA Label Supplement, 2016).

In some embodiments, the methods described herein may further comprise administering at least one additional therapy (e.g., a checkpoint inhibitor, a neoantigen vaccine, a cytokine or cytokine analog, CAR-T, etc.). In some embodiments, the amount of the splicing modulator, antibody-drug conjugate, composition, and/or the at least one additional therapy administered is reduced due to induction of at least one neoantigen and/or a T-cell response, as compared to a standard dosage of the splicing modulator, antibody-drug conjugate, composition, and/or the at least one additional therapy. In some embodiments, the amount of the splicing modulator, antibody-drug conjugate, composition, and/or the at least one additional therapy administered is reduced due to induction of a double-stranded RNA immune response, as compared to a standard dosage of the splicing modulator, antibody-drug conjugate, composition, and/or the at least one additional therapy. In some embodiments, the amount of the splicing modulator, antibody-drug conjugate, composition, and/or the at least one additional therapy administered is reduced due to induction of immunogenic cell death, as compared to a standard dosage of the splicing modulator, antibody-drug conjugate, composition, and/or the at least one additional therapy. In some embodiments, the administered amount of the splicing modulator, antibody-drug conjugate, composition, and/or the at least one additional therapy is reduced by 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 75%, or 90%, as compared to a standard dosage of the splicing modulator, antibody-drug conjugate, composition, and/or the at least one additional therapy. In some embodiments, the splicing modulator, antibody-drug conjugate, composition, and/or the at least one additional therapy is administered at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 75%, or 90% less frequently, as compared to a standard dosing regimen of the splicing modulator, antibody-drug conjugate, composition, and/or the at least one additional therapy. In some embodiments, the administered amount and/or dosage of the splicing modulator, antibody-drug conjugate, composition, and/or the at least one additional therapy results in lower systemic toxicity and/or improved tolerance.

In some embodiments, administration of the splicing modulator, antibody-drug conjugate, or composition is initiated before administration of the at least one additional therapy. In other embodiments, administration of the splicing modulator, antibody-drug conjugate, or composition is initiated after administration of the at least one additional therapy. In still other embodiments, administration of the splicing modulator, antibody-drug conjugate, or composition is initiated concurrently with administration of the at least one additional therapy.

In some embodiments, administration of the splicing modulator, antibody-drug conjugate, or composition is repeated at least once after initial administration. In some embodiments, the amount of the splicing modulator, antibody-drug conjugate, or composition used for repeated administration is reduced as compared to the amount used for initial administration. In some embodiments, the amount of the splicing modulator, antibody-drug conjugate, or composition used for repeated administration is reduced as compared to a standard dosage of the splicing modulator, antibody-drug conjugate, or composition. In some embodiments, the amount of the splicing modulator, antibody-drug conjugate, or composition used for repeated administration is reduced by 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 75%, or 90%, as compared to a standard dosage or initial dosage of the splicing modulator, antibody-drug conjugate, or composition.

In some embodiments, administration of the at least one additional therapy is repeated at least once after initial administration. In some embodiments, the amount of the at least one additional therapy used for repeated administration is reduced as compared to the amount used for initial administration. In some embodiments, the amount of the at least one additional therapy used for repeated administration is reduced as compared to a standard dosage of the at least one additional therapy. In some embodiments, the amount of the at least one additional therapy used for repeated administration is reduced by 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 75%, or 90%, as compared to a standard dosage or initial dosage of the at least one additional therapy.

›Definitions · 60 of 64

In some embodiments, repeated administration of the splicing modulator, antibody-drug conjugate, or composition is concurrent with repeated administration of the at least one additional therapy. In some embodiments, administration of the splicing modulator, antibody-drug conjugate, or composition is sequential or staggered with repeated administration of the at least one additional therapy.

In some embodiments, the at least one additional therapy comprises administering a checkpoint inhibitor, e.g., any checkpoint inhibitor disclosed herein. In some embodiments, the subject is intolerant, non-responsive, or poorly responsive to the checkpoint inhibitor when administered alone. In some embodiments, the checkpoint inhibitor is targeted at PD1/PDL1, CTLA4, OX40, CD40, LAG3, TIM3, GITR, and/or KIR. In some embodiments, the checkpoint inhibitor is targeted at CTLA4, OX40, CD40, and/or GITR. In some embodiments, the checkpoint inhibitor is an antibody having inhibitory or agonist activity to its target. In some embodiments, a checkpoint inhibitor is targeted with an inhibitory antibody or other similar inhibitory molecule. In other embodiments, a checkpoint inhibitor is targeted with an agonist antibody or other similar agonist molecule.

In some other embodiments, the at least one additional therapy comprises administering a neoantigen vaccine, e.g., any neoantigen vaccine disclosed herein. In some embodiments, the splicing modulator, ADC, or composition is administered before administration of the neoantigen vaccine. In some embodiments, the splicing modulator, ADC, or composition is administered after administration of the neoantigen vaccine. In some embodiments, the splicing modulator, ADC, or composition is administered concurrently with administration of the neoantigen vaccine. In some embodiments, administration of the splicing modulator, ADC, or composition is repeated at least once after initial administration. In some embodiments, the amount of the splicing modulator, ADC, or composition used for repeated administration is reduced as compared to the amount used for initial administration.

In some embodiments, the neoantigen vaccine comprises at least one neoantigen peptide. In some embodiments, the at least one neoantigen peptide ranges from about 10 to about 50 amino acids in length. In some embodiments, the at least one neoantigen peptide ranges from about 10 to about 35 amino acids in length. In some embodiments, the at least one neoantigen peptide ranges from about 15 to about 25 amino acids in length. In some embodiments, the at least one neoantigen peptide comprises one or more than one neoantigen sequence.

In some embodiments, the neoantigen sequence comprises an amino acid sequence of any one of SEQ ID NOs: 37-65. In some embodiments, the neoantigen sequence comprises an amino acid sequence of SEQ ID NO: 37. In some embodiments, the neoantigen sequence comprises an amino acid sequence of SEQ ID NO: 39. In some embodiments, the neoantigen sequence comprises an amino acid sequence of any one of SEQ ID NOs: 46-49.

In some other embodiments, the neoantigen sequence comprises an amino acid sequence of any one of SEQ ID NOs: 66-93, or an antigenic portion of any one of SEQ ID NOs: 66-93. In some embodiments, the neoantigen sequence comprises an amino acid sequence of SEQ ID NO: 66, or an antigenic portion of SEQ ID NO: 66. In some embodiments, the neoantigen sequence comprises an amino acid sequence of any one of SEQ ID NOs: 74-77, or an antigenic portion of any one of SEQ ID NOs: 74-77. In some embodiments, the neoantigen sequence and/or antigenic portion ranges from about 10 to about 50 amino acids in length. In some embodiments, the neoantigen sequence and/or antigenic portion ranges from about 10 to about 35 amino acids in length. In some embodiments, the neoantigen sequence and/or antigenic portion ranges from about 15 to about 25 amino acids in length. In some embodiments, the neoantigen sequence and/or antigenic portion ranges from about 10 to about 20 amino acids in length. In some embodiments, the neoantigen sequence and/or antigenic portion does not exclusively overlap or consist of the canonical peptide sequence (e.g., any of the exemplary canonical peptide sequences underlined in Table 21).

The term “antigenic portion” or “antigenic fragment” of a neoantigen sequence, as used herein, refers to one or more fragments of a neoantigen sequence that retain the ability to induce a T-cell response (e.g., antigen-specific expansion and/or maturation of effector T-cell population(s)). An antigenic portion, in some embodiments, may also retain the ability to be internalized, processed, and/or presented by antigen-presenting cells (e.g., dendritic cells). In some embodiments, an antigenic portion also retains T-cell priming function. In some embodiments, an antigenic portion of a neoantigen sequence ranges from about 10 to about 50 amino acids in length. In some embodiments, an antigenic portion of a neoantigen sequence ranges from about 10 to about 35 amino acids in length. In some embodiments, an antigenic portion of a neoantigen sequence ranges from about 15 to about 25 amino acids in length. In some embodiments, an antigenic portion of a neoantigen sequence ranges from about 10 to about 20 amino acids in length. In some embodiments, an antigenic portion of a neoantigen sequence (e.g., an antigenic portion of any one of SEQ ID NOs: 66-93), or its encoding mRNA, is formulated as a neoantigen vaccine.

An exemplary embodiment of an antigenic portion is the region(s) flanking amino acids 45-53 of SEQ ID NO: 66. Another exemplary embodiment of an antigenic portion is the region(s) flanking amino acids 82-90 of SEQ ID NO: 66. In some embodiments, the antigenic portion is capable of binding to at least one HLA allele expressed in a subject (e.g., HLA-A*02:01). In some other embodiments, the antigenic portion is capable of binding to at least one HLA allele expressed in at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, or at least 45% of subjects in a population of subjects suffering from a neoplastic disorder. In some embodiments, the antigenic portion is capable of eliciting a T-cell response against a tumor present in at least 1%, at least 5%, or at least 10% of a population of subjects suffering from a neoplastic disorder.

›Definitions · 61 of 64

In some embodiments, an antigenic portion does not exclusively overlap or consist of a canonical peptide sequence. The term “canonical peptide sequence,” as used herein, refers to any contiguous peptide sequence present in the human proteome in the absence of contact with a splicing modulator (e.g., in the absence of contact with a splicing modulator alone and/or as part of an ADC or composition), and/or to which the immune has previously been exposed. In some embodiments, the canonical peptide sequence is derived from and/or encoded by the canonical transcript open reading frame. Exemplary canonical peptide sequences are underlined in Table 21.

In some embodiments, when a splicing modulator is administered (e.g., alone and/or as part of an ADC or composition), a canonical peptide sequence may be derived from and/or encoded by the immediate 5′ in-frame 24 nucleotides preceding an aberrant splicing event induced by the splicing modulator. Thus, in some embodiments, the canonical peptide sequence comprises or consists of the 8 amino acids immediately N-terminal to the neoantigen sequence induced by the splicing modulator. In some embodiments, when a 5′ exon sequence terminates with a terminal nucleotide of a codon, the canonical peptide sequence terminates at the end of the exon. In some other embodiments, when a 5′ exon sequence terminates with one or two of the three nucleotides of a codon, the canonical peptide sequence is derived from and/or encoded by the 24 nucleotides preceding the incomplete codon. In some embodiments, mRNA sequences 3′ of the aberrant splicing event may be translated in the same open reading frame derived from the 5′ exon until reaching a stop codon, whereupon translation may terminate. In some embodiments, when the aberrant splicing event (e.g., exon skipping) results in a conservation of the canonical transcript open reading frame, the C-terminal sequence may be translated for an additional 24 nucleotides, encoding 8 C-terminal amino acids. In this context, in some embodiments, only the region across the aberrant exon junction may encode a neoantigen sequence. In some embodiments, when the open reading frame is shifted (e.g., intron retention), the complete C-terminal sequence (encoded by the 3′ mRNA) may encode a neoantigen sequence.

In some embodiments, an antigenic portion of a neoantigen sequence is chosen by comparing the neoantigen sequence to the canonical peptide sequence; and selecting a portion of the neoantigen sequence that does not exclusively overlap, consist of, and/or align with the canonical peptide sequence. An antigenic portion of a neoantigen sequence, in some embodiments, can be screened for antigenicity and/or T-cell priming function in the same manner as are full-length neoantigen sequences (e.g., the neoantigen sequence from which the antigenic portion is derived). In some embodiments, an antigenic portion of a neoantigen sequence is evaluated for antigenicity and/or T-cell priming function using a T-cell priming assay, such as the exemplary T-cell priming experiments described herein.

In some embodiments, the neoantigen sequence is a neoantigen sequence specific to the subject. In some embodiments, the neoantigen sequence is a personalized neoantigen vaccine for the subject. In some embodiments, the neoantigen sequence used to create a personalized neoantigen vaccine for a subject is capable of binding to at least one HLA allele expressed in the subject. In some embodiments, a personalized neoantigen vaccine is selected by identifying neoantigens expressed in a subject's tumor, e.g., after administration of a splicing modulator or ADC, and selecting a vaccine comprising a neoantigen sequence observed in the patient's tumor, e.g., a vaccine comprising an amino acid sequence of any one of SEQ ID NOs: 37-65 that is observed in the tumor.

The term “personalized” when used to describe a neoantigen vaccine refers to a vaccine created by identifying one or more neoantigens produced in a patient, preferably one identified in the patient after an exposure to a splicing modulator, ADC, or composition, and then using one or more of those neoantigens as the basis of the vaccine for the same patient. Accordingly, in some embodiments, a patient is given a splicing modulator, ADC, or composition and screened for neoantigens produced by the treatment. In some embodiments, the selected neoantigen vaccine comprises a neoantigen peptide or mRNA disclosed herein and confirmed to be present in the patient after exposure to the splicing modulator, ADC, or composition. In some embodiments, the splicing modulator, ADC, or composition and/or peptide or mRNA vaccine may be administered to the patient once or repeatedly. Subsequently, in some embodiments, one or more of those neoantigens are used to create a personalized vaccine that is given to the patient. In some embodiments, the one or more neoantigens used to create a personalized vaccine possess binding affinity for one or more patient-specific HLA alleles. In some embodiments, the patient expresses one or more MHC1 alleles that bind to the one or more neoantigens. The prediction of whether a given neoantigen will bind to a specific MHC1 allele can be determined using any computational prediction method known in the art. Exemplary computational prediction methods are disclosed, e.g., in Meydan et al. (2013) BMC Bioinformatics 14 (Suppl. 2):S13, which is incorporated herein by reference for such methods.

In some other embodiments, the neoantigen sequence is a universal neoantigen sequence. In some embodiments, the neoantigen sequence is a universal neoantigen vaccine.

The term “universal” when used to describe a neoantigen vaccine refers to a vaccine having a peptide or mRNA sequence that is based on common or known neoantigen(s) observed by sequencing neoantigens produced in multiple patients and/or patient tissue samples, preferably after an exposure to a splicing modulator, ADC, or composition. The peptide or mRNA sequence used in the vaccine need not be present in every patient but rather be observed in at least several patients or patient tissue samples. In some embodiments, the splicing modulator, ADC, or composition and/or peptide or mRNA vaccine may be administered to the patient once or repeatedly. Subsequently, in some embodiments, that peptide or mRNA sequence is used for vaccinating further patients. In some embodiments, a patient is given a splicing modulator, ADC, or composition, and then given a peptide or mRNA vaccine of known neoantigen to enhance immune response to the neoantigens produced by the splicing modulator, ADC, or composition. In some embodiments, a patient is given a universal peptide or mRNA vaccine and then given a splicing modulator, ADC, or composition once or repeatedly. In some embodiments, the neoantigen sequence (or sequences) used to create a universal neoantigen vaccine is selected based on overall MHC1 allele frequency in a given patient population (Maiers et al. (2007) Hum. Immunol. 68(9):779-88).

›Definitions · 62 of 64

In some embodiments, the neoantigen (e.g., a universal neoantigen) sequence is capable of binding to at least one HLA allele expressed in at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, or at least 45% of subjects in a population of subjects suffering from the neoplastic disorder. In some embodiments, the neoantigen sequence is capable of eliciting a T-cell response against a tumor present in at least 1%, at least 5%, or at least 10% of a population of subjects suffering from the neoplastic disorder.

In some embodiments, the neoantigen sequence has been identified by sequencing at least one neoantigen peptide, or its encoding mRNA, induced in the subject by administering an effective amount of the splicing modulator, antibody-drug conjugate, or composition. In some embodiments, the at least one neoantigen peptide comprises a neoantigen sequence induced by contacting a neoplastic cell with an effective amount of the splicing modulator, antibody-drug conjugate, or composition. In some embodiments, the neoplastic cell is present in an in vitro cell culture. In some embodiments, the neoplastic cell is obtained from the subject. In some embodiments, the neoplastic cell is present in the subject.

In some embodiments, the neoantigen vaccine comprises at least one neoantigen peptide and a pharmaceutically acceptable carrier (e.g., any of the exemplary carriers described herein). In some embodiments, the at least one neoantigen peptide is linked to the pharmaceutically acceptable carrier. In some embodiments, the pharmaceutically acceptable carrier is selected from a peptide, a serum albumin, a keyhole limpet hemocyanin, an immunoglobulin, a thyroglobulin, an ovalbumin, a toxoid or an attenuated toxoid derivative, a cytokine, and a chemokine. In some embodiments, the neoantigen peptide and the pharmaceutically acceptable carrier are covalently attached via a linker. In some embodiments, the neoantigen peptide and the pharmaceutically acceptable carrier are expressed as a fusion protein. In some embodiments, the neoantigen vaccine comprises at least one neoantigen peptide and a pharmaceutically acceptable diluent. In some embodiments, the neoantigen vaccine comprises at least one neoantigen peptide and a pharmaceutically acceptable adjuvant.

