Single-domain antibodies to programmed cell death 1 protein (PD-1), encoding nucleic acids and methods of using same

This application is a 35 U.S.C. § 371 National Phase Application of International Application No. PCT/GB2018/050037, filed Jan. 8, 2018, which claims the benefit of GB Application Nos. 1700207.2, filed Jan. 6, 2017, 1700208.0, filed Jan. 6, 2017, and 1700210.6, filed Jan. 6, 2017, the content of each is incorporated herein by reference in its entirety.

The content of the electronically submitted sequence listing (Name: Sequence Listing .txt; Size: 590,279 bytes; and Date of Recordation: Jul. 2, 2019) is herein incorporated by reference in its entirety.

The invention relates to PD-1 binding agents comprising PD-1 binding V H single domain antibodies (sdAb) and the use of such binding agents in the treatment, prevention and detection of disease.

Antibody-based therapeutics have emerged as important components of therapies for an increasing number of human diseases in such fields as oncology, inflammatory and infectious diseases. Indeed, antibodies are one of the best-selling classes of drugs today; five of the top ten best selling drugs are antibodies.

The Programmed Death 1 (PD-1) protein is encoded by the PDCD1 gene and expressed as a 55 kDa type I transmembrane protein (Agata 1996 Int Immunol 8(5):765-72). PD-1 is an immunoglobulin superfamily member (Ishida 1992 EMBO 11(11):3887-95) and it is an inhibitory member of the extended CD28/CTLA-4 family of T cell regulators. Other members of this family include CD28, CTLA-4, ICOS and BTLA. PD-1 exists as a monomer, lacking the unpaired cysteine residue characteristic of other CD28 family members (Zhang 2004 Immunity 20:337-47). Its cytoplasmic domain contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) and an immunoreceptor tyrosine-based switch motif (ITSM) that are phosphorylated during signal transduction (Riley 2009 Immunol Rev 229(1):114-25).

PD-1 is expressed on B cells, T cells, and monocytes (Agata 1996). The role of PD-1 in maintaining immunologic self-tolerance was demonstrated in PDCD1−/− mice, which develop autoimmune disorders (Nishimura 1999 Immunity 11:141-51, Nishimura 2001 Science 291(5502):319-22). The PD-1 pathway therefore regulates antigen responses, balancing autoimmunity and tolerance.

There are two ligands for PD-1 that mediate its regulatory function. PD-L1 (B7-H1) is normally expressed on dendritic cells, macrophages, resting B cells, bone marrow-derived mast cells and T cells as well as non-hematopoietic cell lineages (reviewed in Francisco 2010 Immunol Rev 236:219-42). PD-L2 (B7-DC) is largely expressed on dendritic cells and macrophages (Tseng 2001 J Exp Med 193(7):839-45). Ligand expression is influenced by local mediators and can be upregulated by inflammatory cytokines.

PD-1 is known as an immunoinhibitory protein that negatively regulates TCR signals. The interaction between PD-1 and PD-L1 can act as an immune checkpoint, which can lead to, e.g., a decrease in tumour infiltrating lymphocytes, a decrease in T-cell receptor mediated proliferation, and/or immune evasion by cancerous cells. Immune suppression can be reversed by inhibiting the local interaction of PD-1 with PD-L1 or PD-L2; the effect is additive when the interaction of PD-1 with both PD-L1 and PD-L2 is blocked.

As T cells become activated and co-stimulated by antigen-presenting cells (APCs), T cell expression of PD-1 is induced. PD-1 engagement with ligand on the APC cross-links PD-1 and clusters it into the T cell receptor (TCR) complex within the immunological synapse (Yokosuka 2012 J Exp Med 209(9):1201-17). Within the T cell cytoplasm, PD-1 signalling domains ITIM and ITSM are phosphorylated. This induces Src-homology-2 domain-containing tyrosine phosphatase (SHP1/2) that attenuates various components of the T cell receptor (TCR) signalling. T cell activation is dampened, which leads to a reduction in cytokine response, proliferation and cytolytic activity. This downregulation of T cell function serves to prevent overstimulation, tolerising cells against weakly immunogenic self-antigen.

The PD-1 pathway can be exploited in cancer or infection, whereby tumours or viruses can evade effective immune recognition and T cells demonstrate an ‘exhausted’ phenotype. PD-L1 has also been shown to be expressed in many tumour types including urothelial, ovarian, breast, cervical, colon, pancreatic, gastric, melanoma, glioblastoma and non-small cell lung carcinoma (reviewed in Callahan 2014 J Leukoc Biol 94(1):41-53). The cytokines produced by cancer stromal cells can further upregulate PD-L1 in the tumour microenvironment (He 2015 Nature Scientific Reports 5:13110). As a result, tumour-specific T cells become unresponsive through PD-1 signalling and therefore fail to eliminate their target. T regulatory cells (T regs) have also been shown to express high levels of PD-1 and they suppress the anti-tumour response further (Lowther 2016 JCI Insight 1(5):85935).

Disruption of the PD-1:PD-L1 interaction enhances T cell activity. An anti-PD-1 monoclonal antibody demonstrates blockade of the interaction between PD-1 and its ligands (Wang 2014 Cancer Immunol Res 2(9):846-56). T cell function in-vitro can be enhanced by PD-1 blockade, as demonstrated by improved proliferation and cytokine responses in mixed lymphocyte reactions of T cells and dendritic cells. Cytotoxic lymphocytes (CTLs) derived from melanoma patients has also been shown to be enhanced by PD-1 blockade in vitro using the antibody OPDIVO® (nivolumab), and can become resistant to Treg suppression (Wang 2009 Int Immunol 21(9): 1065-1077). This antibody has been tested in clinical dose escalation studies in melanoma, non-small cell lung carcinoma (NSCLC), renal cell cancer (RCC) and others. It shows improved overall survival rates compared to chemotherapy in NSCLC patients. Another PD-1 blocking antibody, KEYTRUDA® (pembrolizumab), demonstrates responses in NSCLC patients refractory to CTLA-4 blockade. OPDIVO® (nivolumab) and KEYTRUDA® (pembrolizumab) both functionally block the interaction of human PD-1 with its ligands.

It is possible to induce PD-1 signalling by cross-linking it on the membrane with a combination of anti-PD-1 plus anti-CD3 antibodies (Bennett 2003 J Immunol 170:711-18, Keir 2005 J Immunol 175:7372-7379). This function could be detrimental during an anti-tumour response because T cell activity would be suppressed. If suppression of T cell responses were desired, agonistic anti-PD-1 antibodies or those with effector functions could be used to treat immune-related diseases such as rheumatoid arthritis.

The aim of the present invention is to address the need of alternative antibody-based treatments for use in the treatment of disease, in particular in the treatment of cancer.

The invention relates to isolated multifunctional binding agents comprising single domain antibodies that bind to human PD-1 as described herein. In particular, the invention relates to isolated multiparatopic binding molecules that bind to human PD-1 and related methods for treating disease.

Described herein are human variable single domain antibodies generated in vivo in transgenic mice that bind to human PD-1, but do not block the functional interaction between human PD-1 and its ligands. Such anti-PD-1 V H single domain antibodies bind an epitope that is distant from the part of the PD-1 protein that interacts with its ligands PD-L1 and PD-L2 and that is therefore outside the region of binding of known therapeutics targeting PD-1.

Described herein are also human variable single domain antibodies generated in vivo in transgenic mice that bind to human PD-1 and block the functional interaction between human PD-1 and its ligands. The inventors have surprisingly found that when a blocking and a non blocking variable single domain antibody are combined in a single molecule, this results in a synergistic antagonistic effect compared to the effect of a monovalent molecules. Thus, surprisingly, the combination of the two in a single molecule leads to an effect that is greater than the effect provided by either entity alone or the combined effect when in monovalent format. Thus, T-cell effector function can be increased by use of the mulitparatopic molecule.

Therefore, in one aspect, the invention relates to an isolated binding agent comprising a) a first single domain antibody directed against a first epitope of human PD-1 and b) a second single domain antibody directed against a second epitope of human PD-1.

In one embodiment, said domain of the single domain antibody is a human heavy chain variable domain (V H ).

In one embodiment, said first single domain antibody does not block the interaction of human PD-1 with human PD-L1 and/or PD-L2 and wherein said second single domain antibody blocks the interaction of human PD-1 with human PD-L1 and/or PD-L2.

In one embodiment, said first single domain antibody binds to an epitope comprising one or more or all residues selected from R 104 , D 105 , F 106 , M 108 , S 109 and V 110 of human PD-1.

In one embodiment, said first single domain antibody comprises a CDR3 as shown in SEQ ID NO. 3 or 254 or a sequence with at least 90% homology thereto.

In one embodiment, said first single domain antibody comprises a CDR1 as shown in SEQ ID NO. 1 or a sequence with at least 90% homology thereto, a CDR2 as shown in SEQ ID NO. 2 or a sequence with at least 90% homology thereto and a CDR3 as shown in SEQ ID NO. 3 or a sequence with at least 90% homology thereto. In one embodiment, said first single domain antibody comprises a CDR1 as shown in SEQ ID NO. 251 or SEQ ID No. 251 with 1 or 2 amino acid substitutions, a CDR2 as shown in SEQ ID No. 252 or SEQ ID No. 252 with 1 to 5 amino acid substitutions and a CDR3 as shown in SEQ ID No. 253 or SEQ ID No. 253 with 1 to 5 amino acid substitutions.

In one embodiment, said first single domain antibody comprises a CDR1, 2 and 3 selected from Table 1.

In one embodiment, said first single domain antibody comprises a sequence selected from SEQ ID NO. 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, 80, 104, 108, 112, 116, 120, 124, 128, 132, 136, 140, 144, 148, 152, 156, 160, 164, 168, 172, 176, 180, 184, 188, 192, 196, 200, 204, 208, 212, 216, 220, 254, 258, 262, 266, 270, 274, 278, 282, 286, 290, 294, 298, 302, 306, 310, 314, 318, 322, 326, 330, 334, 338, 342, 346, 350, 354, 358, 362, 366, 370, 374, 378, 382, 386, 390, 394, 398, 402, 406, 410, 414, 418, 422, 426, 430, 434, 438, 442, 446, 450, 454, 458 or 462 or a sequence with at least 60%, 70%, 80% or 90% homology thereto.

In one embodiment, said second single domain antibody comprises a CDR3 as shown in SEQ ID NO. 516 or a sequence with at least 90% homology thereto. In one embodiment, said second single domain antibody comprises a CDR1 as shown in SEQ ID No. 516 or SEQ ID No. 516 with 1 or 2 amino acid substitutions, a CDR2 as shown in SEQ ID No. 517 or SEQ ID No. 517 with 1 to 5 amino acid substitutions and a CDR3 as shown in SEQ ID No. 518 or SEQ ID No. 518 with 1 to 5 amino acid substitutions.

In one embodiment, said second single domain antibody comprises a CDR1, 2 and 3 selected from Table 2.

In one embodiment, said second single domain antibody comprises a sequence selected from SEQ ID NO. 519, 523, 527, 531, 535, 539, 543, 547, 551, 555, 559, 563, 567, 571, 575, 579, 583, 587, 591, 595, 599, 603, 607, 611, 615, 619, 623, 627, 631, 635, 639, 643, 647, 651, 655, 659, 663, 667, 671, 675, 679, 683, 687, 691, 695, 699, 703, 707, 711, 715, 719, 723, 727, 731, 735, 739, 743, 747, 751, 755, 759, 763, 767, 771, 775, 779, 783, 787, 791, 795, 799, 803, 807, 884, 888, 892, 896, 900, 904, 908, 912, 916, 920, 924, 928, 932, 936, 940, 944, 948, 952, 956, 960, 964, 968, 972, 976, 980, 984, 988, 992, 996, 1000, 1004, 1008, 1012, 1016, 1020, 1024, 1028, 1032, 1036, 1040, 1044 or 1048 or a sequence with at least 60%, 70%, 80% or 90% homology thereto.

In one embodiment, said first and second single domain antibody are covalently linked by via a peptide. In one embodiment, said peptide linker is between 3 and 50 amino acids in length. In one embodiment, the peptide linker comprises glycine and serine amino acid residues In one embodiment, the peptide linker has the formula (Gly4Ser)n, where n=from 1 to 20. In one embodiment, said binding agent comprises one or more further binding molecules. In one embodiment, said binding molecule is an antibody or fragment thereof. In one embodiment, said binding molecule is a single domain antibody. In one embodiment, said binding molecule binds to an epitope on human PD-1. In one embodiment, said binding molecule binds to a different antigen, i.e. an antigen that is not PD-1. In one embodiment, said binding molecule binds to an immonooncology target.

In one embodiment, at least one single domain antibody is conjugated to a toxin, enzyme, radioisotope, half-life extending moiety, therapeutic molecule or other chemical moiety. In one embodiment, said half-life extending moiety is selected from the group consisting of an albumin binding moiety, a transferrin binding moiety, a polyethylene glycol molecule, a recombinant polyethylene glycol molecule, human serum albumin, a fragment of human serum albumin, and an albumin binding peptide or single domain antibody that binds to human serum albumin.

In one embodiment, said first single domain antibody is located N-terminally and said second single domain antibody is located C-terminally. In one embodiment, said first single domain antibody is located C-terminally and said second single domain antibody is located N-terminally.

In another aspect, the invention relates to an immunoconjugate comprising a binding agent as set out herein linked to a therapeutic agent.

In another aspect, the invention relates to a pharmaceutical composition comprising a binding agent or an immunoconjugate as set out herein and a pharmaceutical carrier.

In another aspect, the invention relates to a method for treating cancer, an immune disorder or viral infection comprising administering a therapeutically effective amount of a comprising a binding agent, an immunoconjugate or a pharmaceutical composition as set out herein.

In another aspect, the invention relates to the use of a binding agent, an immunoconjugate or a pharmaceutical composition as set out herein in the manufacture of a medicament for the treatment of a cancer, an immune disorder, neurological disease, inflammatory disorder, allergy, transplant rejection, viral infection, immune deficiency, and other immune system-related disorder.

