PVRIG-binding protein and its medicinal uses

Abstract

PVRIG-binding proteins and their pharmaceutical uses. Specifically, anti-PVRIG single-domain antibodies and bispecific antibodies against PVRIG and TIGIT, pharmaceutical compositions containing said antibodies, methods for treating cancer, and pharmaceutical uses.

Description

[0001] This disclosure claims priority to Chinese patent application (application number CN202010174835.4) filed on March 13, 2020. Technical Field

[0002] This disclosure relates to PVRIG-binding proteins, such as anti-PVRIG antibodies and bispecific antibodies formed therewith anti-TIGIT antibodies, and their use as drugs for the treatment of cancer. Background Technology

[0003] Cancer remains one of the greatest long-standing health challenges facing humankind. Traditional therapies such as surgery, chemotherapy, and radiotherapy have proven largely ineffective in treating metastatic solid tumors. Tumor immunotherapy is a hot topic in cancer treatment, with T-cell immunotherapy at its core. Tumor immunotherapy fully utilizes and mobilizes the killer T cells in cancer patients to kill tumor cells; it may be the most effective and safest approach to treating cancer. Currently, tumor immunotherapy shows great promise in treating several different types of cancer, including metastatic tumors.

[0004] The activation of T cells in the human body involves a two-signaling pathway system. Besides the first signal provided by antigen-presenting cells (APCs) delivering MHC-antigen peptides, a series of co-stimulatory molecules provide the second signal, enabling T cells to generate a normal immune response. This dual-signaling pathway system plays a crucial role in maintaining the balance of the body's immune system, strictly regulating the different immune responses to self-antigens and non-self-antigens. The absence of the second signal from co-stimulatory molecules can lead to T cell non-response or persistent specific immune responses, resulting in tolerance. Therefore, the second signaling pathway plays a critical regulatory role in the entire process of the body's immune response.

[0005] PVRIG, also known as CD112R, is a cell surface-expressed protein belonging to the B7 / CD28 superfamily, along with TIGIT, CD96, and CD226, and plays an important role in the immune system. It comprises an extracellular domain, a transmembrane domain, and an intracellular domain. When its ligand PVRL2 (also called CD112) binds to PVRIG, it activates the ITIM domain of the intracellular PVRIG, enabling PVRIG to exert its immunosuppressive effect.

[0006] PVRIG is primarily expressed on CD4. + T cells, CD8 +PVRIG and its ligand PVRL2 are highly expressed on the surface of T cells and NK cells. PVRIG and its ligand PVRL2 are highly expressed in many solid tumors, including lung cancer, breast cancer, ovarian cancer, kidney cancer, gastric cancer, endometrial cancer, and head and neck cancer. PVRIG expression in these cancers is highly correlated with TIGIT and PD-1. Similar to PD-1 and TIGIT, PVRIG-positive T cells also show Eomes positivity and Tbet negativity, indicating that PVRIG is associated with T cell exhaustion. Therefore, PVRIG may represent a novel immune checkpoint besides PD-1 and TIGIT, playing a redundant role. In vitro cell experiments and mouse models have shown that knockout or inhibition of PVRIG in mice can effectively suppress tumor growth and works in synergy with PD-1 and TIGIT inhibitors.

[0007] Another target of interest, TIGIT, is highly expressed on lymphocytes, including tumor-infiltrating lymphocytes (TILs) and Tregs infiltrating various types of tumors. TIGIT signaling and binding to its homologous ligand PVR (also known as CD155) have been shown to directly inhibit NK cell cytotoxicity through its cytoplasmic ITIM domain. PVR is also widely expressed in tumors, suggesting that the TIGIT-PVR signaling axis may be a major immune escape mechanism in cancer.

[0008] However, no PVRIG / TIGIT bispecific antibody drugs have yet entered clinical trials. Compugen's COM701 is the world's first FDA-approved humanized hybridoma antibody against PVRIG, currently in Phase I clinical trials for cancer treatment. Surface Oncology also has an anti-PVRIG antibody, SRF-813, under development. Anti-TIGIT antibodies include Genentech's tiragolumab, Ono Pharmaceutical's BMS-986207 (developed in collaboration with BMS), Merck's MK-7684, iTeos Therapeutics' EOS-884448, and Arcus Biosciences' AB-154, all in Phase II clinical trials.

[0009] The prior art still lacks anti-PVRIG antibodies and anti-PVRIG / TIGIT bispecific antibodies with high affinity, high selectivity and high bioactivity that can inhibit the growth of cancer or tumors in the body. This disclosure aims to provide such antibodies that activate the immune system by blocking the inhibitory pathways of PVRIG and / or TIGIT to treat cancer. Summary of the Invention

[0010] This disclosure provides PVRIG binding proteins, anti-PVRIG antibodies (e.g., VHH) and bispecific antibodies to anti-TIGIT antibodies, as well as their encoding nucleic acids, vectors, host cells, pharmaceutical compositions, methods for treating cancer, and pharmaceutical uses.

[0011] Firstly, this disclosure provides a PVRIG-binding protein or an anti-PVRIG antibody.

[0012] In some embodiments, the PVRIG-binding protein comprises at least one immunoglobulin single variable domain, said at least one immunoglobulin single variable domain comprising three complementarity-determining regions CDR1, CDR2, and CDR3, wherein:

[0013] CDR1 is selected from any of the amino acid sequences shown in SEQ ID NO: 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, or amino acid sequences that differ from them by 3, 2, 1 or more amino acids, and / or

[0014] CDR2 is selected from any of the amino acid sequences shown in SEQ ID NO: 8, 11, 14, 17, 20, 23, 26, 29, 32, 35, 38, 41, 44, 47, 50, 53, 56, 59, 62, 65, or amino acid sequences that differ from them by 3, 2, 1 or more amino acids, and / or

[0015] CDR3 is selected from any of the amino acid sequences shown in SEQ ID NO: 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 63, 66, 150, 151, or amino acid sequences that differ from them by 3, 2, 1 or more amino acids.

[0016] Among them, SEQ ID NO: 7-21, 150, 151 are based on the Kabat encoding rule, SEQ ID NO: 22-36 are based on the Chothia encoding rule, SEQ ID NO: 37-51 are based on the IMGT encoding rule, and SEQ ID NO: 52-66 are based on the AbM encoding rule.