In some embodiments, the neoantigen vaccine comprises at least one neoantigen mRNA. In some embodiments, the at least one neoantigen mRNA encodes one or more than one neoantigen sequence. In some embodiments, the neoantigen sequence comprises an amino acid sequence of any one of SEQ ID NOs: 37-65. In some embodiments, the neoantigen sequence comprises an amino acid sequence of SEQ ID NO: 37. In some embodiments, the neoantigen sequence comprises an amino acid sequence of SEQ ID NO: 39. In some embodiments, the neoantigen sequence comprises an amino acid sequence of any one of SEQ ID NOs: 46-49.

In some other embodiments, the neoantigen sequence is a universal neoantigen sequence. In some embodiments, the neoantigen sequence is a universal neoantigen vaccine. In some embodiments, the neoantigen sequence is capable of binding to at least one HLA allele expressed in at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, or at least 45% of subjects in a population of subjects suffering from the neoplastic disorder. In some embodiments, the neoantigen sequence is capable of eliciting a T-cell response against a tumor present in at least 1%, at least 5%, or at least 10% of a population of subjects suffering from the neoplastic disorder.

In some embodiments, the neoantigen sequence has been identified by sequencing the protein sequence of at least one neoantigen. In some embodiments, the neoantigen sequence has been identified by sequencing at least one mRNA encoding a neoantigen induced in the subject by administering an effective amount of the splicing modulator, antibody-drug conjugate, or composition. In some embodiments, the at least one neoantigen mRNA encodes a neoantigen sequence induced by contacting a neoplastic cell with an effective amount of the splicing modulator, antibody-drug conjugate, or composition. In some embodiments, the neoplastic cell is present in an in vitro cell culture. In some embodiments, the neoplastic cell is obtained from the subject. In some embodiments, the neoplastic cell is present in the subject.

In some embodiments, the neoantigen vaccine comprises at least one neoantigen mRNA and a pharmaceutically acceptable carrier (e.g., any of the exemplary carriers described herein). In some embodiments, the at least one neoantigen mRNA is linked to the pharmaceutically acceptable carrier. In some embodiments, the pharmaceutically acceptable carrier is selected from a peptide, a serum albumin, a keyhole limpet hemocyanin, an immunoglobulin, a thyroglobulin, an ovalbumin, a toxoid or an attenuated toxoid derivative, a cytokine, and a chemokine. In some embodiments, the neoantigen vaccine comprises at least one neoantigen mRNA and a pharmaceutically acceptable diluent. In some embodiments, the neoantigen vaccine comprises at least one neoantigen mRNA and a pharmaceutically acceptable adjuvant. In some embodiments, the neoantigen mRNA is encapsulated by an encapsulating agent. In some embodiments, the encapsulating agent is a liposome. In some embodiments, the encapsulating agent is a nanoparticle.

›Definitions · 63 of 64

In some embodiments, the at least one additional therapy comprises administering a cytokine or cytokine analog, e.g., any cytokine or cytokine analog disclosed herein. In some embodiments, the subject is intolerant, non-responsive, or poorly responsive to the cytokine or cytokine analog when administered alone. In some embodiments, the cytokine or cytokine analog comprises a T-cell enhancer. In some embodiments, the cytokine or cytokine analog comprises IL-2, IL-10, IL-12, IL-15, IFNγ, and/or TNFα. In some embodiments, the cytokine or cytokine analog comprises IL-2, IL-10, IL-12, and/or IL-15. In some embodiments, administering the cytokine or cytokine analog enhances T-cell priming following administration of a splicing modulator, antibody-drug conjugate, or composition due to the induction and presentation of neoantigens.

In some embodiments, the at least one additional therapy comprises administering engineered tumor-targeting T-cells (i.e., CAR-T), e.g., any CAR-T therapy disclosed herein.

In some embodiments, the methods described herein may further comprise detecting one or more neoantigens and/or a T-cell response in the subject after administration of the splicing modulator, antibody-drug conjugate, or composition, and, optionally, continuing administration of the splicing modulator, antibody-drug conjugate, or composition if one or more neoantigens and/or a T-cell response is detected. In some embodiments, detecting one or more neoantigens and/or a T-cell response in the subject indicates efficacy of treatment with the splicing modulator, antibody-drug conjugate, or composition. In some embodiments, treatment with the additional therapy, along with splicing modulator, antibody-drug conjugate, or composition, is continued if one or more neoantigens and/or a T-cell response is detected. In some embodiments, treatment is continued at a reduced dosage and/or frequency if one or more neoantigens and/or a T-cell response is detected.

In some embodiments, the methods described herein may further comprise detecting a double-stranded RNA immune response in the subject after administration of the splicing modulator, antibody-drug conjugate, or composition, and, optionally, continuing administration of the splicing modulator, antibody-drug conjugate, or composition if a double-stranded RNA immune response is detected. In some embodiments, detecting a double-stranded RNA immune response in the subject indicates efficacy of treatment with the splicing modulator, antibody-drug conjugate, or composition. In some embodiments, treatment with the additional therapy, along with splicing modulator, antibody-drug conjugate, or composition, is continued if a double-stranded RNA immune response is detected. In some embodiments, treatment is continued at a reduced dosage and/or frequency if a double-stranded RNA immune response is detected.

In some embodiments, the methods described herein may further comprise detecting immunogenic cell death in the subject after administration of the splicing modulator, antibody-drug conjugate, or composition, and, optionally, continuing administration of the splicing modulator, antibody-drug conjugate, or composition if immunogenic cell death is detected. In some embodiments, detecting immunogenic cell death in the subject indicates efficacy of treatment with the splicing modulator, antibody-drug conjugate, or composition. In some embodiments, treatment with the additional therapy, along with splicing modulator, antibody-drug conjugate, or composition, is continued if immunogenic cell death is detected. In some embodiments, treatment is continued at a reduced dosage and/or frequency if immunogenic cell death is detected.

In some embodiments, the subject has a non-synonymous mutational burden of about 150 mutations or less. In some embodiments, the subject has a non-synonymous mutational burden of about 100 mutations or less. In some embodiments, the subject has a non-synonymous mutational burden of about 50 mutations or less. In some embodiments, the subject has or is suspected of having a neoplastic disorder, e.g., a hematological malignancy or a solid tumor. In some embodiments, the hematological malignancy is selected from a B-cell malignancy, a leukemia, a lymphoma, and a myeloma. In some embodiments, the hematological malignancy is selected from acute myeloid leukemia and multiple myeloma. In some embodiments, the solid tumor is selected from breast cancer, gastric cancer, prostate cancer, ovarian cancer, lung cancer, uterine cancer, salivary duct carcinoma, melanoma, colon cancer, cervical cancer, pancreatic cancer, kidney cancer, colorectal cancer, and esophageal cancer. In some embodiments, the solid tumor is selected from HER2-positive breast cancer, gastric adenocarcinoma, prostate cancer, and osteosarcoma.

In various embodiments, the present disclosure further provides a method of treating a subject having or suspected of having a neoplastic disorder, comprising: (a) administering to the subject an effective amount of a splicing modulator, an ADC, or composition comprising a splicing modulator or ADC, wherein administration of the splicing modulator, antibody-drug conjugate, or composition induces at least one neoantigen and/or a T-cell response; (b) detecting one or more neoantigens and/or a T-cell response in the subject after administration of the splicing modulator, antibody-drug conjugate, or composition; and (c) continuing administration of the splicing modulator, antibody-drug conjugate, or composition if one or more neoantigens and/or a T-cell response is detected. In some embodiments, detecting one or more neoantigens and/or a T-cell response in the subject indicates efficacy of treatment with the splicing modulator, antibody-drug conjugate, or composition. In some embodiments, the one or more neoantigens comprise an amino acid sequence of any one of SEQ ID NOs: 37-65. In some embodiments, the one or more neoantigens comprise an amino acid sequence of SEQ ID NO: 37. In some embodiments, the one or more neoantigens comprise an amino acid sequence of SEQ ID NO: 39. In some embodiments, the one or more neoantigens comprise an amino acid sequence of any one of SEQ ID NOs: 46-49.

›Definitions · 64 of 64

Combination of Splicing Modulator/ADC and Immune Checkpoint Inhibition:

In various embodiments, a patient having a cancer as described herein can be treated with a combination of a splicing modulator, ADC, or composition and a checkpoint inhibitor therapy. Immune checkpoints are inhibitory pathways that slow down or stop immune reactions and prevent excessive tissue damage from uncontrolled activity of immune cells. As used herein, the term “checkpoint inhibitor” is meant to refer to any therapeutic agent, including any small molecule chemical compound, antibody, nucleic acid molecule, or polypeptide, or any fragments thereof, that inhibits one or more of the inhibitory pathways, thereby allowing more extensive immune activity.

Treatment of patients with immune checkpoint inhibition has been shown to have robust efficacy in certain clinical indications. Recently, the FDA approved use of a checkpoint inhibitor in patients with tumors exhibiting high microsatellite instability, agnostic to the tissue lineage. This approval was based, in part, on the observation that response rates correlate positively with mutational burden (Rizvi et al. (2015) Science 348(6230):124-8; Hellmann et al. (2018) Cancer Cell 33(5):853-861). Estimates from the literature vary in absolute numbers and by lineage, but generally support that above a threshold of ˜150-250 mutations, the probability of response rises. Analysis of TCGA data shows that a large percentage of adult-onset tumor lineages have comparatively low non-synonymous mutational burden (Vogelstein et al. (2013) Science 339:1549-58). Most lineages have median non-synonymous mutational rates of ˜30-80 per patient, well below the thresholds for improved odds of response to checkpoint inhibitors.

For instance, HER2-positive breast cancer has been shown to have a median of ˜60 non-synonymous mutations present per patient sample. However, the threshold for checkpoint inhibitor treatment efficacy, as mentioned above, is estimated to be in the range of ˜150-250 non-synonymous mutations, i.e., patients above this threshold are more likely to show complete remission, partial remission, and/or stable disease, whereas patients below this threshold are more likely to exhibit progressive disease. Strategies to enhance the apparent number of non-synonymous mutations and/or neoantigens being presented on tumor cells are therefore desirable, and may enhance the overall probability of response, e.g., to checkpoint inhibitor therapies. As cytokines (and analogs thereof) act via a similar mechanism of action, such strategies may also enhance the overall probability of response to cytokine-based therapies.

Current response rates in HER2-positive breast cancer are ˜15-25% (CTI NCT02129556). In various embodiments disclosed herein, treatment with a splicing modulator, ADC, or composition in combination with a checkpoint inhibitor and/or cytokine therapy may improve such response rates. In various embodiments, treatment with a splicing modulator, ADC, or composition in combination with a checkpoint inhibitor and/or cytokine therapy may apply to any adult-onset tumor, particularly those in which the median non-synonymous mutational rate is below the estimated ˜150 mutations threshold. In various embodiments, exemplary cancer types suitable for treatment with a splicing modulator, ADC, or composition of the present disclosure, alone or in combination with an additional therapy (e.g., a checkpoint inhibitor therapy, a cytokine therapy) include but are not limited to esophageal cancer, non-Hodgkin's lymphoma, colorectal cancer, head and neck cancer, gastric cancer, endometrial cancer, pancreatic adenocarcinoma, ovarian cancer, prostate cancer, hepatocellular cancer, glioblastoma, breast cancer (e.g., HER2-positive breast cancer), lung cancer (e.g., non-small cell lung cancer), chronic lymphocytic leukemia, and acute myeloid leukemia. Other exemplary suitable cancer types are identified, e.g., in Vogelstein et al. (2013) Science 339:1549-58, which is incorporated herein by reference in its entirety.

As many checkpoint inhibitor therapies are based on chronic expression of tumor-associated antigens, regular treatment boosts are required for efficacy and for “re-boosting” reactive T-cell populations. The inducible nature of splicing modulator or ADC-derived neoantigens described herein provide for therapeutic dosing regimens that may be designed to enhance the

›Tables in the description — 20

TABLE 1 — Antibodies

mAb	Type	Target
trastuzumab (AB185)	humanized	HER2/NEU
B-B4 (AB205)	murine	CD138 (syndecan-1)
1C1 (AB206)	humanized	EPHA2

TABLE 2 — Amino acid sequences of mAb variable regions SEQ

	IgG	ID
mAb	chain	NO	Amino acid sequence
trastuzumab	Heavy	19	EVQLVESGGGLVQPGGSLRLSCAASGF
(AB185)	chain		NIKDTYIHWVRQAPGKGLEWVARIYPT
			NGYTRYADSVKGRFTISADTSKNTAYL
			QMNSLRAEDTAVYYCSRWGGDGFYAMD
			YWGQGTLVTVSS
trastuzumab	Light	20	DIQMTQSPSSLSASVGDRVTITCRASQ
(AB185)	chain		DVNTAVAWYQQKPGKAPKLLIYSASFL
			YSGVPSRFSGSRSGTDFTLTISSLQPE
			DFATYYCQQHYTTPPTFGQGTKVEIKR
			T
B-B4	Heavy	21	QVQLQQSGSELMMPGASVKISCKATGY
(AB205)	chain		TFSNYWIQRPGHGLEWIGEILPGTGRT
			IYNEKFKGKATFTADISSNTVQMQLSS
			LTSEDSAVYYCARRDYYGNFYYAMDYW
			GQGTSVTVSS
B-B4	Light	22	DIQMTQSTSSLSASLGDRVTISCSASQ
(AB205)	chain		GINNYLNWYQQKPDGTVELLIYYTSTL
			QSGVPSRFSGSGSGTDYSLTISNLEPE
			DIGTYYCQQYSKLPRTFGGGTKLEIK
1C1	Heavy	23	EVQLLESGGGLVQPGGSLRLSCAASGF
(AB206)	chain		TFSHYMMAWVRQAPGKGLEWVSRIGPS
			GGPTHYADSVKGRFTISRDNSKNTLYL
			QMNSLRAEDTAVYYCAGYDSGYDYVAV
			AGPAE-YFQHWGQGTLVTVSS
1C1	Light	24	DIQMTQSPSSLSASVGDRVTITCRASQ
(AB206)	chain		SISTWLAWYQQKPGKAPKLLIYKASNL
			HTGVPSRFSGSGSGTEFSLTISGLQPD
			DFATYYCQQYNSYS-RTFGQGTKVEIK

TABLE 3 — Amino acid sequences of mAb CDRs SEQ

mAb	IgG chain	ID NO	Amino acid sequence
trastuzumab	HCDR1	1	GFNIKDTYIH
(AB185)
trastuzumab	HCDR2	2	RIYPTNGYTRYADSVKG
(AB185)
trastuzumab	HCDR3	3	WGGDGFYAMDV
(AB185)
trastuzumab	LCDR1	4	RASQDVNTAVAW
(AB185)
trastuzumab	LCDR2	5	SASFLES
(AB185)
trastuzumab	LCDR3	6	QQHYTTPPT
(AB185)
B-B4	HCDR1	7	NYWIE
(AB205)
B-B4	HCDR2	8	ILPGTGRTIYNEKFKGKA
(AB205)
B-B4	HCDR3	9	RDYYGNFYYAMDY
(AB205)
B-B4	LCDR1	10	ASQGINNYLN
(AB205)
B-B4	LCDR2	11	TSTLQS
(AB205)
B-B4	LCDR3	12	QQYSKLPRT
(AB205)
1C1	HCDR1	13	HYMMA
(AB206)
1C1	HCDR2	14	RIGPSGGPTHYADSVKG
(AB206)
1C1	HCDR3	15	YDSGYDYVAVAGPAE-YFQH
(AB206)
1C1	LCDR1	16	RASWSISTWLA
(AB206)
1C1	LCDR2	17	KASNLHT
(AB206)
1C1	LCDR3	18	QQYNSYS-RT
(AB206)