In another aspect, the invention relates to a binding agent, an immunoconjugate or a pharmaceutical composition as set out herein for use as medicament.

In another aspect, the invention relates to binding agent, an immunoconjugate or a pharmaceutical composition agent as set out herein for use in the treatment of a cancer, an immune disorder, neurological disease, inflammatory disorder, allergy, transplant rejection, viral infection, immune deficiency, and other immune system-related disorder.

In one embodiment said cancer is selected from bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, breast cancer, brain cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, kidney cancer, sarcoma of soft tissue, cancer of the urethra, cancer of the bladder, renal cancer, lung cancer, leukemia, lymphomas, such as such as Hodgkin's disease, non-Hodgkin's and multiple myelomas.

In another aspect, the invention relates to a method of modulating an immune response comprising administering a binding agent, an immunoconjugate or a pharmaceutical composition as set out herein.

In another aspect, the invention relates to an isolated nucleic acid molecule comprising a nucleotide sequence encoding a binding agent as set out herein. In another aspect, the invention relates to a vector comprising a nucleic acid as herein. In another aspect, the invention relates to host cell comprising a nucleic acid or a vector as set out herein.

In another aspect, the invention relates to method for producing a binding molecule as set out herein comprising expressing a nucleic acid encoding said binding molecule in a host cell and isolating the binding molecule from the host cell.

In another aspect, the invention relates to a kit comprising a binding molecule, an immunoconjugate or a pharmaceutical composition as set out herein.

In another aspect, the invention relates to a method for detecting the presence of human PD-1 in a test sample comprising contacting said sample with a binding molecule as set out herein and at least one detectable label and detecting binding of said single domain antibody to human PD-1.

In another aspect, the invention relates to a combination comprising a) a first single domain antibody directed against a first epitope of human PD-1 and b) a second single domain antibody directed against a second epitope of human PD-1.

In one embodiment, the said domain of the single domain antibody is a human heavy chain variable domain (V H ).

In one embodiment, the said first single domain antibody does not block the interaction of human PD-1 and human PD-L1 and/or PD-L2 and wherein said single domain antibody blocks the interaction of human PD-1 and human PD-L1 and/or PD-L2.

FIGURES

The invention is further described in the following non-limiting figures.

FIG. 1 : a) PDL-2 inhibition assay using blocking Humabody® V H . b) PD-L1 inhibition assay using Humabody® binding V H . c) PDL-2 inhibition assay using binding Humabody® V H .

FIG. 2 : Epitope competition assay. 1.1a did not cross compete with 1.57b-Strep tagged protein for binding to the cells ( FIG. 2 a ). 1.57b and 1.1b did not cross compete with 1.1a-Strep tagged protein and enhanced binding in the assay ( FIG. 2 b ).

FIG. 3 : Example EC 50 data for activity of binding and blocking Humabody® V H in a reporter assay.

FIG. 4 : Species cross reactivity testing a) binding to human PD-1 b) binding to cynomolgus PD-1 recombinant protein c) binding to mouse PD-1 protein.

FIG. 5 : a) FMAT PD1:PDL1 Inhibition assay using different Humabody® V H . b) CHO PD1/PDL1 c) CHO PD1/PDL2.

FIG. 6 : Serum stability of Humabody® V H .

FIG. 7 : a) A biparatopic binding molecule enhances IL-2 secretion. Cells were co-cultured with antibody/Humabody® for 2 days and the IL-2 concentration determined by HTRF (Homogenous Time Resolved Fluorescence assay) and b) A biparatopic binding molecule enhances IL-2 secretion. Cells were co-cultured with antibody/HUMABODY® V H for 2 days and the IL-2 concentration determined by HTRF (Homgenous Time Resolved Fluorescence assay).

FIG. 8 : In Vivo Efficacy of binding agents in HuGEMM PD1 Model with Subcutaneous MC38 Mouse Colon Adenocarcinoma.

FIG. 9 : In Vivo Efficacy of binding agents in hu-CD34 NSG™ mice.

FIG. 10 . Functional reporter gene assay. Different single domain antibodies were tested together with a biparatopic molecule.

Various aspects and embodiments will now be further described. In the following passages, different aspects of the invention are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.

Generally, nomenclatures used in connection with, and techniques of, cell and tissue culture, pathology, oncology, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art. The methods and techniques of the present disclosure are generally performed according to conventional methods well-known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. See, e.g., Green and Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012); Therapeutic Monoclonal Antibodies: From Bench to Clinic, Zhiqiang An (Editor), Wiley, (2009); and Antibody Engineering, 2nd Ed., Vols 1 and 2, Ontermann and Dubel, eds., Springer-Verlag, Heidelberg (2010).

Enzymatic reactions and purification techniques are performed according to manufacturer's specifications, as commonly accomplished in the art or as described herein. The nomenclatures used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

Described herein are human V H single domain antibodies that bind to human PD-1, but do not block the interaction between human PD-1 and its ligands as demonstrated in the functional assays in the examples. By not block the interaction between human PD-1 and its ligands is meant the functional interaction between human PD-1 and its ligands. Described herein are such human V H single domain antibodies which are used in the multifunctional molecules and bind to an epitope that is distinct from the binding site of human PD-1 to PD-L1 or PDL-2. These anti-PD-1 antibodies are particularly useful in combination with an anti-PD-1 single domain antibody that neutralises PD-1.

In one aspect, the invention thus provides an isolated binding agent comprising a) a first single domain antibody directed against a first epitope of human PD-1 and b) a second single domain antibody directed against a second epitope of human PD-1.

In one aspect, the invention relates to an isolated binding agent comprising a) a first single domain antibody directed against a first epitope of human PD-1 which does not block binding to PD-L1 and/or PD-L2 and/or does not block the interaction between human PD-1 and PD-L1 and/or PD-L2 and b) a second single domain antibody directed against a second epitope of human PD-1 which blocks binding to PD-L1 and/or PD-L2. In one embodiment, said binding molecule exhibits one or more or all of the following properties:

(a) binds to human PD-1 with a KD as measured in the examples;

(b) increases IL-2 secretion in an Mixed Lymphocyte Reaction assay;

(c) binds to human PD-1 and cynomolgus monkey PD-1;

(d) does not bind to mouse PD-1;

(e) inhibits the binding of PD-L1 and/or PD-L2 to PD-1;

(f) inhibits tumour cell growth in vivo (e.g. as measured in the examples using hu-CD34 NSG™ mice), compared to a human PD-1 antibody treatment which on average shows no effect;

(g) exhibits better blocking activity, for example in a cell based assay as shown in the examples (Jurkat cells, FIG. 10 ), than the blocking single domain antibody used in a monovalent format. For example, this may be a 10 to 25 fold increase;

(h) has an EC50 or IC50 value as measured in the examples;

(i) elicits tumor regression in vivo (e.g. as measured in the examples using in a HuGEMM PD1 Model with Subcutaneous MC38 Mouse Colon Adenocarcinoma);

(j) induces IL-2 and TNFα secretion in BioMap® Oncology CRC panel compared to a benchmark human PD-1 monoclonal antibody.

Suitable assays to measure the properties as set out above are described in the examples, for example as shown in examples 9, 11, 12 and 13.

The terms “first” and “second” are used to differentiate between the two PD-1 binding single domain antibodies used in the binding molecules of the invention, but are not understood to designate their orientation in the multivalent molecule with respect to the C and N terminus of the protein. For example, the first single domain antibody may be located N-terminally and the second single domain antibody may be located at the C-terminally. Alternatively, the first single domain antibody may be located at the C-terminally of the molecule and the second single domain antibody may be located at the N-terminally. C-terminally can mean that the C-terminus of the molecule or C terminally with respect to the first/second PD-1 binding molecule. N-terminally can mean that the N-terminus of the molecule or C terminally with respect to the first/second PD-1 binding molecule.

The invention also provides a combination and combination therapy comprising a) a first single domain antibody directed against a first epitope of human PD-1 and b) a second single domain antibody directed against a second epitope of human PD-1.

The invention also provides pharmaceutical compositions comprising such binding molecules and combinations, as well as isolated nucleic acids, isolated recombinant expression vectors and isolated host cells for making such binding proteins. Also provided are methods of using the binding agent disclosed herein to detect human PD-1 and methods of treating disease.

In preferred embodiments, the first and second single domain antibody is a single domain antibody wherein the domain is a human variable heavy chain (V H ) domain.

As used herein, the term “do not block or do not inhibit the interaction of human PD-1 with its ligands” refers to the functional interaction of human PD-1 with its ligands. In other words, the binding of such single domain antibodies used in the binding agents of the invention does not abolish or reduce the functional interaction of human PD-1 with its ligands. Thus, the binding of the single domain antibodies used in the binding agents of the invention to human PD-1 does not affect the biological function of the interaction of human PD-1 with its ligands. In one embodiment, the ligand is PD-L1. In one embodiment, the ligand is PD-L2.

The properties of the multifunctional binding agents as described herein can be exploited in therapeutic methods and uses. binding agents as described herein are for example useful in anchoring, associating or bringing into proximity other therapeutic molecule to human PD-1, for example in a targeted therapy to recruit the therapeutic compound to the cell or tissue of interest or to a region of the cell associated with PD-1 localisation. This makes the binding agents as described herein particularly suitable for delivery together with other compounds. This can be done in the same medicament, or by sequential administration of separate compositions.

Binding agents as described herein bind specifically to wild type human PD-1 (UniProt Accession No. Q15116, GenBank Accession No. U64863, SEQ ID No. 1093). Residues 1-20 OF SEQ ID No. 1093 correspond to the pre-sequence, residues 171 and beyond make up the transmembrane helix and the intracellular domain of PD-1.

Unless otherwise specified, the term PD-1 as used herein refers to human PD-1. The terms “Programmed Death 1,” “Programmed Cell Death 1,” “Protein PD-1,” “PD-1,” PD1,” “PDCD1,” “hPD-1” and “hPD-1” are used interchangeably, and include variants, isoforms, species homologs of human PD-1

The terms “PD-1 binding molecule/protein/polypeptide/agent”, “PD-1 antigen binding molecule protein/polypeptide/agent”, “anti-PD-1 single domain antibody”, “anti-PD-1 single immunoglobulin variable domain”, “anti-PD1 heavy chain only antibody” or “anti-PD-1 antibody” all refer to a molecule capable of specifically binding to the human PD-1 antigen. The binding reaction may be shown by standard methods, for example with reference to a negative control test using an antibody of unrelated specificity. The term “PD-1 binding molecule/agent” includes a PD-1 binding protein.

An antibody or binding molecule of the invention, “which binds” or is “capable of binding” an antigen of interest, e.g. PD-1, is one that binds the antigen with sufficient affinity such that the antibody is useful as a therapeutic agent in targeting a cell or tissue expressing the antigen.

Binding molecules of the invention bind specifically to human PD-1. In other words, binding to the PD-1 antigen is measurably different from a non-specific interaction. As demonstrated in the examples, the single domain antibodies of the invention do not cross react with mouse PD-1. Preferably, the single domain antibodies of the invention bind to human PD-1 and also bind to cyno PD-1.

The term “specific binding” or “specifically binds to” or is “specific for” a particular polypeptide or an epitope on a particular polypeptide target as used herein can be exhibited, for example, by a molecule having a KD for the target of at least about 10 −4 M, alternatively at least about 10 −5 M, alternatively at least about 10 −6 M, alternatively at least about 10 −7 M, alternatively at least about 10 −8 M, alternatively at least about 10 −9 M, alternatively at least about 10 −10 M, alternatively at least about 10 −11 M, alternatively at least about 10 −12 M, or greater. In one embodiment, the term “specific binding” refers to binding where a molecule binds to a particular polypeptide or epitope on a particular polypeptide without substantially binding to any other polypeptide or polypeptide epitope. In one embodiment, the binding agent has an affinity in the nanomolar to picomolar range.

In one embodiment, the binding agent specifically binds to human PD-1 with a binding affinity of Kd 10 −9 to 10 −12 M, for example 10 −9 , 10 −10 , 10 11 , 10 −12 M, measured using surface plasmon resonance (Biacore) or bio-light interferometry (for example ForteBio Octet).

In one embodiment, the binding agent inhibits the binding of human PD-L1 or human PD-L2 to human PD-1 with an IC 50 value in the low nanomolar to picomolar range, for example in the subnanomolar range.

The term “antibody” broadly refers to any immunoglobulin (Ig) molecule, or antigen binding portion thereof, comprised of four polypeptide chains, two heavy (H) chains and two light (L) chains, or any functional fragment, mutant, variant, or derivation thereof, which retains the essential epitope binding features of an Ig molecule. Such mutant, variant, or derivative antibody formats are known in the art.

In a full-length antibody, each heavy chain is comprised of a heavy chain variable region or domain (abbreviated herein as HCVR) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, C H 1, C H 2 and C H 3. Each light chain is comprised of a light chain variable region or domain (abbreviated herein as LCVR) and a light chain constant region. The light chain constant region is comprised of one domain, C L .

The heavy chain and light chain variable regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each heavy chain and light chain variable region is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG 1, IgG2, IgG 3, IgG4, IgAl and IgA2) or subclass.

The term “CDR” refers to the complementarity-determining region within antibody variable sequences. There are three CDRs in each of the variable regions of the heavy chain and the light chain, which are designated CDR1, CDR2 and CDR3, for each of the variable regions. The term “CDR set” refers to a group of three CDRs that occur in a single variable region capable of binding the antigen. The exact boundaries of these CDRs can be defined differently according to different systems known in the art.

The Kabat Complementarity Determining Regions (CDRs) are based on sequence variability and are the most commonly used (Kabat et al., (1971) Ann. NY Acad. Sci. 190:382-391 and Kabat, et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242). Chothia refers instead to the location of the structural loops (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). The Kabat numbering system is generally used when referring to a residue in the variable domain (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain).