[0017] In some embodiments, the PVRIG-binding protein comprises at least one immunoglobulin single variable domain, said at least one immunoglobulin single variable domain comprising CDR1, CDR2, CDR3 of any of the sequences shown in SEQ ID NO: 2, 75-79; or comprising CDR1, CDR2, CDR3 of any of the sequences shown in SEQ ID NO: 3, 80-84; or comprising CDR1, CDR2, CDR3 of any of the sequences shown in SEQ ID NO: 4, 86-90; or comprising CDR1, CDR2, CDR3 of any of the sequences shown in SEQ ID NO: 5, 91-95; or comprising CDR1, CDR2, CDR3 of any of the sequences shown in SEQ ID NO: 6, 96-100, wherein CDR1, CDR2, CDR3 are defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering system, and in some specific embodiments, CDRs are determined according to the Kabat numbering rules.

[0018] In some implementations, according to the Kabat coding rules, the immunoglobulin single variable domain of the PVRIG-binding protein contains three complementarity-determining regions, CDR1, CDR2, and CDR3, wherein:

[0019] The amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 7, 8, and 9, respectively;

[0020] The amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 7, 8, and 150, respectively;

[0021] The amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 10, 11, and 12, respectively;

[0022] The amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 10, 11, and 151, respectively;

[0023] The amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 13, 14, and 15, respectively;

[0024] The amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 16, 17, and 18, respectively; or

[0025] The amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 19, 20, and 21, respectively.

[0026] In some implementations, according to the Chothia coding rules, the immunoglobulin single variable domain of the PVRIG-binding protein contains three complementarity-determining regions, CDR1, CDR2, and CDR3, wherein:

[0027] The amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 22, 23, and 24, respectively;

[0028] The amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 25, 26, and 27, respectively;

[0029] The amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 28, 29, and 30, respectively;

[0030] The amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 31, 32, and 33, respectively; or

[0031] The amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 34, 35, and 36, respectively.

[0032] In some implementations, according to the IMGT coding rules, the immunoglobulin single variable domain of the PVRIG-binding protein contains three complementarity-determining regions, CDR1, CDR2, and CDR3, wherein:

[0033] The amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 37, 38, and 39, respectively;

[0034] The amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 40, 41, and 42, respectively;

[0035] The amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 43, 44, and 45, respectively.

[0036] The amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 46, 47, and 48, respectively; or

[0037] The amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 49, 50, and 51, respectively.

[0038] In some implementations, according to the AbM coding rules, the immunoglobulin single variable domain of the PVRIG-binding protein contains three complementarity-determining regions, CDR1, CDR2, and CDR3, wherein:

[0039] The amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 52, 53, and 54, respectively.

[0040] The amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 55, 56, and 57, respectively.

[0041] The amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 58, 59, and 60, respectively;

[0042] The amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 61, 62, and 63, respectively; or

[0043] The amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 64, 65, and 66, respectively.

[0044] In some embodiments, a PVRIG-binding protein is provided, comprising a single immunoglobulin variable domain comprising CDR1, CDR2, and CDR3, wherein the amino acid sequences of CDR1, CDR2, and CDR3 of the single immunoglobulin variable domain are respectively, according to the Kabat numbering system.

[0045] As shown in SEQ ID NO: 7, 8 and 9; or

[0046] As shown in SEQ ID NO: 7, 8 and 150; or

[0047] As shown in SEQ ID NO: 10, 11 and 12; or

[0048] As shown in SEQ ID NO: 10, 11 and 151; or

[0049] As shown in SEQ ID NO: 13, 14 and 15; or

[0050] As shown in SEQ ID NO: 16, 17 and 18; or

[0051] As shown in SEQ ID NO: 19, 20 and 21.

[0052] In some embodiments, the PVRIG-binding protein of this disclosure is an antibody or its antigen-binding fragment, preferably a VHH antibody, more preferably a humanized and / or affinity-matured VHH antibody.

[0053] In some embodiments, the amino acid sequence of the immunoglobulin single variable domain of the PVRIG-binding protein of this disclosure is as shown in any of SEQ ID NO: 2-6, or has at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with it. In some embodiments, the PVRIG-binding protein is a variant protein that has 3, 2, 1, or more amino acid differences in CDR1, and / or 3, 2, 1, or more amino acid differences in CDR2, and / or 3, 2, 1, or more amino acid differences in CDR3.

[0054] In some embodiments, an anti-PVRIG antibody is provided, comprising CDR1, CDR2, and CDR3 of the PVRIG binding protein described above. It may be humanized and / or affinity-matured. In some specific embodiments, the amino acid sequence of the anti-PVRIG antibody is as shown in any one of SEQ ID NO: 2-6, 75-84, 86-100, or has at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with it. In some specific embodiments, the anti-PVRIG single-domain antibody is linked to the Fc region of human IgG1, IgG2, IgG3, or IgG4, for example, to the Fc region of IgG4 with S228P, F234A, L235A, and / or K447A mutations (e.g., as shown in SEQ ID NO: 101 or 153).

[0055] In some embodiments, the immunoglobulin single variable domain in the PVRIG-binding protein of this disclosure is a single-domain antibody (VHH), and in some specific embodiments, the VHH is a humanized and / or affinity-matured VHH.

[0056] In some implementations, the PVRIG-binding protein of this disclosure comprises an antibody.

[0057] In some embodiments, the PVRIG-binding protein of this disclosure is an antibody (e.g., VHH).

[0058] In some implementations, the PVRIG-binding protein disclosed herein is a camel antibody, a humanized antibody, or a fully human antibody.

[0059] In some embodiments, the PVRIG-binding protein of this disclosure or the immunoglobulin single variable domain therein is a camel antibody, wherein the amino acid sequence of VHH is as shown in any of SEQ ID NO: 2-6, or has at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it.

[0060] In some embodiments, the PVRIG binding protein of this disclosure or the immunoglobulin single variable domain therein is a humanized antibody, and the frame region of the antibody is the heavy chain frame region of the human germline template, such as IGHV3-7, specifically IGHV3-7*01 and IGHV3-30*02.

[0061] In some specific embodiments, the amino acid sequence of the anti-PVRIG humanized antibody disclosed herein is shown in any one of SEQ ID NO: 75-84, 86-100, or has at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it.

[0062] In some embodiments, the PVRIG-binding protein of this disclosure comprises or is humanized with an antibody that includes a heavy chain framework region of a human germline template.

[0063] In some implementations, the heavy chain frame region of the ancestral template is IGHV3-7*01 or IGHV3-30*02.

[0064] In some embodiments, the amino acid sequence of the immunoglobulin single variable domain of the humanized antibody is as shown in any of SEQ ID NO: 75-84 and 86-100, or has at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with it.