TABLE 4 — Amino acid sequences of full-length mAb ig chains

mAb	IgG chain	Class	SEQ ID NO	Amino acid sequence
trastuzumab	Heavy chain	IgG1	25	EVQLVESGGGLVQPGGSLRLSCAA
(AB185)				SGFNIKDTYIHWVRQAPGKGLEWV
				ARIYPTNGYTRYADSVKGRFTISA
				DTSKNTAYLQMNSLRAEDTAVYYC
				SRWGGDGFYAMDYWGQGTLVTVSS
				ASTKGPSVFPLAPSSKSTSGGTAA
				LGCLVKDYFPEPVTVSWNSGALTS
				GVHTFPAVLQSSGLYSLSSVVTVP
				SSSLGTQTYICNVNHKPSNTKVDK
				KVEPPKSCDKTHTCPPCPAPELLG
				GPSVFLFPPKPKDTLMISRTPEVT
				CVVVDVSHEDPEVKFNWYVDGVEV
				HNAKTKPREEQYNSTYRVVSVLTV
				LHQDWLNGKEYKCKVSNKALPAPI
				EKTISKAKGQPREPQVYTLPPSRD
				ELTKNQVSLTCLVKGFYPSDIAVE
				WESNGQPENNYKTTPPVLDSDGSF
				FLYSKLTVDKSRWQQGNVFSCSVM
				HEALHNHYTQKSLSLSPGK
trastuzumab	Light chain	kappa	26	DIQMTQSPSSLSASVGDRVTITCR
(AB185)				ASQDVNTAVAWYQQKPGKAPKLLI
				YSASFLYSGVPSRFSGSRSGTDFT
				LTISSLQPEDFATYYCQQHYTTPP
				TFGQGTKVEIKRTVAAPSVFIFPP
				SDEQLKSGTASVVCLLNNFYPREA
				KVQWKVDNALQSGNSQESVTEQDS
				KDSTYSLSSTLTLSKADYEKHKVY
				ACEVTHQGLSSPVTKSFNRGEC
B-B4	Heavy chain	IgG2a	27	QVQLQQSGSELMMPGASVKISCKA
(AB205)				TGYTFSNYWIQRPGHGLEWIGEIL
				PGTGRTIYNEKFKGKATFTADISS
				NTVQMQLSSLTSEDSAVYYCARRD
				YYGNFYYAMDYWGQGTSVTVSSAK
				TTPPSVYPLAPGSAAQTNSMVTLG
				CLVKGYFPEPVTVTWNSGSLSSGV
				HTFPAVLQSDLYTLSSSVTVPSST
				WPSETVTCNVAHPASSTKVDKKIV
				PRDCGCKPCICTVPEVSSVFIFPP
				KPKDVLTITLTPKVTCVVVDISKD
				DPEVQFSWFVDDVEVHTAQTQPRE
				EQFNSTFRSVSELPIMHQDWLNGK
				EFKCRVNSAAFPAPIEKTISKTKG
				RPKAPQVYTIPPPKEQMAKDKVSL
				TCMITDFFPEDITVEWQWNGQPAE
				NYKNTQPIMDTDGSYFVYSKLNVQ
				KSNWEAGNTFTCSVLHEGLHNHHT
				EKSLSHSPG
B-B4	Light chain	kappa	28	DIQMTQSTSSLSASLGDRVTISCS
(AB205)				ASQGINNYLNWYQQKPDGTVELLI
				YYTSTLQSGVPSRFSGSGSGTDYS
				LTISNLEPEDIGTYYCQQYSKLPR
				TFGGGTKLEIKRADAAPTVSIFPP
				SSEQLTSGGASWCFLNNFYPKDI
				NVKWKIDGSERQNGVLNSWTDQDS
				KDSTYSMSSTLTLTKDEYERHNSY
				TCEATHKTSTSPIVKSFNRNEC
1C1	Heavy chain	IgG1	29	EVQLLESGGGLVQPGGSLRLSCAA
(AB206)				SGFTFSHYMMAWVRQAPGKGLEWV
				SRIGPSGGPTHYADSVKGRFTISR
				DNSKNTLYLQMNSLRAEDTAVYYC
				AGYDSGYDYVAVAGPAEYFQHWGQ
				GTLVTVSSASTKGPSVFPLAPSSK
				STSGGTAALGCLVKDYFPEPVTVS
				WNSGALTSGVHTFPAVLQSSGLYS
				LSSVVTVPSSSLGTQTYICNVNHK
				PSNTKVDKKVEPKSCDKTHTCPPC
				PAPELLGGPSVFLFPPKPKDTLMI
				SRTPEVTCVVVDVSHEDPEVKFNW
				YVDGVEVHNAKTKPREEQYNSTYR
				VVSVLTVLHQDWLNGKEYKCKVSN
				KALPAPIEKTISKAKGQPREPQVY
				TLPPSRDELTKNQVSLTCLVKGFY
				PSDIAVEWESNGQPENNYKTTPPV
				LDSDGSFFLYSKLTVDKSRWQQGN
				VFSCSVMHEALHNHYTQKSLSLSP
				G
1C1	Light chain	kappa	30	DIQMTQSPSSLSASVGDRVTITCR
(AB206)				ASQSISTWLAWYQQKPGKAPKLLI
				YKASNLHTGVPSRFSGSGSGTEFS
				LTISGLQPDDFATYYCQQYNSYSR
				TFGQGTKVEIKRTVAAPSVFIFPP
				SDEQLKSGTASVVCLLNNFYPREA
				KVQWKVDNALQSGNSQESVTEQDS
				KDSTYSLSSTLTLSKADYEKHKVY
				ACEVTHQGLSSPVTKSFNRGEC