The system described by Kabat is used herein unless otherwise specified. The terms “Kabat numbering”, “Kabat definitions” and “Kabat labeling” are used interchangeably herein. These terms, which are recognized in the art, refer to a system of numbering amino acid residues which are more variable (i.e., hypervariable) than other amino acid residues in the heavy and light chain variable regions of an antibody, or an antigen binding portion.

A chimeric antibody is a recombinant protein that contains the variable domains including the complementarity determining regions (CDRs) of an antibody derived from one species, preferably a rodent antibody, while the constant domains of the antibody molecule are derived from those of a human antibody. For veterinary applications, the constant domains of the chimeric antibody may be derived from that of other species, such as a cat or dog.

A humanized antibody is a recombinant protein in which the CDRs from an antibody from one species; e.g., a rodent antibody, are transferred from the heavy and light variable chains of the rodent antibody into human heavy and light variable domains (e.g., framework region sequences). The constant domains of the antibody molecule are derived from those of a human antibody. In certain embodiments, a limited number of framework region amino acid residues from the parent (rodent) antibody may be substituted into the human antibody framework region sequences.

The term “antigen binding site” refers to the part of the antibody or antibody fragment that comprises the area that specifically binds to an antigen. An antigen binding site may be provided by one or more antibody variable domains. Preferably, an antigen binding site is comprised within the associated V H and V L of an antibody or antibody fragment.

An antibody fragment is a portion of an antibody, for example as F(ab′) 2 , Fab, Fv, sFv and the like. Functional fragments of a full length antibody retain the target specificity of a full length antibody. Recombinant functional antibody fragments, such as Fab (Fragment, antibody), scFv (single chain variable chain fragments) and single domain antibodies (dAbs) have therefore been used to develop therapeutics as an alternative to therapeutics based on mAbs.

scFv fragments (˜25 kDa) consist of the two variable domains, V H and V L . Naturally, V H and V L domain are non-covalently associated via hydrophobic interaction and tend to dissociate. However, stable fragments can be engineered by linking the domains with a hydrophilic flexible linker to create a single chain Fv (scFv).

The smallest antigen binding fragment is the single variable fragment, namely the V H or V L domain. Binding to a light chain/heavy chain partner respectively is not required for target binding. Such fragments are used in single domain antibodies. A single domain antibody (˜12 to 15 kDa) therefore consists of or comprises either the V H or V L domain.

The terms “single domain antibody, variable single domain or immunoglobulin single variable domain (ISV)” are all well known in the art and describe the single variable fragment of an antibody that binds to a target antigen. These terms are used interchangeably herein. As explained below, preferred embodiments of the various aspects of the invention relate to a binding agent comprising at least two single heavy chain variable domain antibodies/immunoglobulin heavy chain single variable domains which bind a PD-1 antigen in the absence of light chain. One embodiment thus relates to single human heavy chain variable domain (V H ) antibodies. Such binding molecules are also termed Humabody® herein. Humabody® is a registered trademark of Crescendo Biologics Ltd.

Thus, in some preferred embodiments, the isolated binding agents/molecules of the invention comprise at least two single domain antibody wherein said domain is a human heavy chain variable domain. Thus, in one aspect, the binding agents of the invention comprise or consist of at least two human immunoglobulin single variable heavy chain domains; they are devoid of V L domains.

The term “isolated” binding agent refers to binding agent that is substantially free of other binding agents, antibodies or antibody fragments having different antigenic specificities. Moreover, an isolated binding agent may be substantially free of other cellular material and/or chemicals.

A “blocking binding agent, single domain antibody or antibody” or a “neutralizing binding agent, single domain antibody or antibody”, as used herein is intended to refer to an antibody whose binding to PD-1 results in inhibition of at least one biological activity of PD-1. For example, a blocking single domain antibody or multivalent binding agent as described herein may prevent or block PD-1 binding to PD-L1 and/or PD-L2.

Each single V H domain antibody comprises three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Thus, in one embodiment of the invention, the domain is a human variable heavy chain (V H ) domain with the following formula FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

Modifications to the C or N-terminal V H framework sequence may be made to the single domain antibodies used in the invention to improve their properties. For example, the V H domain may comprise C or N-terminal extensions or deletions. C-terminal extensions or deletions can be added to/deleted from the C terminal end of a V H domain which terminates with the residues VTVSS (SEQ ID No. 1091).

In one embodiment, the single domain antibodies used in the invention comprise C-terminal extensions/deletions of from 1 to 50 residues, for example 1 to 25, for example 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 additional amino acids. In one embodiment, the single domain antibodies of the invention comprise additional amino acids of the human C H 1 domain thus that the C-terminal end extends into the C H 1 domain. In one embodiment, said extension comprises at least 1 alanine residue, for example a single alanine residue, a pair of alanine residues or a triplet of alanine residues.

Additional C or N-terminal residues can be linkers that are employed to conjugate the single domain antibodies used in the invention to another moiety, or tags that aid the detection of the molecule. Such tags are well known in the art and include for, example linker His tags, e.g., hexa-His (HHHHHH, SEQ ID No. 1092) or myc tags.

As used herein, the term “homology” generally refers to the percentage of amino acid residues in a sequence that are identical with the residues of the reference polypeptide with which it is compared, after aligning the sequences and in some embodiments after introducing gaps, if necessary, to achieve the maximum percent homology, and not considering any conservative substitutions as part of the sequence identity. Thus, the percent homology between two amino acid sequences is equivalent to the percent identity between the two sequences. Neither N- or C-terminal extensions, tags or insertions shall be construed as reducing identity or homology. Methods and computer programs for the alignment are well known. The percent identity between two amino acid sequences can be determined using well known mathematical algorithms.

According to the various aspects and embodiments of the invention, the variable domain of the single domain antibodies of the binding molecules of the invention is preferably a human variable domain (V H ). As used herein, a human V H domain includes a fully human or substantially fully human V H domain. As used herein, the term human V H domain also includes V H domains that are isolated from heavy chain only antibodies made by transgenic mice expressing fully human immunoglobulin heavy chain loci, in particular in response to an immunisation with an antigen of interest, for example as described in WO2016/062990 and in the examples. In one embodiment, a human V H domain can also include a V H domain that is derived from or based on a human V H domain amino acid or nucleic acid sequence encoding such V H domain. Thus, the term includes variable heavy chain regions derived from or encoded by human germline immunoglobulin sequences. A substantially human V H domain or V H domain that is derived from or based on a human V H domain may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced in vitro, e.g. by random or site-specific mutagenesis, or introduced by somatic mutation in vivo). The term “human V H domain” therefore also includes a substantially human V H domain wherein one or more amino acid residue has been modified. For example, a substantially human V H domain the V H domain may include up to 10, for example 1, 2, 3, 4 or 5 amino acid modifications compared to a fully human sequence.

However, the term “human V H domain” or “substantially human V H domain”, as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. Preferably, the term “human V H domain”, as used herein, is also not intended to include camelized V H domains, that is human V H domains that have been specifically modified, for example in vitro by conventional mutagenesis methods to select predetermined positions in the V H domains sequence and introduce one or more point mutation at the predetermined position to change one or more predetermined residue to a specific residue that can be found in a camelid V HH domain.

The binding agent is biparatopic. Thus, the binding molecule may comprise a first V H single domain antibody (V H (A)) binding to a first epitope on human PD-1 and a second V H single domain antibody (V H (B)) binding to a second epitope on human PD-1 and thus has the following formula: V H (A)-V H (B) or V H (B)-V H (A).

Each V H comprises CDR and FR regions. The order of the immunoglobulin single variable domains A and B is not particularly limited, so that, within a polypeptide of the invention, immunoglobulin single variable domain A may be located N-terminally and immunoglobulin single variable domain B may be located C-terminally, or vice versa. The V H domain antibodies are typically connected via a linker. Suitable linkers include for example a linker with GS residues such as (Gly 4 Ser)n, where n=from 1 to 10 or 1 to 20, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

Thus, the binding molecule may have the following formula: FR1(A)-CDR1(A)-FR2(A)-CDR2(A)-FR3(A)-CDR3(A)-FR4(A)-linker-FR1(B)-CDR1(B)-FR2(B)-CDR2(BA)-FR3(B)-CDR3(B)-FR4(B) or FR1(B)-CDR1(B)-FR2(B)-CDR2(BA)-FR3(B)-CDR3(B)-FR4(B)-linker-FR1 (A)-CDR1(A)-FR2(A)-CDR2(A)-FR3(A)-CDR3(A)-FR4(A)

Examples of first and second human V H single domain antibodies that form PD-1 binding arms of the multivalent binding agents described herein are set out below.

Described are V H domains that bind to PD-1 and do not block ligand binding; these bind to PD-1 at common residues of PD-1, namely R 104 , D 105 , F 106 , H 107 , M 108 , S 109 and V 110 of human PD-1. In one embodiment, the invention relates thus to an isolated single domain antibody that binds to an epitope, epitope part, domain, subunit or conformation of human PD-1 comprising one or more or all of the residues selected from R 104 , D 105 , F 106 , H 107 , M 108 , S 109 and V 110 of human PD-1.

In one embodiment, said epitope, epitope part, domain, subunit or conformation further comprises one or more or all of G 103 , V 111 , R 112 and A 113 .

In one embodiment, said epitope, epitope part, domain, subunit or conformation comprises G 103 . In one embodiment, said epitope, epitope part, domain, subunit or conformation comprises R 104 . In one embodiment, said epitope, epitope part, domain, subunit or conformation comprises S 109 . In one embodiment, said epitope, epitope part, domain, subunit or conformation comprises V 110 . In one embodiment, said epitope, epitope part, domain, subunit or conformation comprises V 111 . In one embodiment, said epitope, epitope part, domain, subunit or conformation comprises R 112 . In one embodiment, said epitope, epitope part, domain, subunit or conformation comprises A 113 . In one embodiment, said epitope, epitope part, domain, subunit or conformation comprises all of G 103 , R 104 , S 109 , V 110 , V 111 , R 112 and A 113 of human PD-1.

In one embodiment, said epitope, epitope part, domain, subunit or conformation further comprises one or more of residues N 102 , D 105 , F 106 , H 107 , M 108 , R 114 and R 115 of human PD-1. In one embodiment, said epitope, epitope part, domain, subunit or conformation comprises N 102 . In one embodiment, said epitope, epitope part, domain, subunit or conformation comprises D 105 . In one embodiment, said epitope, epitope part, domain, subunit or conformation comprises F 106 . In one embodiment, said epitope, epitope part, domain, subunit or conformation comprises H 107 . In one embodiment, said epitope, epitope part, domain, subunit or conformation further comprises M 108 . In one embodiment, said epitope, epitope part, domain, subunit or conformation comprises R 114 . In one embodiment, said epitope, epitope part, domain, subunit or conformation comprises R 115 . In one embodiment, said epitope, epitope part, domain, subunit or conformation comprises all of N 102 , D 105 , F 106 , H 107 , M 108 , R 114 and R 115 of human PD-1. In one embodiment, said epitope, epitope part, domain, subunit or conformation comprises all of N 102 , G 103 , D 105 , F 106 , H 107 , M 108 , S 109 , V 110 , V 111 , R 112 , A 113 , R 114 and R 115 of human PD-1.

In another embodiment, the single domain antibody binds to an epitope comprising one or more or all residues selected from R 104 , D 105 , F 106 , H 107 , M 108 , S 109 and V 110 of human PD-1 and further to one or more or all of S 60 , E 61 , S 62 , F 63 , V 64 , L 65 , N 66 , W 67 , Y 68 , R 69 , M 70 , S 71 , G 90 , Q 91 , D 92 , C 93 , R 94 , F 95 , R 96 , V 97 , T 98 , V 111 , R 112 , A 113 and R 11 .

The term “epitope” or “antigenic determinant” refers to a site on the surface of an antigen (e.g., PD-1) to which an immunoglobulin, antibody or antibody fragment, including a V H single domain antibody specifically binds. Generally, an antigen has several or many different epitopes and reacts with many different antibodies. The term specifically includes linear epitopes and conformational epitopes.

Epitopes within protein antigens can be formed both from contiguous amino acids (usually a linear epitope) or non-contiguous amino acids juxtaposed by tertiary folding of the protein (usually a conformational epitope). Epitopes formed from contiguous amino acids are typically, but not always, retained on exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. An epitope typically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids in a unique spatial conformation. Methods for determining what epitopes are bound by a given antibody or antibody fragment (i.e., epitope mapping) are well known in the art and include, for example, immunoblotting and immunoprecipitation assays, wherein overlapping or contiguous peptides from are tested for reactivity with a given antibody or antibody fragment.

An antibody binds “essentially the same epitope” as a reference antibody, when the two antibodies recognize identical or sterically overlapping epitopes. The most widely used and rapid methods for determining whether two epitopes bind to identical or sterically overlapping epitopes are competition assays, which can be configured in different formats, using either labelled antigen or labelled antibody.

In one embodiment, a first single V H domain antibody that binds to human PD-1, but does not block ligand binding, comprises a CDR3 sequence as shown Table 1 below or a sequence with at least 60%, 70%, 80%, 90%, 95% or more sequence identity thereto. In one embodiment, said sequence homology is at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. In one embodiment, said sequence homology is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.

In one embodiment, the first V H single domain antibody has a CDR3 sequence comprising SEQ ID No. 3, 253, 175, 297 or a sequence having at least 70%, at least 80%, at least 90%, or at least 95% homology thereto.

In one embodiment, the first V H single domain antibody has a CDR1 as shown in SEQ ID No. 1 or SEQ ID No. 1 with 1 or 2 amino acid substitutions, a CDR2 as shown in SEQ ID No. 2 or SEQ ID No. 2 with 1 to 5 amino acid substitutions and a CDR3 as shown in SEQ ID No. 3 or SEQ ID No. 3 with 1 to 5 amino acid substitutions. In one embodiment, the V H single domain antibody has a CDR1 as shown in SEQ ID No. 251 or SEQ ID No. 251 with 1 or 2 amino acid substitutions, a CDR2 as shown in SEQ ID No. 252 or SEQ ID No. 252 with 1 to 5 amino acid substitutions and a CDR3 as shown in SEQ ID No. 253 or SEQ ID No. 253 with 1 to 5 amino acid substitutions.