[0065] In some embodiments, the PVRIG-binding protein of this disclosure further comprises a human immunoglobulin Fc region, such as the Fc region of human IgG1, IgG2, IgG3, or IgG4. In some specific embodiments, the human immunoglobulin Fc region is the Fc region of human IgG4. In some specific embodiments, the human immunoglobulin Fc region is the Fc region of human IgG1. The Fc region may have mutations, such as amino acid mutations in S228P, F234A, L235A, and / or K447A (e.g., as shown in SEQ ID NO: 101 or 153).

[0066] In some embodiments, the PVRIG-binding protein of this disclosure comprises a single variable domain of immunoglobulin capable of specifically binding to PVRIG, which is directly or indirectly linked to the Fc region of immunoglobulin. The linker may be a non-functional amino acid sequence of 1-20, 1-30, 1-40, 1-50 or more amino acids without secondary or higher structures. The linker may be a flexible linker, such as GS, GAP, ASGS, G4S, (G4S)2, (G4S)3, (G4S)4, (G4S)5, (G4S)6, YGNGT, (YGNGT)2, (YGNGT)3, (YGNGT)4, (YGNGT) )5 (YGNGT))6 wait.

[0067] In some specific embodiments, the Fc region of the PVRIG-binding protein of this disclosure allows the PVRIG-binding protein to form a dimer molecule containing two or four PVRIG-binding domains. Such a PVRIG-binding protein is also referred to as a divalent or tetravalent PVRIG-binding protein. The dimer is, for example, a homodimer.

[0068] The PVRIG-binding protein or anti-PVRIG antibody disclosed herein has at least one of the following characteristics:

[0069] (a) K-binding human PVRIG D Value less than 1×10 -7 M;

[0070] (b) Block the interaction between PVRIG and its ligands (e.g., PVRL2);

[0071] (c) Relieve the inhibitory effect of dendritic cells on T cells and activate T cells;

[0072] (d) Relieve the inhibitory effect of tumor cells on NK cells;

[0073] (e) Inhibit tumor growth.

[0074] The present disclosure describes a PVRIG-binding protein or anti-PVRIG antibody that binds to the K+ of PVRIG. D The value can be less than 1×10 -7 M, less than 1×10 -8 M, less than 1×10 -9 M, less than 1×10 -10 M.

[0075] The PVRIG-binding protein or anti-PVRIG antibody disclosed herein can inhibit tumor growth by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, and at least about 80%.

[0076] The PVRIG-binding protein or anti-PVRIG antibody disclosed herein may be monomeric, and / or PEGylated, and / or glycosylated, and / or albumin-conjugated or fused, and / or Fc-fused, and / or hydroxyethylated, and / or de-glycosylated.

[0077] Secondly, this disclosure provides a bispecific antibody against PVRIG.

[0078] In some implementations, a bispecific antibody is provided, comprising a first antigen-binding domain and a second antigen-binding domain, wherein the first antigen-binding domain specifically binds to PVRIG.

[0079] In some embodiments, the first antigen-binding domain of the bispecific antibody of this disclosure specifically binds to PVRIG, the first antigen-binding domain comprising at least one immunoglobulin single variable domain (e.g., VHH), the at least one immunoglobulin single variable domain (e.g., VHH) comprising three complementarity-determining regions CDR1, CDR2, and CDR3, wherein:

[0080] CDR1 is selected from any of the amino acid sequences shown in SEQ ID NO: 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 49, 52, 55, 58, 61, 64, or amino acid sequences that differ from them by 3, 2, 1 or more amino acids, and / or

[0081] CDR2 is selected from any of the amino acid sequences shown in SEQ ID NO: 8, 11, 14, 17, 20, 23, 26, 29, 32, 35, 38, 41, 44, 47, 50, 53, 56, 59, 62, 65, or amino acid sequences that differ from them by 3, 2, 1 or more amino acids, and / or

[0082] CDR3 is selected from any of the amino acid sequences shown in SEQ ID NO: 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45, 48, 51, 54, 57, 60, 63, 66, 150, 151, or amino acid sequences that differ from them by 3, 2, 1 or more amino acids.

[0083] Among them, SEQ ID NO: 7-21 is based on the Kabat encoding rule, SEQ ID NO: 22-36 is based on the Chothia encoding rule, SEQ ID NO: 37-51 is based on the IMGT encoding rule, and SEQ ID NO: 52-66 is based on the AbM encoding rule.

[0084] In some embodiments, the first antigen-binding domain that specifically binds PVRIG in the bispecific antibody includes at least one immunoglobulin single variable domain, wherein the at least one immunoglobulin single variable domain includes CDR1, CDR2, and CDR3 of any of the sequences shown in SEQ ID NO: 2, 75-79; or includes CDR1, CDR2, and CDR3 of any of the sequences shown in SEQ ID NO: 3, 80-84; or includes CDR1, CDR2, and CDR3 of any of the sequences shown in SEQ ID NO: 4, 86-90; or includes CDR1, CDR2, and CDR3 of any of the sequences shown in SEQ ID NO: 5, 91-95; or includes CDR1, CDR2, and CDR3 of any of the sequences shown in SEQ ID NO: 6, 96-100. The CDR1, CDR2, and CDR3 are defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering system. In some specific embodiments, the CDRs are determined according to the Kabat numbering rules.

[0085] In some specific implementations, according to the Kabat coding rules, the first antigen-binding domain (such as VHH) that specifically binds to PVRIG contains three complementarity-determining regions CDR1, CDR2, and CDR3, wherein:

[0086] The amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 7, 8, and 9, respectively;

[0087] The amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 7, 8, and 150, respectively;

[0088] The amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 10, 11, and 12, respectively;

[0089] The amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 10, 11, and 151, respectively;

[0090] The amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 13, 14, and 15, respectively;

[0091] The amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 16, 17, and 18, respectively; or

[0092] The amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 19, 20, and 21, respectively.

[0093] In some specific implementations, according to the Chothia coding rules, the first antigen-binding domain (such as VHH) that specifically binds to PVRIG contains three complementarity-determining regions CDR1, CDR2, and CDR3, wherein:

[0094] The amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 22, 23, and 24, respectively;

[0095] The amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 25, 26, and 27, respectively;

[0096] The amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 28, 29, and 30, respectively;

[0097] The amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 31, 32, and 33, respectively; or

[0098] The amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 34, 35, and 36, respectively.

[0099] In some specific implementations, according to the IMGT coding rules, the first antigen-binding domain (such as VHH) that specifically binds to PVRIG contains three complementarity-determining regions CDR1, CDR2, and CDR3, wherein:

[0100] The amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 37, 38, and 39, respectively;

[0101] The amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 40, 41, and 42, respectively;

[0102] The amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 43, 44, and 45, respectively.

[0103] The amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 46, 47, and 48, respectively; or

[0104] The amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 49, 50, and 51, respectively.