TABLE 5 — Exemplary target antigen amino acid sequences

Antigen	SEQ ID NO	Amino acid sequence
HER2/NEU	31	MELAALCRWGLLLALLPPGAASTQVCTGTDMKLRLPASPETHLDM
		LRHLYQGCQVVQGNLELTYLPTNASLSFLQDIQEVQGYVLIAHNQ
		VRQVPLQRLRIVRGTQLFEDNYALAVLDNGDPLNNTTPVTGASPG
		GLRELQLRSLTEILKGGVLIQRNPQLCYQDTILWKDIFHKNNQLA
		LTLIDTNRSRACHPCSPMCKGSRCWGESSEDCQSLTRTVCAGGCA
		RCKGPLPTDCCHEQCAAGCTGPKHSDCLACLHFNHSGICELHCPA
		LVTYNTDTFESMPNPEGRYTFGASCVTACPYNYLSTDVGSCTLVC
		PLHNQEVTAEDGTQRCEKCSKPCARVCYGLGMEHLREVRAVTSAN
		IQEFAGCKKIFGSLAFLPESFDGDPASNTAPLQPEQLQVFETLEE
		ITGYLYISAWPDSLPDLSVFQNLQVIRGRILHNGAYSLTLQGLGI
		SWLGLRSLRELGSGLALIHHNTHLCFVHTVPWDQLFRNPHQALLH
		TANRPEDECVGEGLACHQLCARGHCWGPGPTQCVNCSQFLRGQEC
		VEECRVLQGLPREYVNARHCLPCHPECQPQNGSVTCFGPEADQCV
		ACAHYKDPPFCVARCPSGVKPDLSYMPIWKFPDEEGACQPCPINC
		THSCVDLDDKGCPAEQRASPLTSIISAVVGILLVVVLGVVFGILI
		KRRQQKIRKYTMRRLLQETELVEPLTPSGAMPNQAQMRILKETEL
		RKVKVLGSGAFGTVYKGIWIPDGENVKIPVAIKVLRENTSPKANK
		EILDEAYVMAGVGSPYVSRLLGICLTSTVQLVTQLMPYGCLLDHV
		RENRGRLGSQDLLNWCMQIAKGMSYLEDVRLVHRDLAARNVLVKS
		PNHVKITDFGLARLLDIDETEYHADGGKVPIKWMALESILRRRFT
		HQSDVWSYGVTVWELMTFGAKPYDGIPAREIPDLLEKGERLPQPP
		ICTIDVYMIMVKCWMIDSECRPRFRELVSEFSRMARDPQRFVVIQ
		NEDLGPASPLDSTFYRSLLEDDDMGDLVDAEEYLVPQQGFFCPDP
		APGAGGMVHHRHRSSSTRSGGGDLTLGLEPSEEEAPRSPLAPSEG
		AGSDVFDGDLGMGAAKGLQSLPTHDPSPLQRYSEDPTVPLPSETD
		GYVAPLTCSPQPEYVNQPDVRPQPPSPREGPLPAARPAGATLERP
		KTLSPGKNGVVKDVFAFGGAVENPEYLTPQGGAAPQPHPPPAFSP
		AFDNLYYWDQDPPERGAPPSTFKGTPTAENPEYLGLDVPV
CD138	32	MRRAALWLWLCALALSLQPALPQIVATNLPPEDQDGSGDDSDNFS
		GSGAGALQDITLSQQTPSTWKDTQLLTAIPTSPEPTGLEATAAST
		STLPAGEGPKEGEAVVLPEVEPGLTAREQEATPRPRETTQLPTTH
		LASTTTATTAQEPATSHPHRDMQPGHHETSTPAGPSQADLHTPHT
		EDGGPSATERAAEDGASSQLPAAEGSGEQDFTFETSGENTAVVAV
		EPDRRNQSPVDQGATGASQGLLDRKEVLGGVIAGGLVGLIFAVCL
		VGFMLYRMKKKDEGSYSLEEPKQANGGAYQKPTKQEEFYA
EPHA2	33	MELQAARACFALLWGCALAAAAAAQGKEVVLLDFAAAGGELGWLT
		HPYGKGWDLMQNIMNDMPIYMYSVCNVMSGDQDNWLRTNWVYRGE
		AERIFIELKFTVRDCNSFPGGASSCKETFNLYYAESDLDYGTNFQ
		KRLFTKIDTIAPDEITVSSDFEARHVKLNVEERSVGPLTRKGFYL
		AFQDIGACVALLSVRVYYKKCPELLQGLAHFPETIAGSDAPSLAT
		VAGTCVDHAVVPPGGEEPRMHCAVDGEWLVPIGQCLCQAGYEKVE
		DACQACSPGFFKFEASESPCLECPEHTLPSPEGATSCECEEGFFR
		APQDPASMPCTRPPSAPHYLTAVGMGAKVELRWTPPQDSGGREDI
		VYSVTCEQCWPESGECGPCEASVRYSEPPHGLTRTSVTVSDLEPH
		MNYTFTVEARNGVSGLVTSRSFRTASVSINQTEPPKVRLEGRSTT
		SLSVSWSIPPPQQSRVWKYEVTYRKKGDSNSYNVRRTEGFSVTLD
		DLAPDTTYLVQVQALTQEGQGAGSKVHEFQTLSPEGSGNLAVIGG
		VAVGVVLLLVLAGVGFFIHRRRKNQRARQSPEDVYFSKSEQLKPL
		KTYVDPHTYEDPNQAVLKFTTEIHPSCVTRQKVIGAGEFGEVYKG
		MLKTSSGKKEVPVAIKTLKAGYTEKQRVDFLGEAGIMGQFSHHNI
		IRLEGVISKYKPMMIITEYMENGALDKFLREKDGEFSVLQLVGML
		RGIAAGMKYLANMNYVHRDLAARNILVNSNLVCKVSDFGLSRVLE
		DDPEATYTTSGGKIPIRWTAPEAISYRKFTSASDVWSFGIVMWEV
		MTYGERPYWELSNHEVMKAINDGFRLPTPMDCPSAIYQLMMQCWQ
		QERARRPKFADIVSILDKLIRAPDSLKTLADFDPRVSIRLPSTSG
		SEGVPFRTVSEWLESIKMQQYTEHFMAAGYTAIEKVVQMTNDDIK
		RIGVRLPGHQ KRIAYSLLGLKDQVNTVGIPI
MSLN	43	MALPTARPLLGSCGTPALGSLLFLLFSLGWVQPSRTLAGETGQEA
		APLDGVLANPPNISSLSPRQLLGFPCAEVSGLSTERVRELAVALA
		QKNVKLSTEQLRCLAHRLSEPPEDLDALPLDLLLFLNPDAFSGPQ
		ACTRFFSRITKANVDLLPRGAPERQRLLPAALACWGVRGSLLSEA
		DVRALGGLACDLPGRFVAESAEVLLPRLVSCPGPLDQDQQEAARA
		ALQGGGPPYGPPSTWSVSTMDALRGLLPVLGQPIIRSIPQGIVAA
		WRQRSSRDPSWRQPERTILRPRFRREVEKTACPSGKKAREIDESL
		IFYKKWELEACVDAALLATQMDRVNAIPFTYEQLDVLKHKLDELY
		PQGYPESVIQHLGYLFLKMSPEDIRKWNVTSLETLKALLEVNKGH
		EMSPQAPRRPLPQVATLIDRFVKGRGQLDKDTLDTLTAFYPGYLC
		SLSPEELSSVPPSSIWAVRPQDLDTCDPRQLDVLYPKARLAFQNM
		NGSEYFVKIQSFLGGAPTEDLKALSQQNVSMDLATFMKLRTDAVL
		PLTVAEVQKLLGPHVEGLKAEERHRPVRDWILRQRQDDLDTLGLG
		LQGGIPNGYLVLDLSMQEALSGTPCLLGPGPVLTVLALLLASTLA
FOLH1	44	MWNLLHETDSAVATARRPRWLCAGALVLAGGFFLLGFLFGWFIKS
		SNEATNITPKHNMKAFLDELKAENIKKFLYNFTQIPHLAGTEQNF
		QLAKQIQSQWKEFGLDSVELAHYDVLLSYPNKTHPNYISIINEDG
		NEIFNTSLFEPPPPGYENVSDIVPPFSAFSPQGMPEGDLVYVNYA
		RTEDFFKLERDMKINCSGKIVIARYGKVFRGNKVKNAQLAGAKGV
		ILYSDPADYFAPGVKSYPDGWNLPGGGVQRGNILNLNGAGDPLTP
		GYPANEYAYRRGIAEAVGLPSIPVHPIGYYDAQKLLEKMGGSAPP
		DSSWRGSLKVPYNVGPGFTGNFSTQKVKMHIHSTNEVTRIYNVIG
		TLRGAVEPDRYVILGGHRDSWVFGGIDPQSGAAVVHEIVRSFGTL
		KKEGWRPRRTILFASWDAEEFGLLGSTEWAEENSRLLQERGVAYI
		NADSSIEGNYTLRVDCTPLMYSLVHNLTKELKSPDEGFEGKSLYE
		SWTKKSPSPEFSGMPRISKLGSGNDFEVFFQRLGIASGRARYTKN
		WETNKFSGYPLYHSVYETYELVEKFYDPMFKYHLTVAQVRGGMVF
		ELANSIVLPFDCRDYAVVLRKYADKIYSISMKHPQEMKTYSVSFD
		SLFSAVKNFTEIASKFSERLQDFDKSNPIVLRMMNDQLMFLERAF
		IDPLGLPDRPFYRHVIYAPSSHNKYAGESFPGIYDALFDIESKVD
		PSKAWGEVKRQIYVAAFTVQAAAETLSEVA
CDH6	45	MRTYRYFLLLFWVGQPYPTLSTPLSKRTSGFPAKKRALELSGNSK
		NELNRSKRSWMWNQFFLLEEYTGSDYQYVGKLHSDQDRGDGSLKY
		ILSGDGAGDLFIINENTGDIQATKRLDREEKPVYILRAQAINRRT
		GRPVEPESEFIIKIHDINDNEPIFTKEVYTATVPEMSDVGTFVVQ
		VTATDADDPTYGNSAKVVYSILQGQPYFSVESETGIIKTALLNMD
		RENREQYQVVIQAKDMGGQMGGLSGTTTVNITLTDVNDNPPRFPQ
		STYQFKTPESSPPGTPIGRIKASDADVGENAEIEYSITDGEGLDM
		FDVITDQETQEGIITVKKLLDFEKKKVYTLKVEASNPYVEPRFLY
		LGPFKDSATVRIVVEDVDEPPVFSKLAYILQIREDAQINTTIGSV
		TAQDPDAARNPVKYSVDRHTDMDRIFNIDSGNGSIFTSKLLDRET
		LLWHNITVIATEINNPKQSSRVPLYIKVLDVNDNAPEFAEFYETF
		VCEKAKADQLIQTLHAVDKDDPYSGHQFSFSLAPEAASGSNFTIQ
		DNKDNTAGILTRKNGYNRHEMSTYLLPVVISDNDYPVQSSTGTVT
		VRVCACDHHGNMQSCHAEALIHPTGLSTGALVAILLCIVILLVTV
		VLFAALRRQRKKEPLIISKEDIRDNIVSYNDEGGGEEDTQAFDIG
		TLRNPEAIEDNKLRRDIVPEALFLPRRTPTARDNTDVRDFINQRL
		KENDTDPTAPPYDSLATYAYEGTGSVADSLSSLESVTTDADQDYD
		YLSDWGPRFKKLADMYGGVDSDKDS
CEACAM5	46	MESPSAPPHRWCIPWQRLLLTASLLTFWNPPTTAKLTIESTPFNV
		AEGKEVLLLVHNLPQHLFGYSWYKGERVDGNRQIIGYVIGTQQAT
		PGPAYSGREIIYPNASLLIQNIIQNDTGFYTLHVIKSDLVNEEAT
		GQFRVYPELPKPSISSNNSKPVEDKDAVAFTCEPETQDATYLWWV
		NNQSLPVSPRLQLSNGNRTLTLFNVTRNDTASYKCETQNPVSARR
		SDSVILNVLYGPDAPTISPLNTSYRSGENLNLSCHAASNPPAQYS
		WFVNGTFQQSTQELFIPNITVNNSGSYTCQAHNSDTGLNRTTVTT
		ITVYAEPPKPFITSNNSNPVEDEDAVALTCEPEIQNTTYLWWVNN
		QSLPVSPRLQLSNDNRTLTLLSVTRNDVGPYECGIQNKLSVDHSD
		PVILNVLYGPDDPTISPSYTYYRPGVNLSLSCHAASNPPAQYSWL
		IDGNIQQHTQELFISNITEKNSGLYTCQANNSASGHSRTTVKTIT
		VSAELPKPSISSNNSKPVEDKDAVAFTCEPEAQNTTYLWWVNGQS
		LPVSPRLQLSNGNRTLTLFNVTRNDARAYVCGIQNSVSANRSDPV
		TLDVLYGPDTPIISPPDSSYLSGANLNLSCHSASNPSPQYSWRIN
		GIPQQHTQVLFIAKITPNNNGTYACFVSNLATGRNNSIVKSITVS
		ASGTSPGLSAGATVGIMIGVLVGVALI
CFC1B	47	MTWRHHVRLLFTVSLALQIINLGNSYQREKHNGGREEVTKVATQK
		HRQSPLNWTSSHFGEVTGSAEGWGPEEPLPYSWAFGEGASARPRC
		CRNGGTCVLGSFCVCPAHFTGRYCEHDQRRSECGALEHGAWTLRA
		CHLCRCIFGALHCLPLQTPDRCDPKDFLASHAHGPSAGGAPSLLL
		LLPCALLHRLLRPDAPAHPRSLVPSVLQRERRPCGRPGLGHRL
ENPP3	48	MESTLTLATEQPVKKNTLKKYKIACIVLLALLVIMSLGLGLGLGL
		RKLEKQGSCRKKCFDASFRGLENCRCDVACKDRGDCCWDFEDTCV
		ESTRIWMCNKFRCGETRLEASLCSCSDDCLQRKDCCADYKSVCQG
		ETSWLEENCDTAQQSQCPEGFDLPPVILFSMDGFRAEYLYTWDTL
		MPNINKLKTCGIHSKYMRAMYPTKTFPNHYTIVTGLYPESHGIID
		NNMYDVNLNKNFSLSSKEQNNPAWWHGQPMWLTAMYQGLKAATYF
		WPGSEVAINGSFPSIYMPYNGSVPFEERISTLLKWLDLPKAERPR
		FYTMYFEEPDSSGHAGGPVSARVIKALQVVDHAFGMLMEGLKQRN
		LHNCVNIILLADHGMDQTYCNKMEYMTDYFPRINFFYMYEGPAPR
		IRAHNIPHDFFSFNSEEIVRNLSCRKPDQHFKPYLTPDLPKRLHY
		AKNVRIDKVHLFVDQQWLAVRSKSNTNCGGGNHGYNNEFRSMEAI
		FLAHGPSFKEKTEVEPFENIEVYNLMCDLLRIQPAPNNGTHGSLN
		HLLKVPFYEPSHAEEVSKFSVCGFANPLPTESLDCFCPHLQNSTQ
		LEQVNQMLNLTQEEITATVKVNLPFGRPRVLQKNVDHCLLYHREY
		VSGFGKAMRMPMWSSYTVPQLGDTSPLPPTVPDCLRADVRVPPSE
		SQKCSFYLADKNITHGFLYPPASNRTSDSQYDALITSNLVPMYEE
		FRKMWDYFHSVLLIKHATERNGVNVVSGPIFDYNYDGHFDAPDEI
		TKHLANTDVPIPTHYFVVLTSCKNKSHTPENCPGWLDVLPFIIPH
		RPTNVESCPEGKPEALWVEERFTAHIARVRDVELLTGLDFYQDKV
		QPVSEILQLKTYLPTFETTI
FOLR1	49	MAQRMTTQLLLLLVWVAVVGEAQTRIAWARTELLNVCMNAKHHKE
		KPGPEDKLHEQCRPWRKNACCSTNTSQEAHKDVSYLYRFNWNHCG
		EMAPACKRHFIQDTCLYECSPNLGPWIQQVDQSWRKERVLNVPLC
		KEDCEQWWEDCRTSYTCKSNWHKGWNWTSGFNKCAVGAACQPFHF
		YFPTPTVLCNEIWTHSYKVSNYSRGSGRCIQMWFDPAQGNPNEEV
		ARFYAAAMSGAGPWAAWPFLLSLALMLLWLLS
HAVCR1	50	MHPQVVILSLILHLADSVAGSVKVGGEAGPSVTLPCHYSGAVTSM
		CWNRGSCSLFTCQNGIVWTNGTHVTYRKDTRYKLLGDLSRRDVSL
		TIENTAVSDSGVYCCRVEHRGWFNDMKITVSLEIVPPKVTTTPIV
		TTVPTVTTVRTSTTVPTTTTVPMTTVPTTTVPTTMSIPTTTTVLT
		TMTVSTTTSVPTTTSIPTTTSVPVTTTVSTFVPPMPLPRQNHEPV
		ATSPSSPQPAETHPTTLQGAIRREPTSSPLYSYTTDGNDTVTESS
		DGLWNNNQTQLFLEHSLLTANTTKGIYAGVCISVLVLLALLGVII
		AKKYFFKKEVQQLSVSFSSLQIKALQNAVEKEVQAEDNIYIENSL
		YATD
KIT	51	MRGARGAWDFLCVLLLLLRVQTGSSQPSVSPGEPSPPSIHPGKSD
		LIVRVGDEIRLLCTDPGFVKWTFEILDETNENKQNEWITEKAEAT
		NTGKYTCTNKHGLSNSIYVFVRDPAKLFLVDRSLYGKEDNDTLVR
		CPLTDPEVTNYSLKGCQGKPLPKDLRFIPDPKAGIMIKSVKRAYH
		RLCLHCSVDQEGKSVLSEKFILKVRPAFKAVPVVSVSKASYLLRE
		GEEFTVTCTIKDVSSSVYSTWKRENSQTKLQEKYNSWHHGDFNYE
		RQATLTISSARVNDSGVFMCYANNTFGSANVTTTLEVVDKGFINI
		FPMINTTVFVNDGENVDLIVEYEAFPKPEHQQWIYMNRTFTDKWE
		DYPKSENESNIRYVSELHLTRLKGTEGGTYTFLVSNSDVNAAIAF
		NVYVNTKPEILTYDRLVNGMLQCVAAGFPEPTIDWYFCPGTEQRC
		SASVLPVDVQTLNSSGPPFGKLVVQSSIDSSAFKHNGTVECKAYN
		DVGKTSAYFNFAFKGNNKEQIHPHTLFTPLLIGFVIVAGMMCIIV
		MILTYKYLQKPMYEVQWKVVEEINGNNYVYIDPTQLPYDHKWEFP
		RNRLSFGKTLGAGAFGKVVEATAYGLIKSDAAMTVAVKMLKPSAH
		LTEREALMSELKVLSYLGNHMNIVNLLGACTIGGPTLVITEYCCY
		GDLLNFLRRKRDSFICSKQEDHAEAALYKNLLHSKESSCSDSTNE
		YMDMKPGVSYVVPTKADKRRSVRIGSYIERDVTPAIMEDDELALD
		LEDLLSFSYQVAKGMAFLASKNCIHRDLAARNILLTHGRITKICD
		FGLARDIKNDSNYVVKGNARLPVKWMAPESIFNCVYTFESDVWSY
		GIFLWELFSLGSSPYPGMPVDSKFYKMIKEGFRMLSPEHAPAEMY
		DIMKTCWDADPLKRPTFKQIVQLIEKQISESTNHIYSNLANCSPN
		RQKPVVDHSVRINSVGSTASSSQPLLVHDDV
MET	52	MKAPAVLAPGILVLLFTLVQRSNGECKEALAKSEMNVNMKYQLPN
		FTAETPIQNVILHEHHIFLGATNYIYVLNEEDLQKVAEYKTGPVL
		EHPDCFPCQDCSSKANLSGGVWKDNINMALVVDTYYDDQLISCGS
		VNRGTCQRHVFPHNHTADIQSEVHCIFSPQIEEPSQCPDCVVSAL
		GAKVLSSVKDRFINFFVGNTINSSYFPDHPLHSISVRRLKETKDG
		FMFLTDQSYIDVLPEFRDSYPIKYVHAFESNNFIYFLTVQRETLD
		AQTFHTRIIRFCSINSGLHSYMEMPLECILTEKRKKRSTKKEVFN
		ILQAAYVSKPGAQLARQIGASLNDDILFGVFAQSKPDSAEPMDRS
		AMCAFPIKYVNDFFNKIVNKNNVRCLQHFYGPNHEHCFNRTLLRN