In one embodiment, the first V H single domain antibody comprises a combination of CDR1, 2 and 3 sequences selected from the CDR1, 2 and 3 sequences shown for a V H single domain antibody in Table 1 or combinations thereof. In one embodiment, the V H single domain antibody comprises a set of CDR1, 2 and 3 sequences selected from the sets of CDR1, 2 and 3 sequences as shown for the any of the clones in Table 1. Thus, in one aspect, the first single domain antibody comprises a CDR1, CDR2 and CDR3 selected from CDRs1-3 of full length sequences SEQ ID No: 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, 80, 104, 108, 112, 116, 120, 124, 128, 132, 136, 140, 144, 148, 152, 156, 160, 164, 168, 172, 176, 180, 184, 188, 192, 196, 200, 204, 208, 212, 216, 220, 254, 258, 262, 266, 270, 274, 278, 282, 286, 290, 294, 298, 302, 306, 310, 314, 318, 322, 326, 330, 334, 338, 342, 346, 350, 354, 358, 362, 366, 370, 374, 378, 382, 386, 390, 394, 398, 402, 406, 410, 414, 418, 422, 426, 430, 434, 438, 442, 446, 450, 454, 458 or 462.

Accordingly, in one embodiment, the V H single domain antibody comprises CDR1 having SEQ ID No. 1, CDR2 having SEQ ID No. 2 and CDR3 having SEQ ID No. 3 (CDRs of SEQ ID NO. 4, i.e. clone 1.1a as shown in table 1), CDR1 having SEQ ID No. 5, CDR2 having SEQ ID No. 6 and CDR3 having SEQ ID No. 7 (CDRs of SEQ ID NO. 8) and so forth. Thus, the first V H single domain antibody comprises one of the following CDR combinations: SEQ ID Nos. 1, 2, 3; SEQ ID Nos. 5, 6, 7; SEQ ID Nos. 9, 10, 11; SEQ ID Nos. 13, 14, 15; SEQ ID Nos. 17, 18, 19; SEQ ID Nos. 21, 22, 23; SEQ ID Nos. 25, 26, 27; SEQ ID Nos. 29, 30, 31; SEQ ID Nos. 33, 34, 35; SEQ ID Nos. 37, 38, 39; SEQ ID Nos. 41, 42, 43, SEQ ID Nos. 45, 46, 47; SEQ ID Nos. 49, 50, 51; SEQ ID Nos. 53, 54, 55; SEQ ID Nos. 57, 58, 59; SEQ ID Nos. 61, 62, 63; SEQ ID Nos. 65, 66, 67; SEQ ID Nos. 69, 70, 71; SEQ ID Nos.73. 74. 75; SEQ ID Nos. 77, 78, 79; SEQ ID Nos. 101, 102, 103; SEQ ID Nos. 105, 106, 107; SEQ ID Nos. 109, 110, 111; SEQ ID Nos. 113, 114, 115; SEQ ID Nos. 117, 118, 119; SEQ ID Nos. 121, 122, 123; SEQ ID Nos. 125, 126, 127; SEQ ID Nos. 129; 130; 131; SEQ ID Nos. 133, 134, 15; SEQ ID Nos. 137, 138, 139; SEQ ID Nos. 141, 142, 143; SEQ ID Nos. 145, 146, 147; SEQ ID Nos. 149, 150, 151; SEQ ID Nos. 153, 154, 155; SEQ ID Nos. 157, 158, 159; SEQ ID Nos. 161, 162, 163; SEQ ID Nos. 165, 166, 167; SEQ ID Nos. 169, 170, 171; SEQ ID Nos. 173, 174, 175; SEQ ID Nos. 177, 178, 179; SEQ ID Nos. 181, 182, 183; SEQ ID Nos. 185, 186, 187; SEQ ID Nos. 189, 190, 191; SEQ ID Nos. 193, 194, 195; SEQ ID Nos. 197, 198, 199; SEQ ID Nos. 201, 202, 203; SEQ ID Nos. 205, 206, 207; SEQ ID Nos. 209, 210, 211; SEQ ID Nos. 213, 214, 215; SEQ ID Nos. 217, 218, 219; SEQ ID Nos. 251, 252, 253; SEQ ID Nos. 255, 256, 257; SEQ ID Nos. 259, 260, 261; SEQ ID Nos. 263, 264, 265; SEQ ID Nos. 267, 268, 269; SEQ ID Nos. 271, 272, 273; SEQ ID Nos. 275, 276, 277; SEQ ID Nos. 279, 280, 281; SEQ ID Nos. 283, 284, 285; SEQ ID Nos. 287, 288, 289; SEQ ID Nos. 291, 292, 293; SEQ ID Nos. 295, 296, 297; SEQ ID Nos. 299, 300, 301; SEQ ID Nos. 303, 304, 305; SEQ ID Nos. 307, 308, 309; SEQ ID Nos. 311, 312, 313; SEQ ID Nos. 315, 316, 317; SEQ ID Nos. 319, 320, 321, SEQ ID Nos. 323, 324, 325; SEQ ID Nos. 327, 328, 329; SEQ ID Nos. 331, 332, 333; SEQ ID Nos. 335, 336, 337; SEQ ID Nos. 339, 340, 341; SEQ ID Nos. 343, 344, 345; SEQ ID Nos. 347, 348, 349; SEQ ID Nos. 351, 352, 353; SEQ ID Nos. 355, 356, 357; SEQ ID Nos. 359, 360, 361; SEQ ID Nos. 363, 364, 365; SEQ ID Nos. 367, 368, 369; SEQ ID Nos. 371, 372, 373; SEQ ID Nos. 375, 376, 377; SEQ ID Nos. 379, 380, 381; SEQ ID Nos. 383, 384, 385; SEQ ID Nos. 387, 388, 389; SEQ ID Nos. 391, 392, 393; SEQ ID Nos. 395, 396, 397; SEQ ID Nos. 399, 400, 401; SEQ ID Nos. 403, 404, 405; SEQ ID Nos. 407, 408, 409; SEQ ID Nos. 411, 412, 413; SEQ ID Nos. 415, 416, 417; SEQ ID Nos. 419, 420, 421; SEQ ID Nos. 423, 424, 425; SEQ ID Nos. 427, 428, 429; SEQ ID Nos. 431, 432, 433; SEQ ID Nos. 435, 436, 437; SEQ ID Nos. 439, 440, 441; SEQ ID Nos. 443, 444, 445; SEQ ID Nos. 447, 448, 449; SEQ ID Nos. 451, 452, 453; SEQ ID Nos. 455, 456, 457 or SEQ ID Nos. 459, 460, 461.

In another embodiment, said CDR1 comprises or consists of the amino acid sequence SEQ ID No. 1 or 251 or a sequence with at least at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% 96%, 97%, 98% or 99% homology thereto. In one embodiment, said CDR2 comprises or consists of the amino acid sequence SEQ ID No. 2 or 252 or a sequence with at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% 96%, 97%, 98% or 99% homology thereto. In one embodiment, said CDR3 comprises or consists of the amino acid sequence SEQ ID No. 3, 176 or 253 or a sequence with at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% 96%, 97%, 98% or 99% homology thereto.

In another embodiment, the first V H single domain antibody comprises or consists of a polypeptide sequence as shown for any one of V H single domain antibodies 1.1a to 1.103a as shown in Table 1 or a sequence with at least 60%, 70%, 80%, 90%, 95% or more sequence homology thereto. Thus, the V H single domain antibody comprises or consists of an amino acid sequence selected from SEQ ID Nos. 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, 80, 104, 108, 112, 116, 120, 124, 128, 132, 136, 140, 144, 148, 152, 156, 160, 164, 168, 172, 176, 180, 184, 188, 192, 196, 200, 204, 208, 212, 216, 220, 254, 258, 262, 266, 270, 274, 278, 282, 286, 290, 294, 298, 302, 306, 310, 314, 318, 322, 326, 330, 334, 338, 342, 346, 350, 354, 358, 362, 366, 370, 374, 378, 382, 386, 390, 394, 398, 402, 406, 410, 414, 418, 422, 426, 430, 434, 438, 442, 446, 450, 454, 458 or 462 or a sequence with at least 60%, 70%, 80%, 90%, 95% or more sequence homology thereto. the first V H single domain antibody comprises or consists of a polypeptide sequence as shown for any one of V H single domain antibodies 1.20a to1.50a or1.51a and 1.62a to 1.103a as shown in Table 1 or a sequence with at least 60%, 70%, 80%, 90%, 95% or more sequence homology thereto. In one embodiment, the V H single domain antibody comprises or consists of SEQ ID No. 4, 176, 254, 298 or 446 a sequence with at least 60%, 70%, 80%, 90%, 95% or more sequence homology thereto.

In one embodiment, said sequence homology as above is at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.

In one embodiment, the second single V H domain antibody that binds to human PD-1 and blocks ligand binding comprises a CDR3 sequence as shown Table 2 below or a sequence with at least 60%, 70%, 80%, 90%, 95% or more sequence identity thereto. In one embodiment, said sequence homology is at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. In one embodiment, said sequence homology is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.

In one embodiment, the second V H single domain antibody has a CDR3 sequence comprising SEQ ID No. 518 or a sequence having at least 60%, 70%, at least 80%, at least 90%, or at least 95% homology to SEQ ID No. 518. In one embodiment, the V H single domain antibody has a CDR3 sequence comprising SEQ ID No. 955 or a sequence having at least 70%, at least 80%, at least 90%, or at least 95% homology to SEQ ID No. 955. In one embodiment, said sequence homology to SEQ ID No. 518 or 955 is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.

In one embodiment, the second V H single domain antibody has a CDR1 as shown in SEQ ID No. 516 or SEQ ID No. 516 with 1 or 2 amino acid substitutions, a CDR2 as shown in SEQ ID No.517 or SEQ ID No. 517 with 1 to 5 amino acid substitutions and a CDR3 as shown in SEQ ID No. 518 or SEQ ID No. 518 with 1 to 5 amino acid substitutions. In one embodiment, the V H single domain antibody has a CDR1 as shown in SEQ ID No. 740 or SEQ ID No. 740 with 1 or 2 amino acid substitutions, a CDR2 as shown in SEQ ID No. 741 or SEQ ID No. 741 with 1 to 5 amino acid substitutions and a CDR3 as shown in SEQ ID No. 742 or SEQ ID No. 742 with 1 to 5 amino acid substitutions.

In one embodiment, the first V H single domain antibody comprises a combination of CDR1, 2 and 3 sequences selected from the CDR1, 2 and 3 sequences of a V H single domain antibody in Table 2 or combinations thereof. In one embodiment, the V H single domain antibody comprises a set of CDR1, 2 and 3 sequences selected from the sets of CDR1, 2 and 3 sequences as shown for the any of the full length V H in Table 2. Thus, in one aspect, the first single domain antibody comprises a CDR1, CDR2 and CDR3 selected from CDRs1-3 of full length sequences SEQ ID No: 519, 523, 527, 531, 535, 539, 543, 547, 551, 555, 559, 563, 567, 571, 575, 579, 583, 587, 591, 595, 599, 603, 607, 611, 615, 619, 623, 627, 631, 635, 639, 643, 647, 651, 655, 659, 663, 667, 671, 675, 679, 683, 687, 691, 695, 699, 703, 707, 711, 715, 719, 723, 727, 731, 735, 739, 743, 747, 751, 755, 759, 763, 767, 771, 775, 779, 783, 787, 791, 795, 799, 803, 807, 884, 888, 892, 896, 900, 904, 908, 912, 916, 920, 924, 928, 932, 936, 940, 944, 948, 952, 956, 960, 964, 968, 972, 976, 980, 984, 988, 992, 996, 1000, 1004, 1008, 1012, 1016, 1020, 1024, 1028, 1032, 1036, 1040, 1044 or 1048.