[0105] In some specific implementations, according to the AbM coding rules, the first antigen-binding domain (such as VHH) that specifically binds to PVRIG contains three complementarity-determining regions CDR1, CDR2, and CDR3, wherein:

[0106] The amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 52, 53, and 54, respectively.

[0107] The amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 55, 56, and 57, respectively.

[0108] The amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 58, 59, and 60, respectively;

[0109] The amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 61, 62, and 63, respectively; or

[0110] The amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 64, 65, and 66, respectively.

[0111] In some embodiments, the first antigen-binding domain (such as VHH) of the bispecific antibody of this disclosure comprises any of the amino acid sequences shown in SEQ ID NO: 2-6, 75-84, 86-100, or sequences having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with it.

[0112] In some embodiments, the bispecific antibody of this disclosure includes:

[0113] The first antigen-binding domain is the first antibody, which is VHH;

[0114] The second antigen-binding domain is the second antibody, which includes a heavy chain (HC) and a light chain (LC).

[0115] The VHH, as the first antibody, is located at the N-terminus and / or C-terminus of the heavy or light chain of the second antibody.

[0116] In some specific embodiments, the bispecific antibody disclosed herein comprises a second antibody and a first antibody with two VHHs; the second antibody comprises two HCs and two LCs, wherein the VH of one HC of the second antibody forms an antigen-binding site with the VL of one LC, and the VH of the other HC forms an antigen-binding site with the VL of the other LC.

[0117] In some specific embodiments, the first antibody of one VHH in the bispecific antibody of this disclosure is located at the N-terminus of the heavy or light chain of the second antibody, and the first antibody of the other VHH is located at the C-terminus of the heavy or light chain of the second antibody.

[0118] In some specific embodiments, the first antibody of each VHH in the bispecific antibody of this disclosure is located at the N-terminus of the two heavy chains or two light chains of the second antibody; or, the first antibody of each VHH is located at the C-terminus of the two heavy chains or two light chains of the second antibody.

[0119] In some specific embodiments, the first antibody of each VHH in the bispecific antibody of this disclosure is located at the N-terminus of the two heavy chains of the first antibody; or, the first antibody of each VHH is located at the C-terminus of the two heavy chains of the first antibody.

[0120] In some specific embodiments, the first antibody of this disclosure may be linked to 1, 2, 3, 4, 5, 6, 7, or 8 second antibodies with VHHs. The second antibodies of the VHHs may be the same or different, and may all be linked to the N-terminus of the heavy chain of the first antibody, or all be linked to the C-terminus of the heavy chain of the first antibody, or all be linked to the N-terminus of the light chain of the first antibody, or all be linked to the C-terminus of the light chain of the first antibody, or any combination of the N-terminus of the heavy chain, the C-terminus of the heavy chain, the N-terminus of the light chain, and the C-terminus of the light chain.

[0121] In some specific embodiments, the first antibody of the VHH in the bispecific antibody of this disclosure is directly or via a linker linked to the N-terminus or C-terminus of each heavy chain of the second antibody. The linker is selected from: such as (G... m S n ) x Or (GGNGT) x Or (YGNGT) x The amino acid sequences shown are provided, where m and n are each independently selected from integers 1-8 (e.g., 1, 2, 3, 4, 5, 6, 7, or 8), and x is independently selected from integers 1-20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20). For example, the amino acid sequences shown with linkers G4S, (G4S)2, (G4S)3, (G4S)4, (G4S)5, and (G4S)6.

[0122] In some embodiments, the heavy chain of the second antibody of the bispecific antibody disclosed herein comprises a heavy chain variable region (VH) and a heavy chain constant region (CH), and the light chain comprises a light chain variable region (VL) and a light chain constant region (CL). The second antibody may be a full-length antibody.

[0123] In some embodiments, the heavy chain of the second antibody of the bispecific antibody disclosed herein is an IgG isotype, such as IgG1, IgG2, IgG3 or IgG4, for example, an IgG1 isotype; and / or, the light chain of the second antibody is a Kappa isotype.

[0124] In some embodiments, the two HCs of the second antibody of the bispecific antibody of this disclosure contain the same CDR and / or the two LCs contain the same CDR. In some specific embodiments, the two HCs of the second antibody contain the same VH and / or the two LCs contain the same VL. In some specific embodiments, the two HCs of the second antibody have the same amino acid sequence and / or the two LCs have the same amino acid sequence.

[0125] In some embodiments, the first antibodies of the two VHHs of the bispecific antibody disclosed herein may have the same or different amino acid sequences. For example, the first antibodies of the two VHHs have the same amino acid sequence.

[0126] In some embodiments, the bispecific antibody of this disclosure comprises two first polypeptide chains and two second polypeptide chains, wherein for each polypeptide chain: a) each of the first polypeptide chains independently comprises the heavy chain (HC) of the first antibody and the second antibody VHH; and b) each of the second polypeptide chains independently comprises the light chain (LC) of the second antibody; wherein VHH is linked to the N-terminus and / or C-terminus of the HC of the first antibody via a linker.

[0127] Alternatively, i) each of the first polypeptide chains independently comprises the heavy chain (HC) of the second antibody; and ii) each of the second polypeptide chains independently comprises the light chain (LC) of the first antibody and the second antibody, VHH; wherein VHH is directly or via a linker to the N-terminus and / or C-terminus of the LC of the second antibody.

[0128] In some specific embodiments, the bispecific antibody disclosed herein comprises two identical first polypeptide chains and two identical second polypeptide chains.

[0129] In some embodiments, the second antigen-binding domain of the bispecific antibody disclosed herein is any anti-TIGIT antibody. The TIGIT antibodies described herein are incorporated herein by reference in their entirety in the following formats: WO2009126688, WO2014089113, WO2015009856, WO2015143343, WO2015174439, WO2016028656, WO2016106302, WO2017053748, WO2017030823, US20160176963, US20130251720, WO2019232484, and WO2019062832. For example, the TIGIT antibody can be any one of CPA.9.083.H4(S241P), CPA.9.086.H4(S241P), CHA.9.547.7.H4(S241P), and CHA.9.547.13.H4(S241P) (see WO2019232484).