		SSGCEARRDEYRTEFTTALQRVDLFMGQFSEVLLTSISTFIKGDL
		TIANLGTSEGRFMQVVVSRSGPSTPHVNFLLDSHPVSPEVIVEHT
		LNQNGYTLVITGKKITKIPLNGLGCRHFQSCSQCLSAPPFVQCGW
		CHDKCVRSEECLSGTWTQQICLPAIYKVFPNSAPLEGGTRLTICG
		WDFGFRRNNKFDLKKTRVLLGNESCTLTLSESTMNTLKCTVGPAM
		NKHFNMSIIISNGHGTTQYSTFSYVDPVITSISPKYGPMAGGTLL
		TLTGNYLNSGNSRHISIGGKTCTLKSVSNSILECYTPAQTISTEF
		AVKLKIDLANRETSIFSYREDPIVYEIHPTKSFISGGSTITGVGK
		NLNSVSVPRMVINVHEAGRNFTVACQHRSNSEIICCTTPSLQQLN
		LQLPLKTKAFFMLDGILSKYFDLIYVHNPVFKPFEKPVMISMGNE
		NVLEIKGNDIDPEAVKGEVLKVGNKSCENIHLHSEAVLCTVPNDL
		LKLNSELNIEWKQAISSTVLGKVIVQPDQNFTGLIAGVVSISTAL
		LLLLGFFLWLKKRKQIKDLGSELVRYDARVHTPHLDRLVSARSVS
		PTTEMVSNESVDYRATFPEDQFPNSSQNGSCRQVQYPLTDMSPIL
		TSGDSDISSPLLQNTVHIDLSALNPELVQAVQHVVIGPSSLIVHF
		NEVIGRGHFGCVYHGTLLDNDGKKIHCAVKSLNRITDIGEVSQFL
		TEGIIMKDFSHPNVLSLLGICLRSEGSPLVVLPYMKHGDLRNFIR
		NETHNPTVKDLIGFGLQVAKGMKYLASKKFVHRDLAARNCMLDEK
		FTVKVADFGLARDMYDKEYYSVHNKTGAKLPVKWMALESLQTQKF
		TTKSDVWSFGVLLWELMTRGAPPYPDVNTFDITVYLLQGRRLLQP
		EYCPDPLYEVMLKCWHPKAEMRPSFSELVSRISAIFSTFIGEHYV
		HVNATYVNVKCVAPYPSLLSSEDNADDEVDTRPASFWETS
MUC16	53	MLKPSGLPGSSSPTRSLMTGSRSTKATPEMDSGLTGATLSPKTST
		GAIVVTEHTLPFTSPDKTLASPTSSVVGRTTQSLGVMSSALPEST
		SRGMTHSEQRTSPSLSPQVNGTPSRNYPATSMVSGLSSPRTRTSS
		TEGNFTKEASTYTLTVETTSGPVTEKYTVPTETSTTEGDSTETPW
		DTRYIPVKITSPMKTFADSTASKENAPVSMTPAETTVTDSHTPGR
		TNPSFGTLYSSFLDLSPKGTPNSRGETSLELILSTTGYPFSSPEP
		GSAGHSRISTSAPLSSSASVLDNKISETSIFSGQSLTSPLSPGVP
		EARASTMPNSAIPFSMTLSNAETSAERVRSTISSLGTPSISTKQT
		AETILTFHAFAETMDIPSTHIAKTLASEWLGSPGTLGGTSTSALT
		TTSPSTTLVSEETNTHHSTSGKETEGTLNTSMTPLETSAPGEESE
		MTATLVPTLGFTTLDSKIRSPSQVSSSHPTRELRTTGSTSGRQSS
		STAAHGSSDILRATTSSTSKASSWTSESTAQQFSEPQHTQWVETS
		PSMKTERPPASTSVAAPITTSVPSVVSGFTTLKTSSTKGIWLEET
		SADTLIGESTAGPTTHQFAVPTGISMTGGSSTRGSQGTTHLLTRA
		TASSETSADLTLATNGVPVSVSPAVSKTAAGSSPPGGTKPSYTMV
		SSVIPETSSLQSSAFREGTSLGLTPLNTRHPFSSPEPDSAGHTKI
		STSIPLLSSASVLEDKVSATSTFSHHKATSSITTGTPEISTKTKP
		SSAVLSSMTLSNAATSPERVRNATSPLTHPSPSGEETAGSVLTLS
		TSAETTDSPNIHPTGTLTSESSESPSTLSLPSVSGVKTTFSSSTP
		STHLFTSGEETEETSNPSVSQPETSVSRVRTTLASTSVPTPVFPT
		MDTWPTRSAQFSSSHLVSELRATSSTSVTNSTGSALPKISHLTGT
		ATMSQTNRDTFNDSAAPQSTTWPETSPRFKTGLPSATTTVSTSAT
		SLSATVMVSKFTSPATSSMEATSIREPSTTILTTETTNGPGSMAV
		ASTNIPIGKGYITEGRLDTSHLPIGTTASSETSMDFTMAKESVSM
		SVSPSQSMDAAGSSTPGRTSQFVDTFSDDVYHLTSREITIPRDGT
		SSALTPQMTATHPPSPDPGSARSTWLGILSSSPSSPTPKVTMSST
		FSTQRVTTSMIMDTVETSRWNMPNLPSTTSLTPSNIPTSGAIGKS
		TLVPLDTPSPATSLEASEGGLPTLSTYPESTNTPSIHLGAHASSE
		SPSTIKLTMASVVKPGSYTPLTFPSIETHIHVSTARMAYSSGSSP
		EMTAPGETNTGSTWDPTTYITTTDPKDTSSAQVSTPHSVRTLRTT
		ENHPKTESATPAAYSGSPKISSSPNLTSPATKAWTITDTTEHSTQ
		LHYTKLAEKSSGFETQSAPGPVSVVIPTSPTIGSSTLELTSDVPG
		EPLVLAPSEQTTITLPMATWLSTSLTEEMASTDLDISSPSSPMST
		FAIFPPMSTPSHELSKSEADTSAIRNTDSTTLDQHLGIRSLGRTG
		DLTTVPITPLTTTWTSVIEHSTQAQDTLSATMSPTHVTQSLKDQT
		SIPASASPSHLTEVYPELGTQGRSSSEATTFWKPSTDTLSREIET
		GPTNIQSTPPMDNTTTGSSSSGVTLGIAHLPIGTSSPAETSTNMA
		LERRSSTATVSMAGTMGLLVTSAPGRSISQSLGRVSSVLSESTTE
		GVTDSSKGSSPRLNTQGNTALSSSLEPSYAEGSQMSTSIPLTSSP
		TTPDVEFIGGSTFWTKEVTTVMTSDISKSSARTESSSATLMSTAL
		GSTENTGKEKLRTASMDLPSPTPSMEVTPWISLTLSNAPNTTDSL
		DLSHGVHTSSAGTLATDRSLNTGVTRASRLENGSDTSSKSLSMGN
		STHTSMTYTEKSEVSSSIHPRPETSAPGAETTLTSTPGNRAISLT
		LPFSSIPVEEVISTGITSGPDINSAPMTHSPITPPTIVWTSTGTI
		EQSTQPLHAVSSEKVSVQTQSTPYVNSVAVSASPTHENSVSSGSS
		TSSPYSSASLESLDSTISRRNAITSWLWDLTTSLPTTTWPSTSLS
		EALSSGHSGVSNPSSTTTEFPLFSAASTSAAKQRNPETETHGPQN
		TAASTLNTDASSVTGLSETPVGASISSEVPLPMAITSRSDVSGLT
		SESTANPSLGTASSAGTKLTRTISLPTSESLVSFRMNKDPWTVSI
		PLGSHPTTNTETSIPVNSAGPPGLSTVASDVIDTPSDGAESIPTV
		SFSPSPDTEVTTISHFPEKTTHSFRTISSLTHELTSRVTPIPGDW
		MSSAMSTKPTGASPSITLGERRTITSAAPTTSPIVLTASFTETST
		VSLDNETTVKTSDILDARKTNELPSDSSSSSDLINTSIASSTMDV
		TKTASISPTSISGMTASSSPSLFSSDRPQVPTSTTETNTATSPSV
		SSNTYSLDGGSNVGGTPSTLPPFTITHPVETSSALLAWSRPVRTF
		STMVSTDTASGENPTSSNSVVTSVPAPGTWTSVGSTTDLPAMGFL
		KTSPAGEAHSLLASTIEPATAFTPHLSAAVVTGSSATSEASLLTT
		SESKAIHSSPQTPTTPTSGANWETSATPESLLVVTETSDTTLTSK
		ILVTDTILFSTVSTPPSKFPSTGTLSGASFPTLLPDTPAIPLTAT
		EPTSSLATSFDSTPLVTIASDSLGTVPETTLTMSETSNGDALVLK
		TVSNPDRSIPGITIQGVTESPLHPSSTSPSKIVAPRNTTYEGSIT
		VALSTLPAGTTGSLVFSQSSENSETTALVDSSAGLERASVMPLTT
		GSQGMASSGGIRSGSTHSTGTKTFSSLPLTMNPGEVTAMSEITTN
		RLTATQSTAPKGIPVKPTSAESGLLTPVSASSSPSKAFASLTTAP
		PTWGIPQSTLTFEFSEVPSLDTKSASLPTPGQSLNTIPDSDASTA
		SSSLSKSPEKNPRARMMTSTKAISASSFQSTGFTETPEGSASPSM
		AGHEPRVPTSGTGDPRYASESMSYPDPSKASSAMTSTSLASKLTT
		LFSTGQAARSGSSSSPISLSTEKETSFLSPTASTSRKTSLFLGPS
		MARQPNILVHLQTSALTLSPTSTLNMSQEEPPELTSSQTIAEEEG
		TTAETQTLTFTPSETPTSLLPVSSPTEPTARRKSSPETWASSISV
		PAKTSLVETTDGTLVTTIKMSSQAAQGNSTWPAPAEETGSSPAGT
		SPGSPEMSTTLKIMSSKEPSISPEIRSTVRNSPWKTPETTVPMET
		TVEPVTLQSTALGSGSTSISHLPTGTTSPTKSPTENMLATERVSL
		SPSPPEAWTNLYSGTPGGTRQSLATMSSVSLESPTARSITGTGQQ
		SSPELVSKTTGMEFSMWHGSTGGTTGDTHVSLSTSSNILEDPVTS
		PNSVSSLTDKSKHKTETWVSTTAIPSTVLNNKIMAAEQQTSRSVD
		EAYSSTSSWSDQTSGSDITLGASPDVTNTLYITSTAQTTSLVSLP
		SGDQGITSLTNPSGGKTSSASSVTSPSIGLETLRANVSAVKSDIA
		PTAGHLSQTSSPAEVSILDVTTAPTPGISTTITTMGTNSISTTTP
		NPEVGMSTMDSTPATERRTTSTEHPSTWSSTAASDSWTVTDMTSN
		LKVARSPGTISTMHTTSFLASSTELDSMSTPHGRITVIGTSLVTP
		SSDASAVKTETSTSERTLSPSDTTASTPISTFSRVQRMSISVPDI
		LSTSWTPSSTEAEDVPVSMVSTDHASTKTDPNTPLSTFLFDSLST
		LDWDTGRSLSSATATTSAPQGATTPQELTLETMISPATSQLPFSI
		GHITSAVTPAAMARSSGVTFSRPDPTSKKAEQTSTQLPTTTSAHP
		GQVPRSAATTLDVIPHTAKTPDATFQRQGQTALTTEARATSDSWN
		EKEKSTPSAPWITEMMNSVSEDTIKEVTSSSSVLRTLNTLDINLE
		SGTTSSPSWKSSPYERIAPSESTTDKEAIHPSTNTVETTGWVTSS
		EHASHSTIPAHSASSKLTSPVVTTSTREQAIVSMSTTTWPESTRA
		RTEPNSFLTIELRDVSPYMDTSSTTQTSIISSPGSTAITKGPRTE
		ITSSKRISSSFLAQSMRSSDSPSEAITRLSNFPAMTESGGMILAM
		QTSPPGATSLSAPTLDTSATASWTGTPLATTQRFTYSEKTTLFSK
		GPEDTSQPSPPSVEETSSSSSLVPIHATTSPSNILLTSQGHSPSS
		TPPVTSVFLSETSGLGKTTDMSRISLEPGTSLPPNLSSTAGEALS
		TYEASRDTKAIHHSADTAVTNMEATSSEYSPIPGHTKPSKATSPL
		VTSHIMGDITSSTSVFGSSETTEIETVSSVNQGLQERSTSQVASS
		ATETSTVITHVSSGDATTHVTKTQATFSSGTSISSPHQFITSTNT
		FTDVSTNPSTSLIMTESSGVTITTQTGPTGAATQGPYLLDTSTMP
		YLTETPLAVTPDFMQSEKTTLISKGPKDVSWTSPPSVAETSYPSS
		LTPFLVTTIPPATSTLQGQHTSSPVSATSVLTSGLVKTTDMLNTS
		MEPVTNSPQNLNNPSNEILATLAATTDIETIHPSINKAVTNMGTA
		SSAHVLHSTLPVSSEPSTATSPMVPASSMGDALASISIPGSETTD
		IEGEPTSSLTAGRKENSTLQEMNSTTESNIILSNVSVGAITEATK
		MEVPSFDATFIPTPAQSTKFPDIFSVASSRLSNSPPMTISTHMTT
		TQTGSSGATSKIPLALDTSTLETSAGTPSVVTEGFAHSKITTAMN
		NDVKDVSQTNPPFQDEASSPSSQAPVLVTTLPSSVAFTPQWHSTS
		SPVSMSSVLTSSLVKTAGKVDTSLETVTSSPQSMSNTLDDISVTS
		AATTDIETTHPSINTVVTNVGTTGSAFESHSTVSAYPEPSKVTSP
		NVTTSTMEDTTISRSIPKSSKTTRTETETTSSLTPKLRETSISQE
		ITSSTETSTVPYKELTGATTEVSRTDVTSSSSTSFPGPDQSTVSL
		DISTETNTRLSTSPIMTESAEITITTQTGPHGATSQDTFTMDPSN
		TTPQAGIHSAMTHGFSQLDVTTLMSRIPQDVSWTSPPSVDKTSSP
		SSFLSSPAMTTPSLISSTLPEDKLSSPMTSLLTSGLVKITDILRT
		RLEPVTSSLPNFSSTSDKILATSKDSKDTKEIFPSINTEETNVKA
		NNSGHESHSPALADSETPKATTQMVITTTVGDPAPSTSMPVHGSS
		ETTNIKREPTYFLTPRLRETSTSQESSFPTDTSFLLSKVPTGTIT
		EVSSTGVNSSSKISTPDHDKSTVPPDTFTGEIPRVFTSSIKTKSA
		EMTITTQASPPESASHSTLPLDTSTTLSQGGTHSTVTQGFPYSEV
		TTLMGMGPGNVSWMTTPPVEETSSVSSLMSSPAMTSPSPVSSTSP
		QSIPSSPLPVTALPTSVLVTTTDVLGTTSPESVTSSPPNLSSITH
		ERPATYKDTAHTEAAMHHSTNTAVTNVGTSGSGHKSQSSVLADSE
		TSKATPLMSTTSTLGDTSVSTSTPNISQTNQIQTEPTASLSPRLR
		ESSTSEKTSSTTETNTAFSYVPTGAITQASRTEISSSRTSISDLD
		RPTIAPDISTGMITRLFTSPIMTKSAEMTVTTQTTTPGATSQGIL
		PWDTSTTLFQGGTHSTVSQGFPHSEITTLRSRTPGDVSWMTTPPV
		EETSSGFSLMSPSMTSPSPVSSTSPESIPSSPLPVTALLTSVLVT
		TTNVLGTTSPEPVTSSPPNLSSPTQERLTTYKDTAHTEAMHASMH
		TNTAVANVGTSISGHESQSSVPADSHTSKATSPMGITFAMGDTSV
		STSTPAFFETRIQTESTSSLIPGLRDTRTSEEINTVTETSTVLSE
		VPTTTTTEVSRTEVITSSRTTISGPDHSKMSPYISTETITRLSTF
		PFVTGSTEMAITNQTGPIGTISQATLTLDTSSTASWEGTHSPVTQ
		RFPHSEETTTMSRSTKGVSWQSPPSVEETSSPSSPVPLPAITSHS
		SLYSAVSGSSPTSALPVTSLLTSGRRKTIDMLDTHSELVTSSLPS
		ASSFSGEILTSEASTNTETIHFSENTAETNMGTTNSMHKLHSSVS
		IHSQPSGHTPPKVTGSMMEDAIVSTSTPGSPETKNVDRDSTSPLT
		PELKEDSTALVMNSTTESNTVFSSVSLDAATEVSRAEVTYYDPTF
		MPASAQSTKSPDISPEASSSHSNSPPLTISTHKTIATQTGPSGVT
		SLGQLTLDTSTIATSAGTPSARTQDFVDSETTSVMNNDLNDVLKT
		SPFSAEEANSLSSQAPLLVTTSPSPVTSTLQEHSTSSLVSVTSVP
		TPTLAKITDMDTNLEPVTRSPQNLRNTLATSEATTDTHTMHPSIN
		TAVANVGTTSSPNEFYFTVSPDSDPYKATSAVVITSTSGDSIVST
		SMPRSSAMKKIESETTFSLIFRLRETSTSQKIGSSSDTSTVFDKA
		FTAATTEVSRTELTSSSRTSIQGTEKPTMSPDTSTRSVTMLSTFA
		GLTKSEERTIATQTGPHRATSQGTLTWDTSITTSQAGTHSAMTHG
		FSQLDLSTLTSRVPEYISGTSPPSVEKTSSSSSLLSLPAITSPSP
		VPTTLPESRPSSPVHLTSLPTSGLVKTTDMLASVASLPPNLGSTS
		HKIPTTSEDIKDTEKMYPSTNIAVTNVGTTTSEKESYSSVPAYSE
		PPKVTSPMVTSFNIRDTIVSTSMPGSSEITRIEMESTFSLAHGLK
		GTSTSQDPIVSTEKSAVLHKLTTGATETSRTEVASSRRTSIPGPD
		HSTESPDISTEVIPSLPISLGITESSNMTIITRTGPPLGSTSQGT
		FTLDTPTTSSRAGTHSMATQEFPHSEMTTVMNKDPEILSWTIPPS
		IEKTSFSSSLMPSPAMTSPPVSSTLPKTIHTTPSPMTSLLTPSLV
		MTTDTLGTSPEPTTSSPPNLSSTSHEILTTDEDTTAIEAMHPSTS
		TAATNVETTSSGHGSQSSVLADSEKTKATAPMDTTSTMGHTTVST
		SMSVSSETTKIKRESTYSLTPGLRETSISQNASFSTDTSIVLSEV
		PTGTTAEVSRTEVTSSGRTSIPGPSQSTVLPEISTRTMTRLFASP
		TMTESAEMTIPTQTGPSGSTSQDTLTLDTSTTKSQAKTHSTLTQR
		FPHSEMTTLMSRGPGDMSWQSSPSLENPSSLPSLLSLPATTSPPP
		ISSTLPVTISSSPLPVTSLLTSSPVTTTDMLHTSPELVTSSPPKL
		SHTSDERLTTGKDTTNTEAVHPSTNTAASNVEIPSSGHESPSSAL
		ADSETSKATSPMFITSTQEDTTVAISTPHFLETSRIQKESISSLS
		PKLRETGSSVETSSAIETSAVLSEVSIGATTEISRTEVTSSSRTS
		ISGSAESTMLPEISTTRKIIKFPTSPILAESSEMTIKTQTSPPGS
		TSESTFTLDTSTTPSLVITHSTMTQRLPHSEITTLVSRGAGDVPR
		PSSLPVEETSPPSSQLSLSAMISPSPVSSTLPASSHSSSASVTSL
		LTPGQVKTTEVLDASAEPETSSPPSLSSTSVEILATSEVTTDTEK
		IHPFSNTAVTKVGTSSSGHESPSSVLPDSETTKATSAMGTISIMG
		DTSVSTLTPALSNTRKIQSEPASSLTTRLRETSTSEETSLATEAN
		TVLSKVSTGATTEVSRTEAISFSRTSMSGPEQSTMSQDISIGTIP
		RISASSVLTESAKMTITTQTGPSESTLESTLNLNTATTPSWVETH