Accordingly, in one embodiment, the second V H single domain antibody comprises CDR1 having SEQ ID No. 516, CDR2 having SEQ ID No. 517 and CDR3 having SEQ ID No. 518 (CDRs of SEQ ID NO. 519, i.e. clone 1.1b as shown in table b) and so forth. Thus, the second V H single domain antibody comprises one of the following CDR combinations: SEQ ID Nos. 516, 517, 518; SEQ ID Nos. 520, 521, 522; SEQ ID Nos. 524, 525, 526; SEQ ID Nos. 528, 529, 530; SEQ ID Nos. 532, 533, 534; SEQ ID Nos. 536, 537, 538; SEQ ID Nos. 540, 541, 542; SEQ ID Nos. 544, 545, 546; SEQ ID Nos.548, 549, 550; SEQ ID Nos. 552, 553, 554; SEQ ID Nos. 556, 557, 558, SEQ ID Nos. 560, 561, 562; SEQ ID Nos. 564, 565, 566; SEQ ID Nos. 568, 569, 570; SEQ ID Nos. 572, 573, 574; SEQ ID Nos. 576, 577, 578; SEQ ID Nos.580, 581, 582; SEQ ID Nos. 584, 584, 586; SEQ ID Nos.588, 589, 590; SEQ ID Nos. 592, 593, 594; SEQ ID Nos. 596, 597, 598; SEQ ID Nos. 600, 601, 602; SEQ ID Nos. 604, 605, 606 SEQ ID Nos. 608, 609, 610; SEQ ID Nos. 612, 613, 614; SEQ ID Nos. 616, 617, 618; SEQ ID Nos. 620, 621, 622; SEQ ID Nos. 624, 625, 626; SEQ ID Nos. 628, 629, 630; SEQ ID Nos. 632, 633, 634; SEQ ID Nos. 636, 637, 638; SEQ ID Nos. 640, 641, 642; SEQ ID Nos. 644, 645, 646; SEQ ID Nos. 648, 649, 650; SEQ ID Nos. 652, 653, 654; SEQ ID Nos. 656, 657, 658; SEQ ID Nos. 660, 661, 662; SEQ ID Nos. 664, 665, 666; SEQ ID Nos. 668, 669, 670; SEQ ID Nos. 672, 673, 674; SEQ ID Nos. 676, 677, 678; SEQ ID Nos. 680, 681, 682; SEQ ID Nos. 684, 685, 686; SEQ ID Nos. 688, 689, 690; SEQ ID Nos. 692, 694, 694; SEQ ID Nos. 696, 697, 698; SEQ ID Nos. 700, 701, 702; SEQ ID Nos. 704, 705, 706; SEQ ID Nos. 708, 709, 710; SEQ ID Nos. 712, 713, 714; SEQ ID Nos. 716, 717, 718; SEQ ID Nos. 720, 721, 722; SEQ ID Nos. 724, 725, 726; SEQ ID Nos. 728, 729, 730; SEQ ID Nos. 732, 733, 734; SEQ ID Nos. 736, 737, 738; SEQ ID Nos. 740, 741, 742; SEQ ID Nos. 744, 745, 746; SEQ ID Nos. 748, 749, 750; SEQ ID Nos. 752, 753, 754; SEQ ID Nos. 756, 757, 758; SEQ ID Nos. 760, 761, 762; SEQ ID Nos. 764, 765, 766; SEQ ID Nos. 768, 769, 770; SEQ ID Nos. 772, 773, 774; SEQ ID Nos. 776, 777, 778; SEQ ID Nos. 780, 781, 782, SEQ ID Nos. 784, 785, 786; SEQ ID Nos. 788, 789, 790; SEQ ID Nos. 792, 793, 794; SEQ ID Nos. 796, 797, 798; SEQ ID Nos. 800, 801, 802; SEQ ID Nos. 804, 805, 806; SEQ ID Nos. 881, 882, 883; SEQ ID Nos. 885, 886, 887; SEQ ID Nos. 889, 890, 891; SEQ ID Nos. 893, 894, 895; SEQ ID Nos. 897, 898, 899; SEQ ID Nos. 901, 902, 903; SEQ ID Nos. 905, 906, 907; SEQ ID Nos. 909, 910, 911; SEQ ID Nos. 913, 914, 915; SEQ ID Nos. 917, 918, 919; SEQ ID Nos. 921, 922, 923; SEQ ID Nos. 925, 926, 927; SEQ ID Nos. 929, 930, 931; SEQ ID Nos. 933, 934, 935; SEQ ID Nos. 937, 938, 939; SEQ ID Nos. 941, 942, 943; SEQ ID Nos. 945, 946, 947; SEQ ID Nos. 949, 950, 951; SEQ ID Nos. 953, 954, 955; SEQ ID Nos. 957, 958, 959; SEQ ID Nos. 961, 962, 963; SEQ ID Nos. 965, 966, 967; SEQ ID Nos. 969, 970, 971; SEQ ID Nos. 973, 974, 975; SEQ ID Nos. 977, 978, 979; SEQ ID Nos. 981, 982, 983; SEQ ID Nos. 985, 986, 987; SEQ ID Nos. 989, 990, 991; SEQ ID Nos. 993, 994, 995; SEQ ID Nos. 997, 998, 999; SEQ ID Nos. 1001, 1002, 1003; SEQ ID Nos. 1005, 1006, 1007; SEQ ID Nos. 1009, 1100, 1011; SEQ ID Nos. 1013, 1014, 1015; SEQ ID Nos. 1017, 1018, 1019; SEQ ID Nos. 1021, 1022, 1023; SEQ ID Nos. 1025, 1026, 027; SEQ ID Nos. 1029, 1030; 1031; SEQ ID Nos. 1033, 1034, 1035; SEQ ID Nos. 1037, 1038, 1039; SEQ ID Nos. 1041, 1042, 1043 or SEQ ID Nos. 1045, 1046, 1047,

In another embodiment, the second V H single domain antibody comprises or consists of a polypeptide sequence as shown for any one of V H single domain antibodies 1.1b to 1.115 bas shown in Table 2 or a sequence with at least 60%, 70%, 80%, 90%, 95% or more sequence homology thereto. Thus, the V H single domain antibody comprises or consists of an amino acid sequence selected from SEQ ID Nos. 519, 523, 527, 531, 535, 539, 543, 547, 551, 555, 559, 563, 567, 571, 575, 579, 583, 587, 591, 595, 599, 603, 607, 611, 615, 619, 623, 627, 631, 635, 639, 643, 647, 651, 655, 659, 663, 667, 671, 675, 679, 683, 687, 691, 695, 699, 703, 707, 711, 715, 719, 723, 727, 731, 735, 739, 743, 747, 751, 755, 759, 763, 767, 771, 775, 779, 783, 787, 791, 795, 799, 803, 807, 884, 888, 892, 896, 900, 904, 908, 912, 916, 920, 924, 928, 932, 936, 940, 944, 948, 952, 956, 960, 964, 968, 972, 976, 980, 984, 988, 992, 996, 1000, 1004, 1008, 1012, 1016, 1020, 1024, 1028, 1032, 1036, 1040, 1044 or 1048 or a sequence with at least 60%, 70%, 80%, 90%, 95% or more sequence homology thereto. In another embodiment, the second V H single domain antibody comprises or consists of a polypeptide sequence as shown for any one of V H single domain antibodies 1.73b to 1.115b as shown in Table 2 or a sequence with at least 60%, 70%, 80%, 90%, 95% or more sequence homology thereto.

In one embodiment, the V H single domain antibody comprises or consists of SEQ ID No. 4519, 743, 896 or 956 a sequence with at least 60%, 70%, 80%, 90%, 95% or more sequence homology thereto.

In one embodiment, said sequence homology as above is at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.

In some embodiments, the first or second V H single domain antibody used in the binding agent of the invention is a variant of any single V H domain antibodies in table 1 or 2 having one or more amino acid substitution, deletion, insertion or other amino acid modification, and which retains a biological function of the single domain antibody in the biparatopic molecule. A variant V H single domain antibody can be sequence engineered. Suitable modifications include one or more substitution, deletion or insertion of one or more codons in a nucleic acid encoding the single domain antibody or polypeptide that results in a change in the amino acid sequence as compared with the native sequence V H single domain antibody or polypeptide. Amino acid substitutions can be the result of replacing one amino acid with another amino acid having similar structural and/or chemical properties, such as the replacement of a leucine with a serine, i.e., conservative amino acid replacements. Insertions or deletions may optionally be in the range of about 1 to 5 amino acids. The variation allowed may be determined by systematically making insertions, deletions or substitutions of amino acids in the sequence and testing the resulting variants for activity exhibited by the full-length or mature native sequence. A variant of a V H single domain antibody described herein has at least 75%, 76%, 77%, 78%, 79%, 8 0%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology to the non-variant molecule, preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology.

In one embodiment, the modification is a conservative sequence modification. As used herein, the term “conservative sequence modifications” is intended to refer to amino acid modifications that do not significantly affect or alter the binding characteristics of the antibody containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into an antibody of the invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues within the CDR regions of a single domain antibody used in the invention can be replaced with other amino acid residues from the same side chain family and the altered antibody can be tested for retained function (i.e., the functions set forth in (c) through (I) above) using the functional assays described herein.

In some embodiments, the first and/or second V H single domain antibody is a variant of a single domain antibody selected from those shown in Table 1 and/or 2 that comprises one or more amino acid sequence modification and has improvements in one or more of a property such as binding affinity, specificity, thermostability, expression level, effector function, glycosylation, reduced immunogenicity, or solubility as compared to the unmodified single domain antibody.

A skilled person will know that there are different ways to identify, obtain and optimise the antigen binding molecules as described herein, including in vitro and in vivo expression libraries. This is further described in the examples. Optimisation techniques known in the art, such as display (e.g., ribosome and/or phage display) and/or mutagenesis (e.g., error-prone mutagenesis) can be used. The invention therefore also comprises sequence optimised variants of the single domain antibodies described herein.

In one embodiment, modifications can be made to decrease the immunogenicity of the single domain antibody. For example, one approach is to revert one or more framework residues to the corresponding human germline sequence. More specifically, a single domain antibody that has undergone somatic mutation may contain framework residues that differ from the germline sequence from which the single domain antibody is derived. Such residues can be identified by comparing the single domain antibody framework sequences to the germline sequences from which the single domain antibody is derived.

To return one or more of the amino acid residues in the framework region sequences to their germline configuration, the somatic mutations can be “backmutated” to the germline sequence by, for example, site-directed mutagenesis or PCR-mediated mutagenesis.

Another type of framework modification involves mutating one or more residues within the framework region, or even within one or more CDR regions, to remove T cell epitopes to thereby reduce the potential immunogenicity of the antibody.

In still another embodiment, the glycosylation of an antibody is modified. For example, an aglycoslated antibody can be made (i.e., the antibody lacks glycosylation). Glycosylation can be altered to, for example, increase the affinity of the antibody for antigen. Such carbohydrate modifications can be accomplished by, for example, altering one or more sites of glycosylation within the antibody sequence. For example, one or more amino acid substitutions can be made that result in elimination of one or more variable region framework glycosylation sites to thereby eliminate glycosylation at that site. Such aglycosylation may increase the affinity of the antibody for antigen.

Thus, these amino acid changes can typically be made without altering the biological activity, function, or other desired property of the polypeptide, such as its affinity or its specificity for antigen. In general, single amino acid substitutions in nonessential regions of a polypeptide do not substantially alter biological activity. Furthermore, substitutions of amino acids that are similar in structure or function are less likely to disrupt the polypeptides' biological activity. Abbreviations for the amino acid residues that comprise polypeptides and peptides described herein, and conservative substitutions for these amino acid residues are shown in Table 3 below.

In one embodiment, the variant first V H single domain antibody is selected from any one of the V H sequences shown in table 1 or 2, but comprises one or more amino acid substitutions, for example 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions. In one embodiment, the one or more amino acid substitution is in one or more of the framework areas. In another embodiment, the one or more amino acid substitution is in one or more of the CDRs. In one embodiment, the amino acid substitutions are in the framework and CDR sequences. In one embodiment, the first V H single domain antibody comprises or consists of SEQ ID No. 4 or a sequence which comprises one or more amino acid substitutions, for example 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions or SEQ ID No. 254 or a sequence which comprises one or more amino acid substitutions, for example 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions.

In one embodiment, the variant first V H single domain antibody comprises SEQ ID No. 4 with amino acid substitutions at one of the following positions: 5L, 32H, 44G, 55S, 66D, 77S and/or 105T. In one embodiment, the first V H single domain antibody comprises SEQ ID No. 4 (1.1a in Table 1) with amino acid substitutions selected from one of the following:

a) 5L→V, 11S→L, 32H→Y, 44G→D, 55S→G, 62D→A, 66D→G and 77S→N or b) 5L→V, 11S→L, 32H→N, 44G→D, 55S→G, 66D→G and 77S→N or c) 1E→Q, 5L→V, 11S→L, 32H→N, 44G→D, 55S→G, 66D→G, 77S→N, 98R→K, 99E→D105T→I and 102M→T.

In one embodiment, the variant first V H single domain antibody comprises SEQ ID No. 136 with amino acid substitutions at one or more or all of the following positions: M34, M58, V116, V102. In one embodiment, the first V H single domain antibody comprises SEQ ID No 136 with amino acid substitutions selected from one of the following:

a) M34→L, M58→Y, V116A (Humabody® 1.39a) b) M34→F, G54→A, M58→Q, V102A (Humabody® 1.50a) c) V116A (Humabody® 1.26a) d) M34→L, M58→Q, V116A (Humabody® 1.40a)

In one embodiment, the variant first V H single domain antibody comprises SEQ ID No. 254 with amino acid substitutions at one or more or all of the following positions: G 109, D66, G55. In one embodiment, the first V H single domain antibody comprises SEQ ID No 254 with amino acid substitutions selected from one of the following:

a) G109→D, D66→G, G55→A (Humabody® 1.62a) b) G109→D, G55→A (Humabody® 1.77a) c) G109→D (Humabody® 2.53) d) G109→D, D66→G (Humabody® 1.79a) e) G109→V, D66→G (Humabody® 1.103a)

In one embodiment, the variant second V H single domain antibody is selected from any one of the sequences shown in table 2, but comprises one or more amino acid substitutions, for example 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions. In one embodiment, the variant V H single domain antibody is selected from SEQ ID No. 519 or 743. In one embodiment, the variant V H single domain antibody is selected from SEQ ID No. 956. In one embodiment, the one or more amino acid substitution is in one or more of the framework areas. In another embodiment, the one or more amino acid substitution is in one or more of the CDRs. In one embodiment, the amino acid substitutions are in the framework and CDR sequences. In one embodiment, the second V H single domain antibody comprises or consists of SEQ ID No. 519, 743 or 771 or a sequence which comprises one or more amino acid substitutions, for example 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions.

In one embodiment, the variant second V H single domain antibody comprises SEQ ID No. 519 (Humabody® 1.1b) but with amino acid substitutions at one or more or all of the following positions: Y32, T33, T53, T56, 157, K58, Y59, T61 and/or W115.

In one embodiment, the variant second V H single domain antibody comprises SEQ ID No. 519 (1.1b as in Table 2) but with the following amino acid substitutions: Y32→N, T33→S, T53→S, T56→G, I57→V, K58→I, Y59→F, W115→S (1.57b as in Table 2).

In one embodiment, the variant second V H single domain antibody comprises SEQ ID No. 743 (Humabody® 1.57b) but with amino acid substitutions at one or more or all of the following positions: D31, N32, 158, A61, T35, S30, G56, S25Q117, M120 and/or Q1.