[0130] In some embodiments, the second antigen-binding domain of the bispecific antibody disclosed herein is used as the second antibody. The anti-TIGIT antibody from WO2019062832 is incorporated herein by reference in its entirety as the second antibody. In the second antibody:

[0131] The heavy chain variable region includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 115, 116, and 117, respectively, and the light chain variable region includes LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 118, 119, and 120, respectively; or

[0132] The heavy chain variable region includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 121, 122, and 123, respectively; the light chain variable region includes LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 124, 125, and 126, respectively; or

[0133] The heavy chain variable region includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 127, 128, and 129, respectively; the light chain variable region includes LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 130, 131, and 132, respectively; or

[0134] The heavy chain variable region includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 133, 134, and 135, respectively; the light chain variable region includes LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 136, 137, and 138, respectively; or

[0135] The heavy chain variable region includes HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 139, 140 and 141 respectively; the light chain variable region includes LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 142, 143 and 144 respectively.

[0136] In some specific embodiments, the first antigen-binding domain or the first antibody (such as VHH) in the bispecific antibody of this disclosure includes CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 7, 8, and 9, or includes CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 7, 8, and 150; the heavy chain variable region of the second antigen-binding domain or the second antibody includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 121, 122, and 123, respectively, and the light chain variable region includes LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 124, 125, and 126, respectively.

[0137] In some specific embodiments, the first antigen-binding domain or the first antibody (such as VHH) in the bispecific antibody of this disclosure includes CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 10, 11, and 12, or includes CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 10, 11, and 151; the heavy chain variable region of the second antigen-binding domain or the second antibody includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 121, 122, and 123, respectively, and the light chain variable region includes LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 124, 125, and 126, respectively.

[0138] In some specific embodiments, the first antibody of the bispecific antibody disclosed herein comprises an amino acid sequence as shown in any one of SEQ ID NO: 6, 79, 81, 92, 98, 99, or has at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it; the second antibody comprises VH as shown in any one of SEQ ID NO: 145-147, VL as shown in any one of SEQ ID NO: 148-149, or HC as shown in SEQ ID NO: 102 and LC as shown in SEQ ID NO: 103, or has at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with said VH, HC, or VL, LC.

[0139] In some specific implementations, the bispecific antibody disclosed herein includes:

[0140] The first polypeptide chain as shown in SEQ ID NO: 104, and the second polypeptide chain as shown in SEQ ID NO: 103;

[0141] The first polypeptide chain as shown in SEQ ID NO: 105, and the second polypeptide chain as shown in SEQ ID NO: 103;

[0142] The first polypeptide chain as shown in SEQ ID NO: 102, and the second polypeptide chain as shown in SEQ ID NO: 106;

[0143] The first polypeptide chain as shown in SEQ ID NO: 102, and the second polypeptide chain as shown in SEQ ID NO: 107;

[0144] The first polypeptide chain as shown in any of SEQ ID NO: 108-112, 114; the second polypeptide chain as shown in SEQ ID NO: 103; or

[0145] Variants having at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the first and / or second polypeptide chains described above.

[0146] In some embodiments, the Fc region of the PVRIG-binding protein, anti-PVRIG antibody, and bispecific antibody to anti-TIGIT disclosed herein contains mutations, including one or more amino acid mutations selected from the following:

[0147] i) Change the number of cysteine ​​residues in the hinge region of CH1 to facilitate the assembly of light and heavy chains or to improve or reduce the stability of the antibody.

[0148] ii) Mutations that enhance binding to FcγRIIIa to induce enhanced ADCC, and mutations that weaken binding to FcγRIIb, such as 236A, 239D, 239E, 332E, 332D, 239D / 332E, 267D, 267E, 328F, 267E / 328F, 236A / 332E, 239D / 332E / 330Y, 239D, 332E / 330L, 299T, 297N or any combination thereof;

[0149] iii) Mutations that prolong biological half-life, such as T252L, T254S, T256F, 428L, 434A, 434S, 428L / 434S, or any combination thereof;

[0150] iv) Mutations of one or more amino acids at positions 234, 235, 236, 237, 297, 318, 320, and 322, or any combination thereof, to alter the antibody’s affinity for the effector ligand while preserving the antigen-binding ability of the parent antibody;

[0151] v) One or more amino acid mutations at positions 329, 331, and 322, or any combination thereof, that result in an antibody having altered C1q binding and / or reduced or eliminated complement-dependent cytotoxicity (CDC).

[0152] vi) One or more amino acid mutations or any combination thereof within 231-239 alter the antibody’s ability to fix complement;

[0153] vii)238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283 ,285,286,289,290,292,293,294,295,296,298,301,303,305,307,309,312,315,320,322,324 One or more amino acid mutations in 326, 327, 329, 330, 331, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378, 382, ​​388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438, or 439, or any combination thereof, to enhance ADCC capability and / or increase antibody affinity for Fcγ receptor;

[0154] viii) Amino acid mutations in S228P, F234A, L235A, and / or K447A;

[0155] Amino acid mutations in S354C, E356D, M358L and / or T366W.

[0156] In some embodiments, antibodies are provided that competitively bind to the same epitopes as the PVRIG-binding protein, PVRIG / TIGIT-binding protein, anti-PVRIG single-domain antibody, and anti-PVRIG and TIGIT bispecific antibody of this disclosure.

[0157] In some embodiments, this disclosure provides a PVRIG / TIGIT binding protein comprising a first antigen-binding domain that specifically binds PVRIG and a second antigen-binding domain that specifically binds TIGIT.

[0158] The first antigen-binding domain that specifically binds to PVRIG includes an immunoglobulin single variable domain, which includes:

[0159] CDR1, CDR2, or CDR3 from any of the sequences shown in SEQ ID NO: 3, 80-84; or

[0160] CDR1, CDR2, or CDR3 from any of the sequences shown in SEQ ID NO: 2, 75-79; or

[0161] CDR1, CDR2, or CDR3 from any of the sequences shown in SEQ ID NO: 4, 86-90; or

[0162] CDR1, CDR2, or CDR3 from any of the sequences shown in SEQ ID NO: 5, 91-95; or

[0163] CDR1, CDR2, and CDR3 from any of the sequences shown in SEQ ID NO: 6, 96-100;

[0164] The CDR1, CDR2, and CDR3 are defined according to the Kabat, IMGT, Chothia, AbM, or Contact numbering system.

[0165] According to the Kabat numbering system, the amino acid sequences of CDR1, CDR2, and CDR3 of the single variable domain of the immunoglobulin are respectively

[0166] As shown in SEQ ID NO: 7, 8 and 9; or

[0167] As shown in SEQ ID NO: 7, 8 and 150; or

[0168] As shown in SEQ ID NO: 10, 11 and 12; or

[0169] As shown in SEQ ID NO: 10, 11 and 151; or

[0170] As shown in SEQ ID NO: 13, 14 and 15; or

[0171] As shown in SEQ ID NO: 16, 17 and 18; or

[0172] As shown in SEQ ID NO: 19, 20 and 21.