		SIVIQGFPHPEMTTSMGRGPGGVSWPSPPFVKETSPPSSPLSLPA
		VTSPHPVSTTFLAHIPPSPLPVTSLLTSGPATTTDILGTSTEPGT
		SSSSSLSTTSHERLTTYKDTAHTEAVHPSTNTGGTNVATTSSGYK
		SQSSVLADSSPMCTTSTMGDTSVLTSTPAFLETRRIQTELASSLT
		PGLRESSGSEGTSSGTKMSTVLSKVPTGATTEISKEDVTSIPGPA
		QSTISPDISTRTVSWFSTSPVMTESAEITMNTHTSPLGATTQGTS
		TLDTSSTTSLTMTHSTISQGFSHSQMSTLMRRGPEDVSWMSPPLL
		EKTRPSFSLMSSPATTSPSPVSSTLPESISSSPLPVTSLLTSGLA
		KTTDMLHKSSEPVTNSPANLSSTSVEILATSEVTTDTEKTHPSSN
		RTVTDVGTSSSGHESTSFVLADSQTSKVTSPMVITSTMEDTSVST
		STPGFFETSRIQTEPTSSLTLGLRKTSSSEGTSLATEMSTVLSGV
		PTGATAEVSRTEVTSSSRTSISGFAQLTVSPETSTETITRLPTSS
		IMTESAEMMIKTQTDPPGSTPESTHTVDISTTPNWVETHSTVTQR
		FSHSEMTTLVSRSPGDMLWPSQSSVEETSSASSLLSLPATTSPSP
		VSSTLVEDFPSASLPVTSLLNPGLVITTDRMGISREPGTSSTSNL
		SSTSHERLTTLEDTVDTEDMQPSTHTAVTNVRTSISGHESQSSVL
		SDSETPKATSPMGTTYTMGETSVSISTSDFFETSRIQIEPTSSLT
		SGLRETSSSERISSATEGSTVLSEVPSGATTEVSRTEVISSRGTS
		MSGPDQFTISPDISTEAITRLSTSPIMTESAESAITIETGSPGAT
		SEGTLTLDTSTTTFWSGTHSTASPGFSHSEMTTLMSRTPGDVPWP
		SLPSVEEASSVSSSLSSPAMTSTSFFSTLPESISSSPHPVTALLT
		LGPVKTTDMLRTSSEPETSSPPNLSSTSAEILATSEVTKDREKIH
		PSSNTPVVNVGTVIYKHLSPSSVLADLVTTKPTSPMATTSTLGNT
		SVSTSTPAFPETMMTQPTSSLTSGLREISTSQETSSATERSASLS
		GMPTGATTKVSRTEALSLGRTSTPGPAQSTISPEISTETITRIST
		PLTTTGSAEMTITPKTGHSGASSQGTFTLDTSSRASWPGTHSAAT
		HRSPHSGMTTPMSRGPEDVSWPSRPSVEKTSPPSSLVSLSAVTSP
		SPLYSTPSESSHSSPLRVTSLFTPVMMKTTDMLDTSLEPVTTSPP
		SMNITSDESLATSKATMETEAIQLSENTAVTQMGTISARQEFYSS
		YPGLPEPSKVTSPVVTSSTIKDIVSTTIPASSEITRIEMESTSTL
		TPTPRETSTSQEIHSATKPSTVPYKALTSATIEDSMTQVMSSSRG
		PSPDQSTMSQDISTEVITRLSTSPIKTESTEMTITTQTGSPGATS
		RGTLTLDTSTTFMSGTHSTASQGFSHSQMTALMSRTPGDVPWLSH
		PSVEEASSASFSLSSPVMTSSSPVSSTLPDSIHSSSLPVTSLLTS
		GLVKTTELLGTSSEPETSSPPNLSSTSAEILAITEVTTDTEKLEM
		TNVVTSGYTHESPSSVLADSVTTKATSSMGITYPTGDTNVLTSTP
		AFSDTSRIQTKSKLSLTPGLMETSISEETSSATEKSTVLSSVPTG
		ATTEVSRTEAISSSRTSIPGPAQSTMSSDTSMETITRISTPLTRK
		ESTDMAITPKTGPSGATSQGTFTLDSSSTASWPGTHSATTQRFPQ
		SVVTTPMSRGPEDVSWPSPLSVEKNSPPSSLVSSSSVTSPSPLYS
		TPSGSSHSSPVPVTSLFTSIMMKATDMLDASLEPETTSAPNMNIT
		SDESLAASKATTETEAIHVFENTAASHVETTSATEELYSSSPGFS
		EPTKVISPVVTSSSIRDNMVSTTMPGSSGITRIEIESMSSLTPGL
		RETRTSQDITSSTETSTVLYKMPSGATPEVSRTEVMPSSRTSIPG
		PAQSTMSLDISDEVVTRLSTSPIMTESAEITITTQTGYSLATSQV
		TLPLGTSMTFLSGTHSTMSQGLSHSEMTNLMSRGPESLSWTSPRE
		VETTRSSSSLTSLPLTTSLSPVSSTLLDSSPSSPLPVTSLILPGL
		VKTTEVLDTSSEPKTSSSPNLSSTSVEIPATSEIMTDTEKIHPSS
		NTAVAKVRTSSSVHESHSSVLADSETTITIPSMGITSAVDDTTVF
		TSNPAFSETRRIPTEPTFSLTPGFRETSTSEETTSITETSAVLYG
		VPTSATTEVSMTEIMSSNRIHIPDSDQSTMSPDIITEVITRLSSS
		SMMSESTQMTITTQKSSPGATAQSTLTLATTTAPLARTHSTVPPR
		FLHSEMTTLMSRSPENPSWKSSLFVEKTSSSSSLLSLPVTTSPSV
		SSTLPQSIPSSSFSVTSLLTPGMVKTTDTSTEPGTSLSPNLSGTS
		VEILAASEVTTDTEKIHPSSSMAVTNVGTTSSGHELYSSVSIHSE
		PSKATYPVGTPSSMAETSISTSMPANFETTGFEAEPFSHLTSGFR
		KTNMSLDTSSVTPTNTPSSPGSTHLLQSSKTDFTSSAKTSSPDWP
		PASQYTEIPVDIITPFNASPSITESTGITSFPESRFTMSVTESTH
		HLSTDLLPSAETISTGTVMPSLSEAMTSFATTGVPRAISGSGSPF
		SRTESGPGDATLSTIAESLPSSTPVPFSSSTFTTTDSSTIPALHE
		ITSSSATPYRVDTSLGTESSTTEGRLVMVSTLDTSSQPGRTSSSP
		ILDTRMTESVELGTVTSAYQVPSLSTRLTRTDGIMEHITKIPNEA
		AHRGTIRPVKGPQTSTSPASPKGLHTGGTKRMETTTTALKTTTTA
		LKTTSRATLTTSVYTPTLGTLTPLNASMQMASTIPTEMMITTPYV
		FPDVPETTSSLATSLGAETSTALPRTTPSVFNRESETTASLVSRS
		GAERSPVIQTLDVSSSEPDTTASWVIHPAETIPTVSKTTPNFFHS
		ELDTVSSTATSHGADVSSAIPTNISPSELDALTPLVTISGTDTST
		TFPTLTKSPHETETRTTWLTHPAETSSTIPRTIPNFSHHESDATP
		SIATSPGAETSSAIPIMTVSPGAEDLVTSQVTSSGTDRNMTIPTL
		TLSPGEPKTIASLVTHPEAQTSSAIPTSTISPAVSRLVTSMVTSL
		AAKTSTTNRALTNSPGEPATTVSLVTHPAQTSPTVPWTTSIFFHS
		KSDTTPSMTTSHGAESSSAVPTPTVSTEVPGVVTPLVTSSRAVIS
		TTIPILTLSPGEPETTPSMATSHGEEASSAIPTPTVSPGVPGVVT
		SLVTSSRAVTSTTIPILTFSLGEPETTPSMATSHGTEAGSAVPTV
		LPEVPGMVTSLVASSRAVTSTTLPTLTLSPGEPETTPSMATSHGA
		EASSTVPTVSPEVPGVVTSLVTSSSGVNSTSIPTLILSPGELETT
		PSMATSHGAEASSAVPTPTVSPGVSGVVTPLVTSSRAVTSTTIPI
		LTLSSSEPETTPSMATSHGVEASSAVLTVSPEVPGMVTSLVTSSR
		AVTSTTIPTLTISSDEPETTTSLVTHSEAKMISAIPTLAVSPTVQ
		GLVTSLVTSSGSETSAFSNLTVASSQPETIDSWVAHPGTEASSVV
		PTLTVSTGEPFTNISLVTHPAESSSTLPRTTSRFSHSELDTMPST
		VTSPEAESSSAISTTISPGIPGVLTSLVTSSGRDISATFPTVPES
		PHESEATASWVTHPAVTSTTVPRTTPNYSHSEPDTTPSIATSPGA
		EATSDFPTITVSPDVPDMVTSQVTSSGTDTSITIPTLTLSSGEPE
		TTTSFITYSETHTSSAIPTLPVSPGASKMLTSLVISSGTDSTTTF
		PTLTETPYEPETTAIQLIHPAETNTMVPRTTPKFSHSKSDTTLPV
		AITSPGPEASSAVSTTTISPDMSDLVTSLVPSSGTDTSTTFPTLS
		ETPYEPETTATWLTHPAETSTTVSGTIPNFSHRGSDTAPSMVTSP
		GVDTRSGVPTTTIPPSIPGVVTSQVTSSATDTSTAIPTLTPSPGE
		PETTASSATHPGTQTGFTVPIRTVPSSEPDTMASWVTHPPQTSTP
		VSRTTSSFSHSSPDATPVMATSPRTEASSAVLTTISPGAPEMVTS
		QITSSGAATSTTVPTLTHSPGMPETTALLSTHPRTETSKTFPAST
		VFPQVSETTASLTIRPGAETSTALPTQTTSSLFTLLVTGTSRVDL
		SPTASPGVSAKTAPLSTHPGTETSTMIPTSTLSLGLLETTGLLAT
		SSSAETSTSTLTLTVSPAVSGLSSASITTDKPQTVTSWNTETSPS
		VTSVGPPEFSRTVTGTTMTLIPSEMPTPPKTSHGEGVSPTTILRT
		TMVEATNLATTGSSPTVAKTTTTFNTLAGSLFTPLTTPGMSTLAS
		ESVTSRTSYNHRSWISTTSSYNRRYWTPATSTPVTSTFSPGISTS
		SIPSSTAATVPFMVPFTLNFTITNLQYEEDMRHPGSRKFNATERE
		LQGLLKPLFRNSSLEYLYSGCRLASLRPEKDSSATAVDAICTHRP
		DPEDLGLDRERLYWELSNLTNGIQELGPYTLDRNSLYVNGFTHRS
		SMPTTSTPGTSTVDVGTSGTPSSSPSPTTAGPLLMPFTLNFTITN
		LQYEEDMRRTGSRKFNTMESVLQGLLKPLFKNTSVGPLYSGCRLT
		LLRPEKDGAATGVDAICTHRLDPKSPGLNREQLYWELSKLTNDIE
		ELGPYTLDRNSLYVNGFTHQSSVSTTSTPGTSTVDLRTSGTPSSL
		SSPTIMAAGPLLVPFTLNFTITNLQYGEDMGHPGSRKFNTTERVL
		QGLLGPIFKNTSVGPLYSGCRLTSLRSEKDGAATGVDAICIHHLD
		PKSPGLNRERLYWELSQLTNGIKELGPYTLDRNSLYVNGFTHRTS
		VPTSSTPGTSTVDLGTSGTPFSLPSPATAGPLLVLFTLNFTITNL
		KYEEDMHRPGSRKFNTTERVLQTLLGPMFKNTSVGLLYSGCRLTL
		LRSEKDGAATGVDAICTHRLDPKSPGVDREQLYWELSQLTNGIKE
		LGPYTLDRNSLYVNGFTHWIPVPTSSTPGTSTVDLGSGTPSSLPS
		PTTAGPLLVPFTLNFTITNLKYEEDMHCPGSRKFNTTERVLQSLL
		GPMFKNTSVGPLYSGCRLTLLRSEKDGAATGVDAICTHRLDPKSP
		GVDREQLYWELSQLTNGIKELGPYTLDRNSLYVNGFTHQTSAPNT
		STPGTSTVDLGTSGTPSSLPSPTSAGPLLVPFTLNFTITNLQYEE
		DMHHPGSRKFNTTERVLQGLLGPMFKNTSVGLLYSGCRLTLLRPE
		KNGAATGMDAICSHRLDPKSPGLNREQLYWELSQLTHGIKELGPY
		TLDRNSLYVNGFTHRSSVAPTSTPGTSTVDLGTSGTPSSLPSPTT
		AVPLLVPFTLNFTITNLQYGEDMRHPGSRKFNTTERVLQGLLGPL
		FKNSSVGPLYSGCRLISLRSEKDGAATGVDAICTHHLNPQSPGLD
		REQLYWQLSQMTNGIKELGPYTLDRNSLYVNGFTHRSSGLTTSTP
		WTSTVDLGTSGTPSPVPSPTTTGPLLVPFTLNFTITNLQYEENMG
		HPGSRKFNITESVLQGLLKPLFKSTSVGPLYSGCRLTLLRPEKDG
		VATRVDAICTHRPDPKIPGLDRQQLYWELSQLTHSITELGPYTLD
		RDSLYVNGFTQRSSVPTTSTPGTFTVQPETSETPSSLPGPTATGP
		VLLPFTLNFTITNLQYEEDMRRPGSRKFNTTERVLQGLLMPLFKN
		TSVSSLYSGCRLTLLRPEKDGAATRVDAVCTHRPDPKSPGLDRER
		LYWKLSQLTHGITELGPYTLDRHSLYVNGFTHQSSMTTTRTPDTS
		TMHLATSRTPASLSGPMTASPLLVLFTINFTITNLRYEENMHHPG
		SRKFNTTERVLQGLLRPVFKNTSVGPLYSGCRLTLLRPKKDGAAT
		KVDAICTYRPDPKSPGLDREQLYWELSQLTHSITELGPYTLDRDS
		LYVNGFTQRSSVPTTSIPGTPTVDLGTSGTPVSKPGPSAASPLLV
		LFTLNFTITNLRYEENMQHPGSRKFNTTERVLQGLLRSLFKSTSV
		GPLYSGCRLTLLRPEKDGTATGVDAICTHHPDPKSPRLDREQLYW
		ELSQLTHNITELGPYALDNDSLFVNGFTHRSSVSTTSTPGTPTVY
		LGASKTPASIFGPSAASHLLILFTLNFTITNLRYEENMWPGSRKF
		NTTERVLQGLLRPLFKNTSVGPLYSGCRLTLLRPEKDGEATGVDA
		ICTHRPDPTGPGLDREQLYLELSQLTHSITELGPYTLDRDSLYVN
		GFTHRSSVPTTSTGVVSEEPFTLNFTINNLRYMADMGQPGSLKFN
		ITDNVMQHLLSPLFQRSSLGARYTGCRVIALRSVKNGAETRVDLL
		CTYLQPLSGPGLPIKQVFHELSQQTHGITRLGPYSLDKDSLYLNG
		YNEPGPDEPPTTPKPATTFLPPLSEATTAMGYHLKTLTLNFTISN
		LQYSPDMGKGSATFNSTEGVLQHLLRPLFQKSSMGPFYLGCQLIS
		LRPEKDGAATGVDTTCTYHPDPVGPGLDIQQLYWELSQLTHGVTQ
		LGFYVLDRDSLFINGYAPQNLSIRGEYQINFHIVNWNLSNPDPTS
		SEYITLLRDIQDKVTTLYKGSQLHDTFRFCLVTNLTMDSVLVTVK
		ALFSSNLDPSLVEQVFLDKTLNASFHWLGSTYQLVDIHVTEMESS
		VYQPTSSSSTQHFYLNFTITNLPYSQDKAQPGTTNYQRNKRNIED
		ALNQLFRNSSIKSYFSDCQVSTFRSVPNRHHTGVDSLCNFSPLAR
		RVDRVAIYEEFLRMTRNGTQLQNFTLDRSSVLVDGYSPNRNEPLT
		GNSDLPFWAVILIGLAGLLGVITCLICGVLVTTRRRKKEGEYNVQ
		QQCPGYYQSHLDLEDLQ
SLC39A6	54	MARKLSVILILTFALSVTNPLHELKAAAFPQTTEKISPNWESGIN
		VDLAISTRQYHLQQLFYRYGENNSLSVEGFRKLLQNIGIDKIKRI
		HIHHDHDHHSDHEHHSDHERHSDHEHHSEHEHHSDHDHHSHHNHA
		ASGKNKRKALCPDHDSDSSGKDPRNSQGKGAHRPEHASGRRNVKD
		SVSASEVTSTVYNTVSEGTHFLETIETPRPGKLFPKDVSSSTPPS
		VTSKSRVSRLAGRKTNESVSEPRKGFMYSRNTNENPQECFNASKL
		LTSHGMGIQVPLNATEFNYLCPAIINQIDARSCLIHTSEKKAEIP
		PKTYSLQIAWVGGFIAISIISFLSLLGVILVPLMNRVFFKFLLSF
		LVALAVGTLSGDAFLHLLPHSHASHHHSHSHEEPAMEMKRGPLFS
		HLSSQNIEESAYFDSTWKGLTALGGLYFMFLVEHVLTLIKQFKDK
		KKKNQKKPENDDDVEIKKQLSKYESQLSTNEEKVDTDDRTEGYLR
		ADSQEPSHFDSQQPAVLEEEEVMIAHAHPQEVYNEYVPRGCKNKC
		HSHFHDTLGQSDDLIHHHHDYHHILHHHHHQNHHPHSHSQRYSRE
		ELKDAGVATLAWMVIMGDGLHNFSDGLAIGAAFTEGLSSGLSTSV
		AVFCHELPHELGDFAVLLKAGMTVKQAVLYNALSAMLAYLGMATG
		IFIGHYAENVSMWIFALTAGLFMYVALVDMVPEMLHNDASDHGCS
		RWGYFFLQNAGMLLGFGIMLLISIFEHKIVFRINF
SLC44A4	55	MGGKQRDEDDEAYGKPVKYDPSFRGPIKNRSCTDVICCVLFLLFI
		LGYIVVGIVAWLYGDPRQVLYPRNSTGAYCGMGENKDKPYLLYFN
		IFSCILSSNIISVAENGLQCPTPQVCVSSCPEDPWTVGKNEFSQT
		VGEVFYTKNRNFCLPGVPWNMTVITSLQQELCPSFLLPSAPALGR
		CFPWTNVTPPALPGITNDTTIQQGISGLIDSLNARDISVKIFEDF
		AQSWYWILVALGVALVLSLLFILLLRLVAGPLVLVLILGVLGVLA
		YGIYYCWEEYRVLRDKGASISQLGFTTNLSAYQSVQETWLAALIV
		LAVLEAILLLMLIFLRQRIRIAIALLKEASKAVGQMMSTMFYPLV
		TFVLLLICIAYWAMTALYLATSGQPQYVLWASNISSPGCEKVPIN
		TSCNPTAHLVNSSCPGLMCVFQGYSSKGLIQRSVFNLQIYGVLGL
		FWTLNWVLALGQCVLAGAFASFYWAFHKPQDIPTFPLISAFIRTL
		RYHTGSLAFGALILTLVQIARVILEYIDHKLRGVQNPVARCIMCC
		FKCCLWCLEKFIKFLNRNAYIMIAIYGKNFCVSAKNAFMLLMRNI
		VRVVVLDKVTDLLLFFGKLLVVGGVGVLSFFFFSGRIPGLGKDFK
		SPHLNYYWLPIMTSILGAYVIASGFFSVFGMCVDTLFLCFLEDLE
		RNNGSLDRPYYMSKSLLKILGKKNEAPPDNKKRKK
STEAP1	56	MESRKDITNQEELWKMKPRRNLEEDDYLHKDTGETSMLKRPVLLH
		LHQTAHADEFDCPSELQHTQELFPQWHLPIKIAAIIASLTFLYTL
		LREVIHPLATSHQQYFYKIPILVINKVLPMVSITLLALVYLPGVI
		AAIVQLHNGTKYKKFPHWLDKWMLTRKQFGLLSFFFAVLHAIYSL
		SYPMRRSYRYKLLNWAYQQVQQNKEDAWIEHDVWRMEIYVSLGIV
		GLAILALLAVTSIPSVSDSLTWREFHYIQSKLGIVSLLLGTIHAL
		IFAWNKWIDIKQFVWYTPPTFMIAVFLPIVVLIFKSILFLPCLRK
		KILKIRHGWEDVTKINKTEICSQL