In one embodiment, the variant second V H single domain antibody comprises SEQ ID No. 743 (1.57b as in Table 2) but with amino acid substitutions selected from one of the following

1) D31→G, N32→S (1.67b as in Table 2); 2) S30→G, D31→G, N32→S, I58→K, A61→T (1.71b as in Table 2); 3) T35→S (1.69b as in Table 2); 4) S30→G, N32→S, G56→A, I58→K, A61→T (1.70b as in Table 2); 5) S25→T, N32→S, I58→R, A61→T (1.73b as in Table 2); 6) N32→S, I58→K, A61→T, N84→D, I114→T (1.66b as in Table 2); 7) S30→G, Q117→R, M120→T (1.68b as in Table 2); 8) N32→S, T35→S (1.72b as in Table 2); 9) N32→SG56→A, I58→K, A61→T (1.62b as in Table 2); 10) N32→S, I58→K (1.65b as in Table 2); 11) Q1→E, N32→S, I58→K (1.64b as in Table 2); 12) Q1→E, N32→S, I58→K, A61→T (1.63b as in Table 2).

In one embodiment, the variant second V H single domain antibody comprises SEQ ID No. 771 (Humabody® 1.64b) but with amino acid substitutions at one or more or all of the following positions: S32, D90 and/or A60.

In one embodiment, the variant second V H single domain antibody comprises SEQ ID No. 771 (Humabody® 1.64b) but with amino acid substitutions selected from one of the following:

1. S32→E, D90→E (Humabody® 1.92b); 2. S32→R, D90→E (Humabody® 1.99b); 3. S32→Y, D90→E (Humabody® 1.89b); 4. S32→E, D90→E, A61→T (Humabody® 1.84b); 5. D85→E A60→T (Humabody® 1.77b); 6. S32→T, D90→E A60→T (Humabody® 1.82b);

The numbering above reflects the actual position of the residue in the protein.

The binding agents of the invention preferably have KD and EC 50 values as further described herein and as shown in the examples.

The term “KD” refers to the “equilibrium dissociation constant” and refers to the value obtained in a titration measurement at equilibrium, or by dividing the dissociation rate constant (Koff) by the association rate constant (Kon). “KA” refers to the affinity constant. The association rate constant, the dissociation rate constant and the equilibrium dissociation constant are used to represent the binding affinity of an antibody to an antigen. Methods for determining association and dissociation rate constants are well known in the art. Using fluorescence-based techniques offers high sensitivity and the ability to examine samples in physiological buffers at equilibrium. Other experimental approaches and instruments such as a BIAcore® (biomolecular interaction analysis) assay can be used.

In one embodiment, a single domain antibody/binding agent according to the invention has a KD value in the nanomolar to picomolar range.

In one embodiment, the biparatopic binding agent of the invention comprising a first non-neutralising V H single domain antibody and a second neutralising V H single domain antibody provides improved properties compared to said monovalent neutralising single domain antibody. For example, the biparatopic binding agent may have an enhanced, additive or synergistic effect. In another embodiment, blocking activity a biparatopic binding molecule is enhanced 10 to 25 fold, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25-fold compared to the monovalent blocking molecule. In another embodiment, tumour reduction can be enhanced, for example compared to a clinically validated benchmark antibody.

As further detailed in the examples, the inventors have observed that a multiparatopic binding agent as described herein exhibits beneficial properties, including the following: The binding agent

elicits average increases in CD4+ T cells, CD8+ T cells, with a decline in T regulatory cells and macrophages in a HuGEMM PD1 Model with Subcutaneous MC38 Mouse Colon Adenocarcinoma compared to a negative (non-PD-1-binding) control; elicits average increases of cytokine release within said tumours, for example, interferon-gamma, IL-1α, IL-1β, IL-5, IL-6 and MIP-1α are increased relative to a negative control and a human PD-1 antibody treatment; does not show average elevation of IL-6, IL-5, IL-12 and IL-1β in the serum relative to a human PD-1 antibody (which shows a mean increase of these cytokines compared to a negative control) treatment following treatment in a HuGEMM PD1 Model with Subcutaneous MC38 Mouse Colon Adenocarcinoma.

The present invention further provides an isolated nucleic acid construct encoding a binding agent of the present invention.

A nucleic acid sequences encoding a first single domain antibody is linked to a second nucleic acid encoding a second single domain antibody using for example a (G4S)n linker as described in the example. Nucleic acid sequences encoding a first single domain antibody are selected from SEQ ID Nos. SEQ ID Nos. 81 to 100 or 221 to 250 or 463 to 515. Nucleic acid sequences encoding a second single domain antibody are selected from SEQ ID Nos. 808 to 880 or 1049 to 1090.

In one embodiment, the nucleic acid sequence has at least 60%, 70%, 80%, 90%, 95% or more sequence homology to one of the nucleic acid sequences above. In one embodiment, said sequence homology is at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.

In one embodiment, the binding molecule is selected from one of the following: 1.1b-(G4S)n-1.1a (SEQ ID NO. 519 linked to SEQ ID NO.4 with G4S linker); 1.57b-(G4S)n-1.1a (SEQ ID NO. 743 linked to SEQ ID NO.4 with G4S linker); 1.92b-(G4S)n-1.51a (SEQ ID NO. 956 linked to SEQ ID NO.254 with G4S linker); 1.92b-(G4S)n-1.62a (SEQ ID NO. 956 linked to SEQ ID NO. 298 with G4S linker); 1.92b-(G4S)n-1.99a (SEQ ID NO. 956 linked to SEQ ID NO.446 with G4S linker); 1.77b-(G4S)n-1.21a (SEQ ID NO. 896 linked to SEQ ID NO. 104 with G4S linker). For example, n=1, 2, 3, 4, 5, 6, 7, 8, 9, 10.

In one embodiment, the binding molecule is selected from one of the following: 1.1b-2GS-1.1a; 1.1b-4GS-1.1a; 1.1b-6G5-1.1a; 1.57b-4GS-1.1a; 1.57b-4GS-1.1a-4GS-MSA binder; 1.57-4GS-MSA binder-4GS-1.1a; 1.92b-4GS-1.51a; 1.92b-4GS-1.62a SEQ ID NO. 1114); 1.92b-4GS-1.39a (SEQ ID NO. 1112); 1.92b-4GS-1.99a; 1.77b-4GS-1.21a (SEQ ID NO. 1116); 1.77b-1GS-1.21a (SEQ ID NO. 1118) or 1.77b-2GS-1.21a (SEQ ID NO. 1120).

A nucleic acid according to the present invention may comprise DNA or RNA and may be wholly or partially synthetic or recombinantly produced. Reference to a nucleotide sequence as set out herein encompasses a DNA molecule with the specified sequence, and encompasses a RNA molecule with the specified sequence in which U is substituted for T, unless context requires otherwise.

Furthermore, the invention relates to a nucleic acid construct comprising at least one nucleic acid as defined above. The construct may be in the form of a plasmid, vector, transcription or expression cassette.

The invention also relates to an isolated recombinant host cell comprising one or more nucleic acid construct as described above. The host cell may be a bacterial, viral, yeast, insect, plant mammalian or other suitable host cell. In one embodiment, the cell is an E. coli cell. In another embodiment, the cell is a yeast cell. In another embodiment, the cell is a Chinese Hamster Ovary (CHO) cell.

In an embodiment, a method of making an anti-PD-1 binding agent as described herein is provided, wherein the method comprises culturing the host cell under conditions suitable for expression of the polynucleotide encoding the binding agent, and isolating the binding agent.

In another aspect, the invention provides a binding molecule comprising a) a PD-1 binding agent that binds to the same epitope on human PD-1 as any of the PD-1 first single domain antibodies described herein and b) a PD-1 binding agent that binds to the same epitope on human PD-1 as any of the PD-1 second single domain antibodies described herein.

Cross-competing antibodies can be identified based on their ability to cross-compete with a first single domain antibody selected from 1.1a to 1.103a in Table 1 or a second single domain antibody selected from 1.1b to 1.115b in Table 2 respectively in standard PD-1 binding assays. For example, BIAcore analysis, ELISA assays or flow cytometry may be used to demonstrate cross-competition with the single domain antibodies of the current invention.

In one aspect, the invention also relates to a combination or composition comprising a first single domain antibody as described herein and a second single domain antibody as described herein. In one aspect, the invention also relates to a combination therapy comprising administering first single domain antibody as described herein and a second single domain antibody as described herein. Administration may be simultaneously, separately or sequentially.

In one aspect, the binding agent of the invention comprises at least one further moiety. In one embodiment, the further moiety is a binding molecule, for example selected from an antibody or antibody fragment (e.g., a Fab, F(ab′)2, Fv, a single chain Fv fragment (scFv) or single domain antibody, for example a V H domain) or antibody mimetic protein. In one embodiment, the further moiety is a V H domain. In one embodiment, the binding agent of the invention can be linked to an antibody Fc region or fragment thereof, comprising one or both of C H 2 and C H 3 domains, and optionally a hinge region.

In one embodiment, the PD-1 inhibitor is a V H single domain antibody.

Multiparatopic, for example biparatopic, and multivalent binding molecules of the present invention can be constructed using methods known in the art.

In certain embodiments, the binding agent is in the form of a multispecific, for example bispecific, binding agent providing multiple functionalities. Such multispecific agent comprises a single domain antibody that has a first binding specificity to PD-1 and at least one further binding molecule with a second binding specificity. Said further binding molecule can be selected from an antibody, an antibody fragment or antibody mimetic. In one embodiment, said antibody fragment is selected from F(ab′) 2 , Fab, Fv, sFv or domain antibody. In one embodiment, said antibody fragment is a V H single domain antibody.

In one embodiment, the binding agent is bispecific and comprises a biparatopic binding agent according to the invention that has a first binding and second specificity to PD-1 and further comprises a third binding molecule with a third binding specificity to a target other than PD-1. In one embodiment, the third binding molecule binds to an immunomodulatory agent, a checkpoint modulator, an agent involved in T-cell activation, a tumor microenvironment modifier (TME) or a tumour-specific target.

For example, the immunomodulator can be an inhibitor of an immune checkpoint molecule selected from an inhibitor of one or more of PD-L1, PD-L2, CTLA-4, TIM-3, LAG-3, CEACAM, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 or TGFR beta. In another embodiment, the immunomodulator can be an activator of a costimulatory molecule selected from an agonist of one or more of IL-2, II-12, OX40, OX40L, CD2, CD3, CD27, CDS, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD30, CD40, BAFFR, HVEM, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3, B7-H4 or CD83 ligand, CD3, CD8, CD28, CD4 or ICAM-1.

In one embodiment, the binding agent described above comprises further binding molecules. Thus, the binding agent can for example be trispecific or tetraspecific. Additional specificities are also envisaged. Any combination of the aforesaid molecules can be made in a multispecific binding agent, for example, a trispecific binding agent that includes a binding agent of the invention and a second or third binding specificity.

In another embodiment, the further moiety may serve to prolong the half-life of the binding molecule. The further moiety may comprise a protein, for example an antibody, or part thereof that binds a serum albumin, e.g., human serum albumin (HSA) or mouse serum albumin (MSA). The further moiety may comprise a V H domain that binds serum albumin, e.g., human serum albumin (HSA) or mouse serum albumin (MSA).

The further moiety may comprise a serum albumin, e.g. a human serum albumin (HSA) or a variant thereof such as HSA C34S. Further provided is binding molecule as described herein comprising a V H domain and an Fc domain, e.g., wherein the V H domain is fused to an Fc domain. Further provided is a binding molecule that comprises a third variable domain that specifically binds a third antigen, where the third antigen is an antigen other than human PD-1. The third antigen may be a cluster of differentiation (CD) molecule or a Major Histocompatibility Complex (MHC) Class II molecule.

In one embodiment, the binding agents of the invention are labelled with a detectable or functional label. A label can be any molecule that produces or can be induced to produce a signal, including but not limited to fluorophores, fluorescers, radiolabels, enzymes, chemiluminescers, a nuclear magnetic resonance active label or photosensitizers. Thus, the binding may be detected and/or measured by detecting fluorescence or luminescence, radioactivity, enzyme activity or light absorbance.

In still other embodiments, the binding agents of the invention are coupled to at least one therapeutic moiety, such as a drug, an enzyme or a toxin. In one embodiment, the therapeutic moiety is a toxin, for example a cytotoxic radionuclide, chemical toxin or protein toxin.

In another aspect, the multivalent binding agents of the invention are modified to increase half-life, for example by a chemical modification, especially by PEGylation, or by incorporation in a liposome or using a serum albumin protein.

Half-life may be increased by at least 1.5 times, preferably at least 2 times, such as at least 5 times, for example at least 10 times or more than 20 times, greater than the half-life of the corresponding V H single domain antibodies of the invention. For example, increased half-life may be more than 1 hours, preferably more than 2 hours, more preferably more than 6 hours, such as more than 12 hours, or even more than 24, 48 or 72 hours, compared to the corresponding V H single domain antibodies of the invention.

To generate a multivalent binding agents as described above, two binding molecules are connected by a linker, for example a polypeptide linker. Suitable linkers include for example a linker with GS residues such as (Gly 4 Ser)n, where n=from 1 to 20 or 1 to 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In one embodiment n is 1, 2 or 3. In one embodiment n is 4 or more.

In one embodiment, the binding molecules can be used to induce PD-1 agonism. PD-1 signalling is induced by PD-L1 and leads to downregulation of T cell activity. One could say that something that induces PD-1 signalling is a ‘PD1 agonist’, which has contrasting effect to a T cell agonist. We provide evidence of a PD-1 engager that is capable of causing PD1 agonism in absence of a CD3 or T cell receptor clustering antibody.

An exemplary system to measure PD-1 agonism is a reporter cell line that shows a response when PD-1 signalling occurs. Thus, a binding molecule as described herein can provide agonistic function. Another aspect therefore relates to the use of a binding molecule described herein as an agonist.