[0173] In a specific implementation, the first antigen-binding domain of the PVRIG / TIGIT binding protein comprises any one of SEQ ID NO: 2-6, 75-84, and 86-100 or has at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with it.

[0174] In a specific implementation scheme, the second antigen-binding domain of the PVRIG / TIGIT binding protein comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein:

[0175] The heavy chain variable region includes HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 115, 116 and 117 respectively, and the light chain variable region includes LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 118, 119 and 120 respectively.

[0176] The heavy chain variable region includes HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 121, 122 and 123 respectively, and the light chain variable region includes LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 124, 125 and 126 respectively.

[0177] The heavy chain variable region includes HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 127, 128 and 129 respectively, and the light chain variable region includes LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 130, 131 and 132 respectively.

[0178] The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 133, 134, and 135, respectively, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 136, 137, and 138, respectively; or

[0179] The heavy chain variable region includes HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 139, 140 and 141 respectively, and the light chain variable region includes LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 142, 143 and 144 respectively.

[0180] In a specific implementation scheme, the second antigen-binding domain of the PVRIG / TIGIT binding protein comprises a full-length heavy chain (HC) and a full-length light chain (LC).

[0181] Specifically, the full-length heavy chain is an IgG1 or IgG4 isotype, and the full-length light chain is a Kappa isotype;

[0182] More specifically, the heavy chain sequence is shown in SEQ ID NO: 102 or has at least 90% sequence identity with it, and the light chain sequence is shown in SEQ ID NO: 103 or has at least 90% sequence identity with it.

[0183] In a specific embodiment of the PVRIG / TIGIT binding protein disclosed herein, the VHH of the first antigen-binding domain that specifically binds PVRIG is located at the N-terminus of the heavy chain variable region or the full-length heavy chain of the second antigen-binding domain that specifically binds TIGIT.

[0184] The VHH of the first antigen-binding domain that specifically binds to PVRIG is located in the heavy chain variable region or the C-terminus of the full-length heavy chain of the second antigen-binding domain that specifically binds to TIGIT.

[0185] The VHH of the first antigen-binding domain that specifically binds to PVRIG is located at the N-terminus of the light chain variable region or the full-length light chain of the second antigen-binding domain that specifically binds to TIGIT; and / or

[0186] The VHH of the first antigen-binding domain that specifically binds to PVRIG is located at the C-terminus of the light chain variable region or the full-length light chain of the second antigen-binding domain that specifically binds to TIGIT.

[0187] In a specific embodiment of the PVRIG / TIGIT binding protein disclosed herein, the VHH of the first antigen-binding domain that specifically binds PVRIG is directly or indirectly connected to the second antigen-binding domain that specifically binds TIGIT via a linker.

[0188] Preferably, the connector has a shape such as (G4S). x The amino acid sequence shown is given, where x is independently selected from integers from 1 to 20;

[0189] More preferably, the linker is an amino acid sequence as shown in (G4S)2 and (G4S)3.

[0190] In some embodiments, the PVRIG / TIGIT binding protein provided in this disclosure comprises a first polypeptide chain and a second polypeptide chain, wherein:

[0191] The first polypeptide chain comprises an amino acid sequence as shown in any of SEQ ID NO: 108-112 and 114, and the second polypeptide chain comprises an amino acid sequence as shown in SEQ ID NO: 103; or

[0192] The first polypeptide chain contains the amino acid sequence shown in SEQ ID NO: 104 or 105, and the second polypeptide chain contains the amino acid sequence shown in SEQ ID NO: 103; or

[0193] The first polypeptide chain contains an amino acid sequence as shown in SEQ ID NO: 102, and the second polypeptide chain contains an amino acid sequence as shown in SEQ ID NO: 106 or 107.

[0194] Thirdly, this disclosure provides a polynucleotide encoding the aforementioned PVRIG-binding protein, PVRIG / TIGIT-binding protein, anti-PVRIG antibody (such as VHH), or bispecific antibody against PVRIG and TIGIT. The polynucleotide may be DNA or RNA.

[0195] In some embodiments, a polynucleotide composition is provided comprising:

[0196] The first nucleic acid encodes VH or HC containing the bispecific antibody against PVRIG and TIGIT disclosed herein; and the second nucleic acid encodes VL or LC containing the bispecific antibody against PVRIG and TIGIT disclosed herein.

[0197] Fourthly, this disclosure provides an expression vector or expression vector composition containing the polynucleotides or polynucleotide compositions described above, wherein the expression vector may be a eukaryotic expression vector, a prokaryotic expression vector, or a viral vector.

[0198] In some embodiments, an expression vector composition is provided comprising:

[0199] A first expression vector comprising the first nucleic acid in the above-described polynucleotide composition; and

[0200] The second expression vector contains the second nucleic acid in the above-mentioned polynucleotide composition.

[0201] Fifthly, this disclosure provides host cells transformed with or containing the expression vector or expression vector composition as described above, which may be eukaryotic cells or prokaryotic cells.

[0202] In some embodiments, the host cell is a bacterium, yeast, or mammalian cell. In some specific embodiments, the host cell is *Escherichia coli*, *Pichia pastoris*, Chinese hamster ovary (CHO) cells, or human embryonic kidney (HEK) 293 cells.

[0203] In a sixth aspect, this disclosure provides a preparation method comprising: expressing a PVRIG-binding protein, an anti-PVRIG antibody (such as VHH), and a bispecific antibody against PVRIG and TIGIT in a host cell as described above, and isolating and recovering the PVRIG-binding protein, the anti-PVRIG antibody (such as VHH), and the bispecific antibody against PVRIG and TIGIT from the host cell.

[0204] In specific embodiments, this disclosure provides methods for preparing PVRIG-binding proteins, PVRIG / TIGIT-binding proteins, or anti-PVRIG antibodies or their antigen-binding fragments, including:

[0205] The polynucleotides disclosed herein in the host cell, and

[0206] PVRIG-binding protein, PVRIG / TIGIT-binding protein, or anti-PVRIG antibody or its antigen-binding fragment, isolated and expressed from the host cell.

[0207] In a seventh aspect, this disclosure provides compositions (such as pharmaceutical compositions) containing a therapeutically effective amount of the PVRIG-binding protein, anti-PVRIG antibody (such as VHH), bispecific antibody against PVRIG and TIGIT, or PVRIG / TIGIT-binding protein as described above, and pharmaceutically acceptable excipients, diluents, or carriers.

[0208] In some embodiments, the composition (such as a pharmaceutical composition) contains the PVRIG-binding protein or anti-PVRIG antibody (such as VHH) of this disclosure, and an anti-TIGIT antibody. The TIGIT antibody may be any anti-TIGIT antibody as described above, such as the anti-TIGIT antibodies listed in Tables 23 and 24 of this disclosure. Furthermore, the composition may contain pharmaceutically acceptable excipients, diluents, or carriers.