TABLE 6

Original Residue	Exemplary Substitutions
Ala	Ser
Arg	Lys
Asn	Gln, His
Asp	Glu
Cys	Ser
Gln	Asn
Glu	Asp
Gly	Pro
His	Asn, Gln
Ile	Leu, Val
Leu	Ile, Val
Lys	Arg, Gln, Glu
Met	Leu, Ile
Phe	Met, Leu, Tyr
Ser	Thr
Thr	Ser
Trp	Tyr
Tyr	Trp, Phe
Val	Ile, Leu

TABLE 10 — Characterization of exemplary anti-HER2 ADCs

								CTGlo	CTGlo	CTGlo
							Concen-	GMean	GMean	Mean
SMLA	Payload				Percent	Free	tration	GI50 (AM)	LD50(IM)	MinResponse %
Batch ID	Class	Linker	Linker Name	DAR	Monomer	Drug (%)	(mg/ml)	HCC1954.1	HCC1954.1	HCC1954.1
AB185-	Plad D	ADL0010-	maleimido	2.000	99.000	<1	0.730	>200.000	>200.000	58.601
ADL10-D8		01	caproyl
AB185-	Plad D	ADL0010-	maleimido	3.600	97.000	<1	3.600	25.502	>200.000	23.963
ADL10-D1		01	caproyl
AB185-	ArylPlad	ADL0005-	mc-Val-Ala-	3.960	98.000	<1	1.400	2.668	>160.000	14.873
ADL5-D17		01	PAB (Qamine)
AB185-	ArylPlad	ADL0001-	mc-Val-Cit	4.400	95.000	<1	0.550	2.086	158.245
ADL1-D16		01	PABC
AB185-	ArylPlad	ADL0005-	mc-Val-Ala-	4.400	98.000	<1	1.200	0.943	>200.000
ADL5-D26		01	PAB (Qamine)
AB185-	ArylPlad	A0L0001-	mc-Val-Cit	4.340	88.000	<1	4.200	0.925	>200.000	1.271
ADL1-D14		01	PABC
AB185-	ArylPlad	ADL0005-	mc-Val-Ala-	4.000	98.000	<1	0.780	0.912	>200.000	−45.288
ADL5-D15		01	PAB (Qamine)
AB185-	Plad D	ADL0005-	mc-Val-Ala-	3.700	96.000	<1	0.880	0.806	>93.000	−30.929
ADL5-D11		01	PAB (Qamine)
AB185-	ArylPlad	ADL0001-	mc-Val-Cit	8.000	95.000	<1	0.570	0.466	>200.000	−25.859
ADL1-D33		01	PABC
AB185-	Plad D	ADL0001-	mc-Val-Cit	3.000	99.000	<1	0.840	0.443	>200.000	−7.520
ADL1-D22		01	PABC
AB185-	Plad D	ADL0012-	mal-(CH2)6-	3.600	98.600	<1	1.060	0.432	>200.000	18.569
ADL12-D2		01	(non-cleavable)
AB185-	Plad B	ADL0001-	mc-Val-Cit	5.000	98.000	<1	1.200	0.289	18.948	−94.860
ADL1-D13		01	PABC
AB185-	Plad B	ADL0005-	mc-Val-Ala-	6.600	97.000	<1	3.230	0.278	>200.000	−47.381
ADL5-D19		01	PAB (Qamine)
AB185-	Plad D	ADL0005-	mc-Val-Ala-	4.020	97.000	<1	0.650	0.254	>200.000	−2.273
ADL5-D10		01	PAB (Qamine)
AB185-	Plad D	ADL0006-	mc-Val-Ala-	5.200	98.000	<1	0.500	0.244	4.206	−94.511
ADL6-D9		01	PABC
AB185-	Plad D	ADL0005-	mc-Val-Ala-	4.140	99	<1	4.5	0.206	>200.000	−12.239
ADL5-D2		01	PAB (Qamine)
AB185-	Plad D	ADL0001-	mc-Val-Cit	7.200	97.000	<1	3.410	0.188	>200.000	−55.079
ADL1-D8		01	PABC
AB185-	Plad D	ADL0005-	mc-Val-Ala-	4.300	99.000	<1	5.440	0.179	>200.000	−18.586
ADL5-D2		01	PAB (Qamine)
AB185-	Plad D	ADL0015-	mal-CH2CH2—	3.300	98.800	<1	1.600	0.170	>200.000	−23.513
ADL15-D2		01	O—CH2CH2—
			(non-cleavable)
AB185-	Plad D	ADL0012-	mal-(CH2)6-	3.200	98.600	<1	1.390	0.142	>200.000	−57.399
ADL12-D20		01	(non-cleavable)
AB185-	Plad D	ADL0012-	mal-(CH2)6-	3.300	98.800	< 1	1.620	0.126	>200.000	−34.649
ADL12-D1		01	(non-cleavable)
T-DM1	DM1	ADL0019-	SMCC	3.300	98.000	<1	3.300	0.118	2.759	−47.952
		01
AB185-	Plad D	ADL0005-	mc-Val-Ala-	8.500	98	<1	5.1	0.086	2.918	−63.909
ADL5-D2		01	PAB (Qamine)
AB185-	Plad D	ADL0006-	mc-Val-Ala-	8.000	97.000	<1	1.200	0.081	2.877	−85.692
ADL6-D1		01	PABC
AB185-	Plad D	ADL0012-	mal-(CH2)6-	3.000	98.000	<1	2.860	0.081	2.148	−68.999
ADL12-D1		01	(non-cleavable)
AB185-	Plad D	ADL0012-	mal(CH2)6-	4.300	97.000	<1	0.120	0.078	>80.000	28.071
ADL12-D22		01	(non-cleavable)
AB185-	Plad D	ADL0001-	mc-Val-Cit	8.000	98.000	<1	3.600	0.073	1.949	−67.199
ADL1-D4		01	PABC
AB185-	Plad D	ADL0015-	mal-CH2CH2—	3.200	98.800	<1	0.980	0.062	>200.000	−57.829
ADL15-D1		01	O—CH2CH2—
			(non-cleavable)
AB185-	Plad D	ADL0014-	mal-CH2CH	3.500	98.600	<1	0.740	0.055	>200.000	−56.889
ADL14-D1		01	(non-cleavable)
AB185-	Plad D	ADL0001-	mc-Val-Cit	2.800	98.000	<1	1.200	0.050	>200.000	−57.212
ADL1-D4		01	PABC
AB185-	Plad D	ADL0001-	mc-Val-Cit	3.900	99	<1	5.2	0.046	0.374	−78.519
ADL1-D1		01	PABC
AB185-	Plad D	ADL0001-	mc-Val-Cit	4.000	98.000	<1	3.500	0.042	0.447	−77.970
ADL1-D1		01	PABC
AB185-	Plad D	ADL0005-	mc-Val-Ala-	6.000	98.000	<1	0.170	0.038	>113.000	−46.265
ADL5-D25	Zwitt	01	PAB (Qamine)
AB185-	Plad D	ADL0001-	mc-Val-Cit	7.700	98.000	<1	4.200	0.034	0.120	−87.001
ADL1-D1		01	PABC

TABLE 11 — Exemplary anti-HER2 ADCs - HCC1954 cells

	ADC	Payload
Sample	DAR	GI50 (nM)	LD50 (nM)	Rmin (%)	GI50 (nM)	LD50 (nM)	Rmin (%)
AB185-ADL12-D1	3.1	0.064	4.86	−78.393	1.853	5.485	−75.579
AB185-ADL1-D8	7.2	0.075	1.01	−72.765	2.638	11.175	−80.463
T-DM1	3.3	0.083	>200	−64.227
AB185-ADL1-D1	4.04	0.089	2.078	−74.165	1.853	5.485	−75.579
AB185-ADL1-D4	4.9	0.145	1.217	−75.638	1.699	6.374	−81.34
AB185-ADL5-D2	4.1	0.18	>200	−36.513	0.6	2.756	−78.732
AB185-ADL5-D15	4	0.503	>200	−20.387	0.358	2.037	−84.412
AB185-ADL1-D18	3.8	5.994	>200	−8.011	0.208	0.787	−90.055
AB185-ADL12-D2	3.6	6.129	>200	18.763	0.6	2.758	−78.732
AB185-ADL10-D1	3.5	6.642	>200	8.138	1.853	5.485	−75.579
AB185-ADL5-D25	6.3	0.038	>113.000	−51.503	167.54	>200	26.882
AB185	N/A	>200	>200	76.916

TABLE 12 — Exemplary anti-HER2 ADCs - N87 cells

	SMLA	Payload
Sample	DAR	GI50 (nM)	LD50 (nM)	Rmin (%)	GI50 (nM)	LD50 (nM)	Rmin (%)
AB185-ADL1-D4	4.9	0.093	>200	−40.056	3.241	38.774	−81.775
AB185-ADL5-D15	4	0.106	>200	−50.886	0.41	>200	−68.045
AB185-ADL5-D25	6.3	0.11	133	−43.398	>200	>200	40.364
AB185-ADL12-D1	3.1	0.111	>200	−41.228	7.041	>200	−40.041
AB185-ADL1-D18	3.8	0.113	>200	−39.509	0.275	2.993	−92.127
AB185-ADL1-D1	4.04	0.163	>200	−41.075	7.041	>200	−40.041
T-DM1	3.3	0.168	>200	−20.785
AB185-ADL1-D8	7.2	0.172	>200	−31.708	8.844	>200	−34.052
AB185-ADL5-D2	4.1	0.206	>200	−51.328	0.749	>200	−72.706
AB185-ADL12-D2	3.6	0.243	>200	−9.79	0.749	>200	−72.706
AB185-ADL1-D14	4.3	0.296	>200	−26.143	2.925	>200	−52.662
AB185-ADL10-D1	3.5	1.531	>200	30.759	7.041	>200	−40.041
AB185	N/A	>200	>200	77.029

TABLE 15 — Characterization of exemplary anti-CD138 ADCs

	Payload			Percent	Free	Concentration
SMLA Batch ID	Class	Linker	DAR	Monomer	Drug (%)	(mg/mL)
AB205-ADL1-D14	Aryl Plad	ADL0001	2.900	92.000	<2	1.010
AB205-ADL5-D15	Aryl Plad	ADL0005	2.800	95.000	<2	1.060
AB205-ADL5-D19	Plad B	ADL0005	4.900	95.000	<2	0.880
AB205-ADL1-D1	Plad D	ADL0001	5.000	98.000	<2	0.620
AB205-ADL1-D4	Plad D	ADL0001	4.800	98.000	<2	1.010
AB205-ADL5-D25	Plad D	ADL0005	6.400	98.000	<2	0.890
	Zwitt
AB205-ADL10-D1	Plad D	ADL0010	3.400	96.000	<2	0.750
AB205-ADL12-D1	Plad D	ADL0012	3.800	97.000	<2	0.830
AB205-ADL12-D2	Plad D	ADL0012	3.300	97.000	<2	0.970

TABLE 16 — Exemplary anti-CD138 ADCs - MOLP8 cells

	SMLA	Payload
Sample	DAR	GI50 (nM)	LD50 (nM)	Rmin (%)	GI50 (nM)	LD50 (nM)	Rmin (%)
AB205-ADL1-D1	5	<0.061	0.656	−100.099	6.885	25.712	−100.248
AB205-ADL5-D15	2.8	0.258	>400	2.419	0.776	9.178	−100.198
AB205-ADL5-D19	4.9	<0.061	>400	−46.23	0.84	4.58	−62.2
AB205-ADL10-D1	3.4	0.08	>400	−25.099	6.885	25.712	−100.248
AB205-ADL12-D1	3.8	<0.061	0.168	−99.851	6.855	25.712	−100.248
AB205-ADL12-D2	3.3	<0.061	>400	−53.125	0.316	5.035	−100.198
AB205-ADL1-D14	3	<0.061	>400	−74.008	1.9	8.5	−61
AB205-ADL1-D4	4.8	<0.061	0.535	−99.306	1.151	12.99	−101.935
AB205-ADL5-D25	6.4	<0.061	11.669	−76.736	149.874	>400	5.94

TABLE 17 — Characterization of exemplary anti-EPHA2 ADCs

	Payload			Percent	Free	Concentration
SMLA Batch ID	Class	Linker	DAR	Monomer	Drug(%)	(mg/ml)
AB206-ADL1-D1	Plad D	ADL0001	5.600	99.000	<2	2.060
AB206-ADL5-D2	Plad D	ADL0005	6.300	99.000	<2	1.900
AB206-ADL1-D4	Plad D	ADL0001	6.000	95.000	<2	1.140
AB206-ADL1-D8	Plad D	ADL0001	6.500	95.000	<2	1.660
AB206-ADL1-D14	Aryl Plad	ADL0001	3.600	80.000	<2	1.620
AB206-ADL5-D19	Plad B	ADL0005	4.600	97.000	<2	0.960
AB206-ADL5-D15	Ary Plad	ADL0005	4.200	95.000	<2	0.820
AB206-ADL5-D25	Plad D	ADL0005	5.000	96.000	<2	1.130
	Zwitt
AB206-ADL10-D1	Plad D	ADL0010	2.700	97.000	<2	1.620
AB206-ADL12-D1	Plad D	ADL0012	6.400	90 000	<2	0.670
AB206-ADL12-D2	Plad D	ADL0012	6.300	90.000	<2	0.850

TABLE 18 — Exemplary anti-EPHA2 ADCs - PC3 cells

	SMLA	Payload
Sample	DAR	GI50 (nM)	LD50 (nM)	Rmin (%)	GI50 (nM)	LD50 (nM)	Rmin (%)
AB206-ADL1-D8	6.5	0.175	95.51	−68.678	16.622	177.783	−68.37
AB206-ADL1-D4	6	<0.061	2.732	−69.096	11.057	>400	−55.97
AB206-ADL1-D14	3.6	3.046	>400	−49.267	3.877	>400	−79.835
AB206-ADL12-D1	6.4	0.808	>400	−64.516	13.291	>400	−71.987
AB206-ADL12-D2	6.3	78.977	>400	17.376	2.391	>400	−68.803
AB206-ADL5-D19	4.6	71.73	>400	19.765	1.41	>400	−29.33
AB206-ADL10-D1	2.7	>400	>400	43.796	13.291	>400	−71.987
AB206-ADL5-D15	4.2	9.959	>400	2.441	3.877	>400	−79.835
AB206-ADL5-D25	5	28.078	>400	20.041	>400	>400	77.524
AB206-ADL1-D1	5.6	<0.061	>400	−59.643	13.291	>400	−71.987
AB206-ADL5-D2	6.3	1.003	>400	18.466	2.391	>400	−68.803

TABLE 19 — Accelerated stability testing of select anti-HER2 ADCs

				Concentration	DAR
ADC	Storage Buffer	Days @ 37 C.	Aggregation (%)	(mg/mL)	HIC	RP-HPLC
AB185-ADL14-D1	DPBS(pH 7.5)	0	0.68	4.99	4.3	4.32
		1	1.18	4.98	4.29	4.31
		2	1.31	5.12	4.33	4.32
		4	2.08	5.02	4.28	4.27
AB185-ADL5-D25	DPBS(pH7.5)	0	0.84	5.05	4.54	4.22
		1	1.14	4.16	4 44	4.19
		2	1.41	4.52	4.74	4.19
		4	2.43	3.98	4.64	4.12

TABLE 20 — Neopeptides Junction

Neopeptide	SEQ ID NO	(HG19)	Gene	Event type	Observed in
1	SPTLPPRSL	37	chr12:	TUBA1C	Intron retention	H1568
			49663470-49663610: +
2	HPSIKRGLSSL	38	chr12:	PPHLN1	Exon skipping	H1568
			42729776-42781257: +
3	LLLPHHVL	39	chr12:	TUBA1C	Intron retention	H1568
			49663470-49663610: +
4	RTAPGVRPPF	40	chr14:	CFL2	Intron retention	H1568
			35182767-35183743: −
5	RPQKSIQAL	41	chr10:	WAC	Intron retention	H1568
			28822963-28823162: +
6	APAPPPLPA	42	chr17:	GPS1	Intron retention	H1568
			80009840-80011149: +
7	RPRPSFPVSL	43	chr7:	EGFR	Intron retention	H1568
			55087058-55134942: +
8	RPKHGDGFSL	44	chr11:	MED19	Intron retention	H1568
			57472287-57472444: −
9	GPAPGKTGL	45	chr7:	HSBP1	Intron retention	H1568
			75932393-75933118: +
10
EAARKGNSL
46	chr1:	SCP2	Exon skipping	H1568
			53480715-53504588: +
11
RIKEKIEEL
47	chr9:	SMC5	Exon skipping	H1568
			72897499-72912881: +
12
EIKKRFRQF
48	chr1:	DNAJC8	Exon skipping	H1568
			28531860-28541450: −
13
HESAAMAET
49	chr11:	TMEM123	Exon skipping	HCC1954
			102272937-102323254: −
14	ALKLKQVGV	50	chr1:	CHTOP	Exon skipping	H1568
			153610924-153617539: +
15	DLKKRHITF	51	chr13:	MRPS31	Exon skipping	H1568
			41323417-41331008: −
16	DVKRNDIAM	52	chr1:	NFYC	Exon skipping	H1568
			41213277-41218822: +
17	IPSDHILTPA	53	chr6:	TAB2	Exon skipping	H1568
			149718900-149720239: +
18	TVFSTSSLK	54	chr11:	SDHAF2	Exon skipping	H1568
			61197654-61213412: +
19	ITSCLLNF	55	chr5:	HSPA9	Intron retention	H1568
			137892555-137893090: −
20	RASPVRGQL	56	chr7:	MDH2	Intron retention	H1568
			75677544-75677893: +
21	VVRKPVIAL	57	chr1:	MRPS15	Exon skipping	H1568
			36923582-36929406: −
22	LLSEKKKIS	58	chr6:	VARS	Intron retention	H1568
			31750622-31750872: −
23	APASKPRPRL	59	chr19:	HMG20B	Intron retention	H1568
			3573798-3574380: +
24	RYGQLSEKF	60	chr19:	PDCD5	Exon skipping	HCC1954
			33076813-33078158: +
25	VYISNVSKL	61	chr3:	SELK	Exon skipping	HCC1954
			53920961-53925796: −
26	LPTKETPSF	62	chr2:	TMSB10	Alt 3’ss	HCC1954
			85133241-85133394: +
27	GEAPPPPPA	63	chr17:	CSNK1D	Intron retention	HCC1954
			80223672-80231181: −
28	LEEISKQEI	64	chr17:	TAOK1	Exon skipping	HCC1954
			27804724-27807385: +
29	IYNHITVKI	65	chr4:	ADD1	Exon skipping	HCC1954
			2886393-2896308: +