A biparatopic molecule can enhance receptor cross-linking. A biparatopic molecule binds two epitopes on a single PD-1 monomer. Alternatively it can engage one arm with one PD1 molecule and the other arm with a second molecule. The second molecule still has an available epitope for a further biparatopic to engage and so a ‘chain’ of PD1 molecules can be clustered together. A shorter linker than (Gly 4 Ser) 4 can prevent binding to a single molecule and can encourage cross-linking, thereby permitting PD1 agonism. In one embodiment, we provide a multivalent molecule, ideally with non-antagonistic function, joined by linkers of less than (Gly 4 Ser) 4 , preferably with at least 2 epitopes. In one embodiment n is 1, 2 or 3.

Our data shows a biparatopic format containing an antagonist and an anchor. Although this can induce PD-1 signalling, there is sufficient masking of the ligand-binding epitope to allow it to block ligand-induced signalling and have an antagonistic function.

A PD-1 agonist as described above is useful in the treatment of autoimmune and/or inflammatory and/or infectious diseases. Thus, another aspect relates to a PD-1 agonist as described above for use in the treatment of autoimmune and/or inflammatory and/or infectious diseases as well as methods for use in the treatment of autoimmune and/or inflammatory and/or infectious diseases comprising administration of a PD-1 agonist as described above.

Thus, there is provided an agonistic molecule comprising a V H single domain antibody which does not block the interaction between PD-1 and its ligands as described herein and a V H single domain antibody which blocks the interaction between PD-1 and its ligands as described herein connected with a linker selected from (Gly 4 Ser)n wherein n is 1, 2 or 3.

A binding agent described herein can be obtained by obtaining V H single domain antibodies from a transgenic rodent that expresses heavy chain only antibodies upon stimulation with a PD-1 antigen. The transgenic rodent, for example a mouse, preferably has a reduced capacity to express endogenous antibody genes. Thus, in one embodiment, the rodent has a reduced capacity to express endogenous light and/or heavy chain antibody genes. The rodent may therefore comprise modifications to disrupt expression of endogenous kappa and lambda light and/or heavy chain antibody genes so that no functional light and/or heavy chains are produced, for example as further explained below.

Human heavy chain only antibodies capable of binding human PD-1 that can be used in the biding agents of the invention can be produced by a method comprising

a) immunising a transgenic rodent with an PD-1 antigen wherein said rodent expresses a nucleic acid construct comprising unrearranged human heavy chain V genes and is not capable of making functional endogenous light or heavy chains, b) isolating human heavy chain only antibodies.

A single V H domain antibody capable of binding human PD-1 that can be used in the binding agents of the invention can be produced by a method comprising

c) immunising a transgenic rodent with an PD-1 antigen wherein said rodent expresses a nucleic acid construct comprising unrearranged human heavy chain V genes and is not capable of making functional endogenous light or heavy chains, d) generating a library of sequences comprising V H domain sequences from said mouse and e) isolating sequences comprising V H domain sequences from said libraries.

Further steps may include identifying a single V H domain antibody or heavy chain only antibody that binds to human PD-1 but does not block/blocks the interaction of PD-1 and PD-L1 and isolating said antibody, for example by using functional assays as shown in the examples.

Methods for preparing or generating the polypeptides, nucleic acids, host cells, products and compositions described herein using in vitro expression libraries can comprise the steps of:

a) providing a set, collection or library of nucleic acid sequences encoding amino acid sequences;

b) screening said set, collection or library for amino acid sequences that can bind to/have affinity for PD-1 and

c) isolating the amino acid sequence(s) that can bind to/have affinity for PD-1.

In the above method, the set, collection or library of amino acid sequences may be displayed on a phage, phagemid, ribosome or suitable micro-organism (such as yeast), such as to facilitate screening. Suitable methods, techniques and host organisms for displaying and screening (a set, collection or library of) amino acid sequences will be clear to the person skilled in the art (see for example Phage Display of Peptides and Proteins: A Laboratory Manual, Academic Press; 1st edition (Oct. 28, 1996) Brian K. Kay, Jill Winter, John M cCafferty).

Libraries, for example phage libraries, are generated by isolating a cell or tissue expressing an antigen-specific, heavy chain-only antibody, cloning the sequence encoding the VH domain(s) from mRNA derived from the isolated cell or tissue and displaying the encoded protein using a library. The V H domain(s) can be expressed in bacterial, yeast or other expression systems.

In the various aspects and embodiment of the invention as out herein, the term rodent may relate to a mouse or a rat.

In one embodiment, the rodent is a mouse. The mouse may comprise a non-functional endogenous lambda light chain locus. Thus, the mouse does not make a functional endogenous lambda light chain. In one embodiment, the lambda light chain locus is deleted in part or completely or rendered non-functional through insertion, inversion, a recombination event, gene editing or gene silencing. For example, at least the constant region genes C1, C2 and C3 may be deleted or rendered non-functional through insertion or other modification as described above. In one embodiment, the locus is functionally silenced so that the mouse does not make a functional lambda light chain.

Furthermore, the mouse may comprise a non-functional endogenous kappa light chain locus. Thus, the mouse does not make a functional endogenous kappa light chain. In one embodiment, the kappa light chain locus is deleted in part or completely or rendered non-functional through insertion, inversion, a recombination event, gene editing or gene silencing. In one embodiment, the locus is functionally silenced so that the mouse does not make a functional kappa light chain.

The mouse having functionally-silenced endogenous lambda and kappa L-chain loci may, for example, be made as disclosed in WO 2003/000737, which is hereby incorporated by reference in its entirety.

Furthermore, the mouse may comprise a non-functional endogenous heavy chain locus. Thus, the mouse does not make a functional endogenous heavy chain. In one embodiment, the heavy chain locus is deleted in part or completely or rendered non-functional through insertion, inversion, a recombination event, gene editing or gene silencing. In one embodiment, the locus is functionally silenced so that the mouse does not make a functional heavy chain.

For example, as described in WO 2004/076618 (hereby incorporated by reference in its entirety), all 8 endogenous heavy chain constant region immunoglobulin genes (μ, δ, γ3, γ1, γ2a, γ2b, ε and α) are absent in the mouse, or partially absent to the extent that they are non-functional, or genes δ, γ3, γ1, γ2a, γ2b and ε are absent and the flanking genes μ and α are partially absent to the extent that they are rendered non-functional, or genes μ, δ, γ3, γ1, γ2a, γ2b and ε are absent and α is partially absent to the extent that it is rendered non-functional, or δ, γ3, γ1, γ2a, γ2b, ε and α are absent and μ is partially absent to the extent that it is rendered non-functional. By deletion in part is meant that the endogenous locus gene sequence has been deleted or disrupted, for example by an insertion, to the extent that no functional endogenous gene product is encoded by the locus, i.e., that no functional product is expressed from the locus. In another embodiment, the locus is functionally silenced.

In one embodiment, the mouse comprises a non-functional endogenous heavy chain locus, a non-functional endogenous lambda light chain locus and a non-functional endogenous kappa light chain locus. The mouse therefore does not produce any functional endogenous light or heavy chains. Thus, the mouse is a triple knockout (TKO) mouse.

The transgenic mouse may comprise a vector, for example a Yeast Artificial Chromosome (YAC) for expressing a heterologous, preferably a human, heavy chain locus. YACs are vectors that can be employed for the cloning of very large DNA inserts in yeast. As well as comprising all three cis-acting structural elements essential for behaving like natural yeast chromosomes (an autonomously replicating sequence (ARS), a centromere (CEN) and two telomeres (TEL)), their capacity to accept large DNA inserts enables them to reach the minimum size (150 kb) required for chromosome-like stability and for fidelity of transmission in yeast cells. The construction and use of YACs is well known in the art (e.g., Bruschi, C. V. and Gjuracic, K. Yeast Artificial Chromosomes, Encyclopedia of Life Sciences, 2002 Macmillan Publishers Ltd, Nature Publishing Group).

For example, the YAC may comprise a plethora of unrearranaged human V H , D and J genes in combination with mouse immunoglobulin constant region genes lacking C H 1 domains, mouse enhancer and regulatory regions. The human V H , D and J genes are human V H , D and J loci and they are unrearranged genes that are fully human. An example of such a YAC is provided in the example section.

Alternative methods known in the art may be used for deletion or inactivation of endogenous mouse or rat immunoglobulin genes and introduction of human V H , D and J genes in combination with mouse immunoglobulin constant region genes lacking C H 1 domains, mouse enhancer and regulatory regions.

Transgenic mice can be created according to standard techniques as illustrated in the examples. The two most characterised routes for creating transgenic mice are via pronuclear microinjection of genetic material into freshly fertilised oocytes or via the introduction of stably transfected embryonic stem cells into morula or blastocyst stage embryos. Regardless of how the genetic material is introduced, the manipulated embryos are transferred to pseudo-pregnant female recipients where pregnancy continues and candidate transgenic pups are born.

The main differences between these broad methods are that ES clones can be screened extensively before their use to create a transgenic animal. In contrast, pronuclear microinjection relies on the genetic material integrating to the host genome after its introduction and, generally speaking, the successful incorporation of the transgene cannot be confirmed until after pups are born.

There are many methods known in the art to both assist with and determine whether successful integration of transgenes occurs. Transgenic animals can be generated by multiple means including random integration of the construct into the genome, site-specific integration, or homologous recombination. There are various tools and techniques that can be used to both drive and select for transgene integration and subsequent modification including the use of drug resistance markers (positive selection), recombinases, recombination-mediated cassette exchange, negative selection techniques, and nucleases to improve the efficiency of recombination. Most of these methods are commonly used in the modification of ES cells. However, some of the techniques may have utility for enhancing transgenesis mediated via pronuclear injection.

Further refinements can be used to give more efficient generation of the transgenic line within the desired background. As described above, in preferred embodiments, the endogenous mouse immunoglobulin expression is silenced to permit sole use of the introduced transgene for the expression of the heavy-chain only repertoire that can be exploited for drug discovery. Genetically-manipulated mice, for example TKO mice that are silenced for all endogenous immunoglobulin loci (mouse heavy chain, mouse kappa chain and mouse lambda chain) can be used as described above. The transfer of any introduced transgene to this TKO background can be achieved via breeding, either conventional or with the inclusion of an IVF step to give efficient scaling of the process. However, it is also possible to include the TKO background during the transgenesis procedure. For example, for microinjection, the oocytes may be derived from TKO donors. Similarly, ES cells from TKO embryos can be derived for use in transgenesis.

Triple knock-out mice into which transgenes have been introduced to express immunoglobulin loci are referred to herein as TKO/Tg.

In one embodiment, the mouse is as described in WO2016/062990.

In another aspect of the present invention, there is provided a pharmaceutical composition comprising a binding agent or composition according to the present invention and optionally a pharmaceutically acceptable carrier. A binding agent of the present invention or the pharmaceutical composition of the invention can be administered by any convenient route, including but not limited to oral, topical, parenteral, sublingual, rectal, vaginal, ocular, intranasal, pulmonary, intradermal, intravitrial, intramuscular, intraperitoneal, intravenous, subcutaneous, intracerebral, transdermal, transmucosal, by inhalation, or topical, particularly to the ears, nose, eyes, or skin or by inhalation.

Parenteral administration includes, for example, intravenous, intramuscular, intraarterial, intraperitoneal, intranasal, rectal, intravesical, intradermal, topical or subcutaneous administration. Preferably, the compositions are administered parenterally.

The pharmaceutically acceptable carrier or vehicle can be particulate, so that the compositions are, for example, in tablet or powder form. The term “carrier” refers to a diluent, adjuvant or excipient, with which a drug antibody conjugate of the present invention is administered. Such pharmaceutical carriers can be liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. The carriers can be saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea, and the like. In addition, auxiliary, stabilizing, thickening, lubricating and coloring agents can be used. In one embodiment, when administered to an animal, the single domain antibody of the present invention or compositions and pharmaceutically acceptable carriers are sterile. Water is a preferred carrier when the drug antibody conjugates of the present invention are administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical carriers also include excipients such as starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The present compositions, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.

The pharmaceutical composition of the invention can be in the form of a liquid, e.g., a solution, emulsion or suspension. The liquid can be useful for delivery by injection, infusion (e.g., IV infusion) or sub-cutaneously.

When intended for oral administration, the composition is preferably in solid or liquid form, where semi-solid, semi-liquid, suspension and gel forms are included within the forms considered herein as either solid or liquid.

As a solid composition for oral administration, the composition can be formulated into a powder, granule, compressed tablet, pill, capsule, chewing gum, wafer or the like form. Such a solid composition typically contains one or more inert diluents. In addition, one or more of the following can be present: binders such as carboxymethylcellulose, ethyl cellulose, microcrystalline cellulose, or gelatin; excipients such as starch, lactose or dextrins, disintegrating agents such as alginic acid, sodium alginate, corn starch and the like; lubricants such as magnesium stearate; glidants such as colloidal silicon dioxide; sweetening agents such as sucrose or saccharin; a flavoring agent such as peppermint, methyl salicylate or orange flavoring; and a coloring agent. When the composition is in the form of a capsule (e. g. a gelatin capsule), it can contain, in addition to materials of the above type, a liquid carrier such as polyethylene glycol, cyclodextrin or a fatty oil.

The composition can be in the form of a liquid, e. g. an elixir, syrup, solution, emulsion or suspension. The liquid can be useful for oral administration or for delivery by injection. When intended for oral administration, a composition can comprise one or more of a sweetening agent, preservatives, dye/colorant and flavor enhancer. In a composition for administration by injection, one or more of a surfactant, preservative, wetting agent, dispersing agent, suspending agent, buffer, stabilizer and isotonic agent can also be included.

Compositions can take the form of one or more dosage units.

In specific embodiments, it can be desirable to administer the composition locally to the area in need of treatment, or by intravenous injection or infusion.

The amount of the binding agent of the present invention that is effective/active in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques. In addition, in vitro or in vivo assays can optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the compositions will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. Factors like age, body weight, sex, diet, time of administration, rate of excretion, condition of the host, drug combinations, reaction sensitivities and severity of the disease shall be taken into account.