[0209] In some specific embodiments, the anti-TIGIT antibody contains HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 121, 122, and 123, and the light chain variable region contains LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 124, 125, and 126, respectively.

[0210] In some specific embodiments, the pharmaceutical composition may contain 0.01 to 99% by weight of PVRIG-binding protein, anti-PVRIG antibody (such as VHH), and bispecific antibody against PVRIG and TIGIT per unit dose, or the amount of PVRIG-binding protein, anti-PVRIG antibody (such as VHH), and bispecific antibody against PVRIG and TIGIT per unit dose may be 0.1-2000 mg or 1-1000 mg.

[0211] Eighthly, the present disclosure provides any one or any combination of the following: a PVRIG-binding protein, an anti-PVRIG antibody (such as VHH), a bispecific antibody against PVRIG and TIGIT, a PVRIG / TIGIT-binding protein, and a polynucleotide encoding any one or any combination thereof, for the diagnosis, treatment, and prevention of diseases, and for the preparation of pharmaceuticals and pharmaceutical compositions (e.g., for the treatment or prevention of proliferative diseases (such as cancer or tumors) or for delaying the progression of related diseases).

[0212] Some implementations provide methods for treating or alleviating symptoms in a subject, including administering to the subject the PVRIG-binding protein of this disclosure, an anti-PVRIG antibody (such as VHH), or a bispecific antibody against PVRIG and TIGIT, wherein the symptoms are cancer.

[0213] In some embodiments, a method for activating cytotoxic T cells (CTLs) of a subject is provided, comprising administering to the subject a PVRIG-binding protein of the present disclosure, an anti-PVRIG antibody (such as VHH), a bispecific antibody against PVRIG and TIGIT, wherein a subgroup of the subject's CTLs is activated.

[0214] In some embodiments, a method for activating NK cells in a subject is provided, comprising administering to the subject a PVRIG-binding protein of the present disclosure, an anti-PVRIG antibody (such as VHH), a bispecific antibody against PVRIG and TIGIT, wherein a subgroup of the subject's NK cells is activated.

[0215] In some embodiments, a method for activating γδT cells of a subject is provided, comprising administering to the subject a PVRIG-binding protein of the present disclosure, an anti-PVRIG antibody (such as VHH), a bispecific antibody against PVRIG and TIGIT, wherein a subgroup of the subject's γδT cells is activated.

[0216] In some embodiments, a method for activating Th1 cells in a subject is provided, comprising administering to the subject a PVRIG-binding protein of the present disclosure, an anti-PVRIG antibody (such as VHH), a bispecific antibody against PVRIG and TIGIT, wherein a subgroup of the subject's Th1 cells is activated.

[0217] In some embodiments, a method is provided to activate, reduce or eliminate the number and / or activity of at least one of regulatory T cells (Tregs) in a subject, including administering to the subject the PVRIG-binding protein of this disclosure, an anti-PVRIG antibody (such as VHH), a bispecific antibody against PVRIG and TIGIT.

[0218] Some embodiments provide methods for increasing interferon-γ production and / or pro-inflammatory cytokine secretion in a subject, including administering to the subject the PVRIG-binding protein of this disclosure, an anti-PVRIG antibody (such as VHH), or a bispecific antibody against PVRIG and TIGIT.

[0219] Some implementations provide a method for inhibiting the interaction between PVRIG and PVLR2 in a subject, including administering to the subject the PVRIG-binding protein of this disclosure, an anti-PVRIG antibody (such as VHH), or a bispecific antibody against PVRIG and TIGIT.

[0220] Some embodiments provide a method of treating a subject, including administering to the subject or a subject the PVRIG-binding protein of this disclosure, an anti-PVRIG antibody (such as VHH), or a bispecific antibody against PVRIG and TIGIT.

[0221] In some specific implementations, the aforementioned subjects' conditions are proliferative disorders (e.g., cancer or tumors) or they have proliferative disorders (e.g., cancer or tumors). The cancer or tumor is selected from the following or combinations thereof: prostate cancer, liver cancer (HCC), colorectal cancer, ovarian cancer, endometrial cancer, breast cancer, triple-negative breast cancer, pancreatic cancer, stomach / gastric cancer, cervical cancer, head and neck cancer, thyroid cancer, testicular cancer, urothelial carcinoma, lung cancer (small cell lung cancer, non-small cell lung cancer), melanoma, non-melanoma skin cancer (squamous and basal cell carcinoma), glioma, renal cancer (RCC), lymphoma (NHL or HL), acute myeloid leukemia (AML), T-cell acute lymphoblastic leukemia (T-ALL), diffuse large B-cell lymphoma, testicular germ cell tumors, mesothelioma, esophageal cancer, Merkel cell cancer, high MSI cancer, KRAS-mutant tumors, adult T-cell leukemia / lymphoma, and myelodysplastic syndromes (MDS). The aforementioned conditions may be associated with abnormal expression of PVRIG and / or TIGIT. In some specific embodiments, the cancer or tumor is selected from the following cancers or combinations thereof: triple-negative breast cancer, gastric cancer, lung cancer (small cell lung cancer, non-small cell lung cancer), Merck cell carcinoma, high MSI cancer, KRAS-mutant tumors, adult T-cell leukemia / lymphoma, and myelodysplastic syndromes (MDS). In some specific embodiments, the cancer or tumor is selected from the following cancers or combinations thereof: triple-negative breast cancer, gastric cancer, lung cancer (small cell lung cancer, non-small cell lung cancer), Merck cell carcinoma, and high MSI cancer.

[0222] In some implementations, the subjects have a condition associated with PVRIG and / or TIGIT. In some specific implementations, the subject's condition includes cancer expressing or not expressing PVRIG, and further includes non-metastatic or non-invasive as well as invasive or metastatic cancer, wherein PVRIG expression on immune cells, stromal cells, or diseased cells suppresses antitumor and anti-invasive immune responses. The methods disclosed herein are particularly suitable for treating vascularized tumors.

[0223] In some embodiments, methods for treating or preventing infection or sepsis in a subject are provided, including administering to the subject or a subject a PVRIG-binding protein of this disclosure, an anti-PVRIG antibody (such as VHH), or a bispecific antibody against PVRIG and TIGIT. In some specific embodiments, the infection is a pathogen infection characterized by varying degrees of functional impairment of virus-specific T-cell responses, such as HIV, HCV, or HBV. In some specific embodiments, the sepsis includes severe sepsis, septic shock, systemic inflammatory response syndrome (SIRS), bacteremia, sepsis, toxemia, and septic syndrome.