TABLE 22 — T-cell priming data

	ELISpot		SEQ ID	HLA
Donor	response	Gene	NO	(predicted binding)
1	+++	DNAJC8	76	A31:01/B40:13
1	+	TMEM123	77	A*02:01
2	+++	TMEM123	77	A*02:01
2	+	SCP2	74	B*39:06
6	+	DNAJC8	76	C07:01/B27
7	+++	DNAJC8	76	C07:02/C06:02
8	+++	DNAJC8	76	C*07:01
8	+	SMC5	75	C07:01/B08:02
8	+++	SCP2	74	C*03:03

TABLE 24 — Efficacy of exemplary anti-HER2 ADCs in NCI-N87 Day 22

	(in vivo)	NCI-N87 (in vitro)
	TGI	T/C			R min
	(%)	(%)	GI 50 (nM)	LD 50 (nM)	%
Vehicle/IV/Q7D × 2	—	—	—	—	—
TDM1/10 mg/kg/IV/Q7D × 2	71	29	0.1	40	−37
trastuzumab/10 mg/kg/IV/	4	96	>200	>200	98
Q7D × 2
AB185-ADL5-D2/10 mg/kg/	73	27	0.2	>200	0
IV/Q7D × 2
AB185-ADL1-D4/10 mg/kg/	68	32	0.1	>200	−22
IV/Q7D × 2
AB185-ADL5-D15/10 mg/kg/	67	33	0.1	>200	−37
IV/Q7D × 2
AB185-ADL14-D1/10 mg/kg/	−7	107	0.1	>200	−40
IV/Q7D × 2

TABLE 25 — Characterization of exemplary anti-HER2 ADCs and corresponding payloads Properties—HER2 ADCs antiproliferative activity antigen (+) cell #1

							CTGlo	CTGlo
							GMean	GMean
		Lethality/					GI50 (nM)	LD50 (nM)
		Splicing					HER2	HER2
		Category					SMLA	SMLA
		(HCC1954,		Percent	Free		screen	screen
ADC Batch Id	Linker	N87)	DAR	Monomer	Drug	Conc.	HCC1954.1	HCC1954.1
AB185-ADL1-D12	mc-Val-Cit-PABC	1, 3	6.25	99%	<1%	0.82	0.032	0.09
AB185-ADL13-D4	b-glucuronide	1, 1	4.47	99%	<1%	0.78	0.059	0.275
AB185-ADL1-D5	mc-Val-Cit-PABC	1, 3	6.14	99%	<1%	1.1	0.079	0.298
AB185-ADL23-D4	mc-Glu-Val-Cit-	2, 3	5.84	99%	<1%	1.03	0.175	1.526
	PABC
AB185-ADL1-D3	mc-Val-Cit-PABC	2, 3	6.75	99%	<1%	1.13	0.117	0.741
AB185-ADL22-D4	mal-PEG2-Val-Cit	1, 3	6.12	99%	<1%	1.01	0.134	0.797
	PABC
AB185-ADL21-D1	mc-Ala-Ala-Asn-	2, 3	5.51	99%	<1%	1.35	0.207	2.65
	PABC
AB185-ADL21-D8	mc-Ala-Ala-Asn-	4, 3	6.4	99%	<1%	1.67	0.493	>100.000
	PABC
AB185-ADL21-D4	mc-Ala-Ala-Asn-		1.55	99%	<1%	1.11	1.174	>100.000
	PABC
AB185-ADL15-D2	mal-CH2CH2—	3, ND	3.25	98.80%	<1%	1.6	0.24	>200.000
	O—CH2CH2-
	(non-cleavable)
		Properties—HER2 ADCs
		anti-
		proliferative
		activity
		antigen (+)	antiproliferative activity	antiproliferative activity
		cell #1	antigen (+) cell #2	antigen (−) cell
		CTGlo	CTGlo	CTGlo	CTGlo	CTGlo	CTGlo	CTGlo
		Mean	GMean	GMean	Mean	GMean	GMean	Mean
		MinResp %	GI50 (nM)	LD50 (nM)	MinResp %	GI50 (nM)	LD50 (nM)	MinResp %
		HER2	HER2	HER2	HER2	HER2	HER2	HER2
		SMLA	SMLA	SMLA	SMLA	SMLA	SMLA	SMLA
		screen	screen	screen	screen	screen	screen	screen
	ADC Batch Id	HCC1954.1	NCIN87	NCIN87	NCIN87	MCF7	MCF7	MCF7
	AB185-ADL1-D12	−78.471	<0.015	>100.000	−35.162	>100.000	>100.000	53.606
	AB185-ADL13-D4	−80.145	0.029	3.436	−55.322	>100.000	>100.000	87.451
	AB185-ADL1-D5	−81.189	0.04	>100.000	−32.663	>100.000	>100.000	93.099
	AB185-ADL23-D4	−68.762	0.049	>100.000	−49.316	>100.000	>100.000	91.248
	AB185-ADL1-D3	−77.982	0.049	>100.000	−15.629	>100.000	>100.000	91.495
	AB185-ADL22-D4	−75.134	0.057	>100.000	−25.808	>100.000	>100.000	79.996
	AB185-ADL21-D1	−72.894	0.072	>100.000	−46.965	>100.000	>100.000	74.018
	AB185-ADL21-D8	−37.547	0.138	>100.000	−40.873	>100.000	>100.000	94.772
	AB185-ADL21-D4	14.652	0.235	>100.000	−36.848	>100.000	>100.000	96.18
	AB185-ADL15-D2	−13.023	0.115	>100.000	−15.056
							Properties—HER2 ADCs
							splicing PD
							antigen (+) cell #1
							qPCR-	qPCR-
							CellScreen	CellScreen
							GMean IC50	Mean
							(nM) HER2	MinResp %
		Lethality/					SMLA	HER2 SMLA
		Splicing					screen	screen
		Category					SLC25A19	SLC25A19
		(HCC1954,		Percent	Free		HCC1954.1	HCC1954.1
ADC Batch Id	Linker	N87)	DAR	Monomer	Drug	Conc.	null	null
AB185-ADL1-D12	mc-Val-Cit-PABC	1, 3	6.25	99%	<1%	0.82	0.144	−85.744
AB185-ADL13-D4	b-glucuronide	1, 1	4.47	99%	<1%	0.78	0.45	−72.455
AB185-ADL1-D5	mc-Val-Cit-PABC	1, 3	6.14	99%	<1%	1.1	0.624	−65.799
AB185-ADL23-D4	mc-Glu-Val-Cit-	2, 3	5.84	99%	<1%	1.03	>100.000	−40.549
	PABC
AB185-ADL1-D3	mc-Val-Cit-PABC	2, 3	6.75	99%	<1%	1.13	>100.000	−38.621
AB185-ADL22-D4	mal-PEG2-Val-Cit	1, 3	6.12	99%	<1%	1.01	2.302	−58.063
	PABC
AB185-ADL21-D1	mc-Ala-Ala-Asn-	2, 3	5.51	99%	<1%	1.35	>100.000	−37.396
	PABC
AB185-ADL21-D8	mc-Ala-Ala-Asn-	4, 3	6.4	99%	<1%	1.67	>100.000	−27.319
	PABC
AB185-ADL21-D4	mc-Ala-Ala-Asn-		1.55	99%	<1%	1.11	>100.000	−16.088
	PABC
AB185-ADL15-D2	mal-CH2CH2—	3, ND	3.25	98.80%	<1%	1.6
	O—CH2CH2-
	(non-cleavable)
					Properties—HER2 ADCs
					splicing PD	splicing PD
					antigen (+) cell #2	antigen (−) cell
						qPCR-	qPCR-	qPCR-
					qPCR-	CellScreen	CellScreen	CellScreen
					CellScreen	Mean	GMean	Mean
					GMean IC50	MinResp %	IC50 (nM)	MinResp %
					(nM) HER2	HER2	HER2	HER2
					SMLA	SMLA	SMLA	SMLA
					screen	screen	screen	screen
					SLC25A19	SLC25A19	SLC25A19	SLC25A19
			ADC Batch Id	NCIN87 null	NCIN87 null	MCF7 null	MCF7 null
			AB185-ADL1-D12	0.095	−93.185	>100.000	−12.254
			AB185-ADL13-D4	0.177	−93.984	>100.000	−28.389
			AB185-ADL1-D5	0.095	−91.243	>100.000	−10.455
			AB185-ADL23-D4	0.249	−89.841	>100.000	−16.033
			AB185-ADL1-D3	0.199	−78.218	>100.000	−24.342
			AB185-ADL22-D4	0.17	−91.266	>100.000	−23.433
			AB185-ADL21-D1	0.201	−84.588	>100.000	−15.463
			AB185-ADL21-D8	0.577	−70.144	>100.000	−12.798
			AB185-ADL21-D4	5.097	−54.535	>100.000	−12.096
			AB185-ADL15-D2	0.296	−83.148
	Properties—Corresponding
	Free Payloads
	target	biochemical
	binding	splicing
							SPA-ATS	assay
							GMean	qPCR-IVS-
		Lethality/					IC50	ATS
		Splicing					(nM)	GMean
		Category					SF3B1	IC50 (nM)
		(HCC1954,		Percent	Free		(WT)	Ad2.1
ADC Batch Id	Linker	N87)	DAR	Monomer	Drug	Conc.	HELA.2	HELA.2
AB185-ADL1-D12	mc-Val-Cit-PABC	1, 3	6.25	99%	<1%	0.82		16.528
AB185-ADL13-D4	b-glucuronide	1, 1	4.47	99%	<1%	0.78	8.778	20.887
AB185-ADL1-D5	mc-Val-Cit-PABC	1, 3	6.14	99%	<1%	1.1		17.392
AB185-ADL23-D4	mc-Glu-Val-Cit-	2, 3	5.84	99%	<1%	1.03	8.778	20.887
	PABC
AB185-ADL1-D3	mc-Val-Cit-PABC	2, 3	6.75	99%	<1%	1.13		9.326
AB185-ADL22-D4	mal-PEG2-Val-Cit	1, 3	6.12	99%	<1%	1.01	8.778	20.887
	PABC
AB185-ADL21-D1	mc-Ala-Ala-Asn-	2, 3	5.51	99%	<1%	1.35	6.085	17.568
	PABC
AB185-ADL21-D8	mc-Ala-Ala-Asn-	4, 3	6.4	99%	<1%	1.67	5.921	11.403
	PABC
AB185-ADL21-D4	mc-Ala-Ala-Asn-		1.55	99%	<1%	1.11	8.778	20.887
	PABC
AB185-ADL15-D2	mal-CH2CH2—	3, ND	3.25	98.80%	<1%	1.6
	O—CH2CH2-
	(non-cleavable)
						cellular
				cellular potency	cellular potency	potency
		biochemical	cell line #1	cell line #2	cell line #3
		splicing assay	CTGlo-	CTGlo-	CTGlo-	CTGlo-	CTGlo-
		qPCR-IVS-		ATS	ATS	ATS	ATS	ATS
		ATS		GMean	GMean	GMean	GMean	GMean
		GMean		GI50	LD50	GI50	LD50	GI50
		IC50 (nM)		(nM)	(nM)	(nM)	(nM)	(nM)
		Ad2.2	Ratio	72 h	72 h	72 h	72 h	72 h
	ADC Batch Id	HELA.2	Ad2.1/2.2	NCIH1650.1	NCIH1650.1	NCIH1568.1	NCIH1568.1	THP1.1
	AB185-ADL1-D12	25.09	0.659	257.925	>10000.00	163.604	1193.037	202.487
	AB185-ADL13-D4	17.321	1.206	11.941	1960.398	6.369	45.485	10.943
	AB185-ADL1-D5	23.943	0.726
	AB185-ADL23-D4	17.321	1.206	11.941	1960.398	6.369	45.485	10.943
	AB185-ADL1-D3	10.928	0.853
	AB185-ADL22-D4	17.321	1.206	11.941	1960.398	6.369	45.485	10.943
	AB185-ADL21-D1	15.853	1.108	12.121	>10000.00	7.363	65.737	11.085
	AB185-ADL21-D8	9.034	1.262	19.15	>10000.00	6.568	68.125	8.745
	AB185-ADL21-D4	17.321	1.206	11.941	1960.398	6.369	45.485	10.943
	AB185-ADL15-D2
					Properties—Corresponding Free Payloads
					cellular
					potency
					cell line #3	cellular permeability
					CTGlo-	Perm.	Perm.
					ATS	Mean	Mean
					GMean	Caco	Caco
					LD50	2 A-B	2 B-A
					(nM)	Perm	Perm
					72 h	(10e−6	(10e−6
			ADC Batch Id	THP1.1	cm/s)	cm/s)	ER
			AB185-ADL1-D12	900.227	0.17	0.23	1.353
			AB185-ADL13-D4	33.329	0.1	1.79	17.9
			AB185-ADL1-D5
			AB185-ADL23-D4	33.329	0.1	1.79	17.9
			AB185-ADL1-D3
			AB185-ADL22-D4	33.329	0.1	1.79	17.9
			AB185-ADL21-D1	41.408	0.14	2.05	14.64
			AB185-ADL21-D8	38.969	0.128	2.027	15.88
			AB185-ADL21-D4	33.329	0.1	1.79	17.9
			AB185-ADL15-D2

TABLE 26 — Exemplary anti-HER2 ADCs—HCC1954 cells Properties—HER2 ADCs antiproliferative activity antigen (+) cell #1

					CTGlo	CTGlo
					GMean	Mean
					GI50 (nM)	MinResp %
			Permeability	pH 5.5	HER2 SMLA	HER2 SMLA
			Caco-2 A-B	Stability	screen	screen
ADC Batch Id	Linker	DAR	(10e−6 cm/s)	(t1/2, 37 C.)	HCC1954.1	HCC1954.1
AB185-ADL5-D19	QVal-Ala	6.600	16	0.8	0.278	−47.381
AB185-ADL5-D25	QVal-Ala	6.000	0.14	3.4	0.038	−46.265
AB185-ADL5-D2	QVal-Ala	4.140	0.6	4	0.206	−12.239
AB185-ADL1-D4	Val-Cit	2.800	0.1	4.5	0.050	−57.212
AB185-ADL5-D10	QVal-Ala	4.020	1.8	5	0.254	−2.273
AB185-ADL1-D16	Val-Cit	4.400	0.72	7	2.086
AB185-ADL5-D17	QVal-Ala	3.960	20	7	2.668	14.873
AB185-ADL5-D34	QVal-Ala		26	7	3.325	−38.285
AB185-ADL5-D15	QVal-Ala	4.000	3.4	7	0.912	−45.388

TABLE 27 — Exemplary target antigens and indications

Target Antigen	Indication(s)
MSLN	ovarian cancer
	cervical cancer
	pancreatic cancer
	lung adenocarcinoma
EPHA2	esophageal cancer
FOLH1	prostate cancer
CDH6	kidney cancer
CEACAM5	colorectal cancer
CFC1B	pancreatic cancer
ENPP3	kidney cancer
FOLR1	ovarian cancer
HAVCR1	kidney cancer
	esophageal cancer
KIT	kidney cancer
MET	kidney cancer
	esophageal cancer
MUC16	ovarian cancer
	cervical cancer
	breast cancer
SLC39A6	breast cancer
	prostate cancer
SLC44A4	prostate cancer
STEAP1	prostate cancer

description truncated at 500,000 characters. 13 of 80 part labels are ours — the grant heads the rest

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Claims as granted

24 claims

Classifications

21 codes

IPC · International Patent Classification

Section A — Human necessities

A61K9/127
A61K9/51
A61K31/365
A61K31/496
A61K39/00
A61K39/395
A61K45/06
A61K47/60
A61K47/68
A61P35/00

Section C — Chemistry; metallurgy

C07D405/12
C07D313/00
C07D405/06
C07K16/24
C07K16/28
C07K16/30
C07K16/32
C07K16/40
C12Q1/6886

Section G — Physics

G01N33/50
G01N33/574

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