Typically, the amount is at least about 0.01% of a single domain antibody of the present invention by weight of the composition. When intended for oral administration, this amount can be varied to range from about 0.1% to about 80% by weight of the composition. Preferred oral compositions can comprise from about 4% to about 50% of the sdAb of the present invention by weight of the composition.

Preferred compositions of the present invention are prepared so that a parenteral dosage unit contains from about 0.01% to about 2% by weight of the single domain antibody of the present invention.

For administration by injection, the composition can comprise from about typically about 0.1 mg/kg to about 250 mg/kg of the animal's body weight, preferably, between about 0.1 mg/kg and about 20 mg/kg of the animal's body weight, and more preferably about 1 mg/kg to about 10 mg/kg of the animal's body weight. In one embodiment, the composition is administered at a dose of about 1 to 30 mg/kg, e.g., about 5 to 25 mg/kg, about 10 to 20 mg/kg, about 1 to 5 mg/kg, or about 3 mg/kg. The dosing schedule can vary from e.g., once a week to once every 2, 3, or 4 weeks.

The invention provides methods of treating PD-1-mediated diseases or disorders in a mammal, e.g., a human patient, comprising administering an effective amount of a binding agent or pharmaceutical composition of the present invention to a mammal in need thereof. In particular, the invention furthermore relates to a method for the prevention and/or treatment of a disorder selected from cancer, an immune disorder, neurological disease, inflammatory disorder, allergy, transplant rejection, viral infection, immune deficiency, and other immune system-related disorder said method comprising administering, to a subject in need thereof, a pharmaceutically active amount of a binding agent or pharmaceutical composition of the invention.

As used herein, “treat”, “treating” or “treatment” means inhibiting or relieving a disease or disorder. For example, treatment can include a postponement of development of the symptoms associated with a disease or disorder, and/or a reduction in the severity of such symptoms that will, or are expected, to develop with said disease. The terms include ameliorating existing symptoms, preventing additional symptoms, and ameliorating or preventing the underlying causes of such symptoms. Thus, the terms denote that a beneficial result is being conferred on at least some of the mammals, e.g., human patients, being treated. Many medical treatments are effective for some, but not all, patients that undergo the treatment.

The term “subject” or “patient” refers to an animal which is the object of treatment, observation, or experiment. A subject includes, but is not limited to, a mammal, including, but not limited to, a human or a non-human mammal, such as a non-human primate, murine, bovine, equine, canine, ovine, or feline subject.

As used herein, the term “effective amount” means an amount of an anti-PD-1 antibody, that when administered alone or in combination with an additional therapeutic agent to a cell, tissue, or subject, is effective to achieve the desired therapeutic or prophylactic effect under the conditions of administration.

The invention also relates to a binding agent or pharmaceutical composition of the invention for use in the treatment or prevention of a disease.

In another aspect, the invention relates to a binding agent or pharmaceutical composition of the invention for use in the treatment or prevention of cancer, an immune disorder, neurological disease, inflammatory disorder, allergy, transplant rejection, viral infection, immune deficiency, and other immune system-related disorder.

In another aspect, the invention relates to the use of a binding agent or pharmaceutical composition of the invention in the treatment or prevention of a disease.

In another aspect, the invention relates to the use of a binding agent or pharmaceutical composition of the invention in the manufacture of a medicament for the treatment or prevention of cancer, an immune disorder, neurological disease, inflammatory disorder, allergy, transplant rejection, viral infection, immune deficiency, and other immune system-related disorder.

The cancer can be selected from a solid or non-solid tumor. For example, the cancer may be selected from bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, breast cancer, brain cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, kidney cancer, sarcoma of soft tissue, cancer of the urethra, cancer of the bladder, renal cancer, lung cancer, non-small cell lung cancer, thymoma, urothelial carcinoma leukemia, prostate cancer, mesothelioma, adrenocortical carcinoma, lymphomas, such as such as Hodgkin's disease, non-Hodgkin's, gastric cancer, and multiple myelomas.

In one embodiment, the tumor is a solid tumor. Examples of solid tumors which may be accordingly treated include breast carcinoma, lung carcinoma, colorectal carcinoma, pancreatic carcinoma, glioma and lymphoma. Some examples of such tumors include epidermoid tumors, squamous tumors, such as head and neck tumors, colorectal tumors, prostate tumors, breast tumors, lung tumors, including small cell and non-small cell lung tumors, pancreatic tumors, thyroid tumors, ovarian tumors, and liver tumors. Other examples include Kaposi's sarcoma, CNS, neoplasms, neuroblastomas, capillary hemangioblastomas, meningiomas and cerebral metastases, melanoma, gastrointestinal and renal carcinomas and sarcomas, rhabdomyosarcoma, glioblastoma, preferably glioblastoma multiforme, and leiomyosarcoma. Examples of vascularized skin cancers for which the antagonists of this invention are effective include squamous cell carcinoma, basal cell carcinoma and skin cancers that can be treated by suppressing the growth of malignant keratinocytes, such as human malignant keratinocytes.

In one embodiment, the tumor is a non-solid tumor. Examples of non-solid tumors include leukemia, multiple myeloma and lymphoma.

In one aspect, the cancer is identified as a PD-L1 positive cancer. In one aspect, the cancer is locally advanced unresectable, metastatic, or recurrent cancer.

Preferred cancers whose growth may be inhibited using the binding agent include cancers typically responsive to immunotherapy. Non-limiting examples of preferred cancers for treatment include melanoma (e.g., metastatic malignant melanoma), renal cancer (e.g. clear cell carcinoma), prostate cancer (e.g. hormone refractory prostate adenocarcinoma), breast cancer, colon cancer and lung cancer (e.g. non-small cell lung cancer).

In one embodiment, the cancer has progressed after another treatment, for example chemotherapy.

The binding agents and pharmaceutical compositions of the present invention are particularly useful for the treatment of cancers that are associated with cells (e.g., exhausted T cells, B cells, monocytes, etc.) that express abnormally high levels of PD-1. Other preferred cancers include those characterized by elevated expression of PD-1 and/or its ligands PD-L1 and/or PD-L2. In one embodiment, the cancer is selected from a cancer that has high levels of cancer-associated genetic mutations and/or high levels of expression of tumour antigens. In another embodiment, the cancer is selected from a cancer known to be immunogenic or that is able to become immunogenic upon treatment with other cancer therapies.

The immune disorder can be selected from graft vs. host disease, arthritis, such as rheumatoid arthritis, alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune diseases of the adrenal gland, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune oophoritis and orchitis, autoimmune thrombocytopenia, Behcet's disease, bullous pemphigoid, cardiomyopathy, celiac sprue-dermatitis, chronic fatigue immune dysfunction syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, Churg-Strauss syndrome, cicatrical pemphigoid, CREST syndrome, cold agglutinin disease, Crohn's disease, discoid lupus, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, glomerulonephritis, Graves' disease, Guillain-Barre, Hashimoto's thyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenia purpura (ITP), IgA neuropathy, juvenile arthritis, lichen planus, lupus erthematosus, Meniere's disease, mixed connective tissue disease, multiple sclerosis, Neuromyelitis optica (NMO), type 1 or immune-mediated diabetes mellitus, myasthenia gravis, pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polychrondritis, polyglandular syndromes, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, Raynauld's phenomenon, Reiter's syndrome, rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, stiff-man syndrome, systemic lupus erythematosus, lupus erythematosus, takayasu arteritis, temporal arteristis/giant cell arteritis, transverse myelitis, ulcerative colitis, uveitis, vasculitides such as dermatitis herpetiformis vasculitis, vitiligo, and Wegener's granulomatosis.

The neurological disease can be selected from Alzheimer's disease, epilepsy, Parkinson's disease, dementia, multiple sclerosis, peripheral neuropathy or post-herpetic neuralgia.

The binding agent or pharmaceutical composition of the invention may be administered as the sole active ingredient or in combination with one or more other therapeutic agent. A therapeutic agent is a compound or molecule which is useful in the treatment of a disease. Examples of therapeutic agents include antibodies, antibody fragments, drugs, toxins, nucleases, hormones, immunomodulators, pro-apoptotic agents, anti-angiogenic agents, boron compounds, photoactive agents or dyes and radioisotopes. An antibody molecule includes a full antibody or fragment thereof (e.g., a Fab, F(ab′)2, Fv, a single chain Fv fragment (scFv) or a single domain antibody, for example a V H domain) or antibody mimetic protein.

In one embodiment, the binding agent is used in combination with an existing therapy or therapeutic agent, for example an anti-cancer therapy. Thus, in another aspect, the invention also relates to a combination therapy comprising administration of a single domain antibody or pharmaceutical composition of the invention and an anti-cancer therapy. The anti-cancer therapy may include a therapeutic agent or radiation therapy and includes gene therapy, viral therapy, RNA therapy bone marrow transplantation, nanotherapy, targeted anti-cancer therapies or oncolytic drugs. Examples of other therapeutic agents include other checkpoint inhibitors, antineoplastic agents, immunogenic agents, attenuated cancerous cells, tumor antigens, antigen presenting cells such as dendritic cells pulsed with tumor-derived antigen or nucleic acids, immune stimulating cytokines (e.g., IL-2, IFNa2, GM-CSF), targeted small molecules and biological molecules (such as components of signal transduction pathways, e.g. modulators of tyrosine kinases and inhibitors of receptor tyrosine kinases, and agents that bind to tumor-specific antigens, including EGFR antagonists), an anti-inflammatory agent, a cytotoxic agent, a radiotoxic agent, or an immunosuppressive agent and cells transfected with a gene encoding an immune stimulating cytokine (e.g., GM-CSF), chemotherapy. Binding agents and pharmaceutical compositions of the invention can also be combined with surgery.

In one embodiment, the binding agent or pharmaceutical composition of the invention is administered together with an immunomodulator, a checkpoint modulator, an agent involved in T-cell activation, a tumour microenvironment modifier (TME) or a tumour-specific target. For example, the immunomodulator can be an inhibitor of an immune checkpoint molecule selected from an inhibitor of one or more of PD-1, PD-L1, PD-L2, CTLA-4, TIM-3, LAG-3, CEACAM, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 or TGFR beta. In another embodiment, the immunomodulator can be an activator of a costimulatory molecule selected from an agonist of one or more of OX40, OX40L, CD2, CD27, CDS, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD30, CD40, BAFFR, HVEM, CD7, LIGHT, NKG2C, SLAMF7, NKp80, CD160, B7-H3 or CD83 ligand, CD3, CD8, CD28, CD4 or ICAM-1.

In one embodiment, the PD-1 inhibitor is an anti-PD-1 antibody chosen from Nivolumab®, Pembrolizumab® or Pidilizumab®.

In one embodiment, the composition is administered concurrently with a chemotherapeutic agent or with radiation therapy. In another specific embodiment, the chemotherapeutic agent or radiation therapy is administered prior or subsequent to administration of the composition of the present invention, preferably at least an hour, five hours, 12 hours, a day, a week, a month, more preferably several months (e. g. up to three months), prior or subsequent to administration of composition of the present invention.

In some embodiments, the binding agent may be administered with two or more therapeutic agents. In some embodiments, the binding agents of the invention may be administered with two or more therapeutic agents.

The binding agent or pharmaceutical composition may be administered at the same time or at a different time as the other therapy or therapeutic compound or therapy, e.g., simultaneously, separately or sequentially.

In another aspect, the invention relates to an immunoconjugate comprising a binding agent of the invention conjugated to at least one therapeutic and/or diagnostic agent.

In another aspect, the invention relates to a method for increasing T-cell effector function by administration of a multiparatopic binding agent as described herein.

In another aspect, the invention relates to a method for rescuing T-cell from exhaustion by administration of a multiparatopic binding agent as described herein.

In another aspect, the invention provides a kit for the treatment or prevention of a disease or an immune response and/or for detecting PD-1 for diagnosis, prognosis or monitoring disease comprising a binding agent of the invention. Such a kit may contain other components, packaging, instructions, or material to aid in the detection of PD-1 protein. The kit may include a labeled binding agent of the invention as described above and one or more compounds for detecting the label.

The invention in another aspect provides a binding agent of the invention packaged in lyophilized form, or packaged in an aqueous medium.

The invention also relates to a binding agent as described herein with reference to the figures and examples.

In another aspect, a binding agent of the invention is used for non-therapeutic purposes, such as diagnostic tests and assays. A method for detecting the presence of human PD-1 in a test sample comprises contacting said sample with a binding agent according to the invention and at least one detectable label and detecting binding of said binding agent to human PD-1.

In one embodiment, the invention relates to a method of diagnosing a PD-1-mediated adaptive immune resistance in a patient who has cancer. The method comprises contacting a sample with a compound disclosed herein that has been labelled with a detectable moiety; and detecting expression of PD-1 on immune cells, e.g., CD8+ T cells; B cells; and macrophages. The sample may be tumour tissue.

Modifications of antibodies for diagnostic purposes are well known in the art. For example, antibodies may be modified with a ligand group such as biotin, or a detectable marker group such as a fluorescent group, a radioisotope, or an enzyme. Compounds of the invention can be labelled using conventional techniques. Suitable detectable labels include but are not limited to fluorophores, chromophores, radioactive atoms, electron-dense reagents, enzymes, and ligands having specific binding partners.

Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. While the foregoing disclosure provides a general description of the subject matter encompassed within the scope of the present invention, including methods, as well as the best mode thereof, of making and using this invention, the following examples are provided to further enable those skilled in the art to practice this invention and to provide a complete written description thereof. However, those skilled in the art will appreciate that the specifics of these examples should not be read as limiting on the invention, the scope of which should be apprehended from the claims and equivalents thereof appended to this disclosure. Various further aspects and embodiments of the present invention will be apparent to those skilled in the art in view of the present disclosure.

All documents mentioned in this specification are incorporated herein by reference in their entirety, including references to gene accession numbers and references to patent publications.

“and/or” where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example “A and/or B” is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein. Unless context dictates otherwise, the descriptions and definitions of the features set out above are not limited to any particular aspect or embodiment of the invention and apply equally to all aspects and embodiments which are described.

The invention is further described in the non-limiting examples.