[0224] In some embodiments, the PVRIG-binding protein, PVRIG / TIGIT-binding protein, anti-PVRIG antibody or its antigen-binding fragment, polynucleotide, or combination thereof described above in this disclosure are provided for treating or delaying a disease, preferably a proliferative disease;

[0225] Furthermore, the proliferative disease in question is cancer;

[0226] More preferably, the cancer is selected from lung cancer, prostate cancer, breast cancer, head and neck cancer, esophageal cancer, stomach cancer, colon cancer, colorectal cancer, bladder cancer, cervical cancer, uterine cancer, ovarian cancer, liver cancer, melanoma, kidney cancer, squamous cell carcinoma, hematologic malignancies, or any other disease or condition characterized by uncontrolled cell growth.

[0227] Some implementation schemes provide methods for diagnosing diseases, including:

[0228] a) Contact tissue from the subject with the PVRIG-binding protein or anti-PVRIG antibody disclosed herein; and

[0229] b) Determine the presence of PVRIG overexpression in the tissue as an indication of the presence of disease or condition.

[0230] The tissue may be a blood sample or a solid tumor biopsy sample. The PVRIG-binding protein or anti-PVRIG antibody may be labeled, and further, a second labeled antibody bound to the PVRIG-binding protein or anti-PVRIG antibody may be contacted with the aforementioned sample. In some embodiments, the PVRIG-binding protein or anti-PVRIG antibody is labeled, including with radioactive isotopes, dyes (such as those having a biotin-antibiotic streptokinin complex), contrast agents, fluorescent compounds or molecules, and enhancement agents for magnetic resonance imaging (MRI) (e.g., paramagnetic ions). In some embodiments, the disease or condition is cancer or tumor, infection, or sepsis as described above.

[0231] Ninthly, this disclosure provides for the detection of PVRIG-binding proteins.

[0232] This disclosure provides compositions for detecting PVRIG, said compositions comprising a PVRIG-binding protein or an anti-PVRIG antibody. This disclosure also provides methods, systems, or apparatus for detecting PVRIG in vivo or in vitro, including the use of a PVRIG-binding protein or an anti-PVRIG antibody.

[0233] In some implementations, the in vitro detection method, system, or apparatus may include, for example, (1) contacting a sample with a PVRIG-binding protein or an anti-PVRIG antibody; (2) detecting a complex formed between the PVRIG-binding protein or anti-PVRIG antibody and the sample; and / or (3) contacting a reference sample (e.g., a control sample) with the antibody; and (4) determining the extent of complex formation between the antibody and the sample by comparison with the reference sample. A change in complex formation in the sample or subject (e.g., a statistically significant change) compared to a control sample or subject indicates the presence of PVRIG in the sample.

[0234] In other embodiments, the in vivo detection method, system, or apparatus may include: (1) administering a PVRIG-binding protein or anti-PVRIG antibody to a subject; and (2) detecting the formation of a complex between the PVRIG-binding protein or anti-PVRIG antibody and the subject. Detection may include determining the location or time of complex formation. The antibody binding PVRIG may be labeled directly or indirectly with a detectable substance to facilitate the detection of the bound or unbound antibody. Suitable detectable substances include a variety of enzymes, cofactors, fluorescent substances, luminescent substances, and radioactive substances. The formation of the complex between the PVRIG-binding protein or anti-PVRIG antibody and PVRIG can be detected by measuring or visualizing the antibody that binds or does not bind to PVRIG. Conventional detection assays may be used, such as enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), or tissue immunohistochemistry. In some embodiments, the presence of PVRIG in a sample is analyzed by a competitive immunoassay using a standard labeled with a detectable substance and an unlabeled PVRIG-binding protein or anti-PVRIG antibody. The live samples that can be tested or measured can be tissue cells, blood, plasma, serum, pancreatic juice, urine, feces, tissue fluid, or culture medium.

[0235] In some implementations, for detection purposes, the PVRIG-binding protein or anti-PVRIG antibody of this disclosure may be labeled with fluorophores and chromophores.

[0236] In some embodiments, a kit is also provided containing a PVRIG-binding protein or an anti-PVRIG antibody, and may also include diagnostic instructions for use. The kit may also contain at least one additional reagent, such as a marker or additional diagnostic agent. For in vivo use, the antibody may be formulated as a pharmaceutical composition.

[0237] The PVRIG antibody, anti-PVRIG and TIGIT bispecific antibody provided in this disclosure have high specificity and high affinity for PVRIG and / or TIGIT; the immunogenicity of the humanized antibody is greatly reduced, while its excellent in vitro and in vivo activity is fully retained; it has good metabolic kinetics in rats and humans; it has a long half-life and high bioavailability; it has good long-term stability, no obvious abnormal chemical modifications, no obvious aggregation at high concentrations, and high purity and thermal stability; it has good effects in enhancing T cell and NK cell activity and inhibiting tumor development and progression. Attached Figure Description

[0238] Figure 1 The results of the PVRIG reporter gene activity test for anti-PVRIG antibody.

[0239] Figure 2 The results show the activity of anti-PVRIG antibody in activating NK cells during NK cell killing experiments.

[0240] Figure 3 The results show the activity of anti-PVRIG antibody in activating T cells during the MLR experiment.

[0241] Figures 4A-4B The results of the detection of humanized anti-PVRIG antibody activity in PVRIG reporter cells.

[0242] Figures 5A-5B The results show the activity of humanized anti-PVRIG antibody in NK cell killing assays to activate NK cells.

[0243] Figures 6A-6E The results show the binding activity of the humanized anti-PVRIG / TIGIT bispecific antibody against recombinant human PVRIG protein, human PVRIG-overexpressing cells, cynomolgus monkey recombinant PVRIG protein, and cynomolgus monkey PVRIG-overexpressing cells, as well as the activity of blocking the binding of human PVRIG to human PVRL2.

[0244] Figures 7A-7E The results show the binding activity of the humanized anti-PVRIG / TIGIT bispecific antibody against recombinant human TIGIT protein, human TIGIT-overexpressing cells, cynomolgus monkey TIGIT recombinant protein, and cynomolgus monkey TIGIT-overexpressing cells, as well as the activity of blocking the binding of human TIGIT to human PVR.

[0245] Figure 8 The results show the activity of humanized anti-PVRIG / TIGIT bispecific antibody in activating T cells during MLR experiments.

[0246] Figures 9A-9B The effects of anti-PVRIG / TIGIT bispecific antibodies on mouse body weight and tumor volume in a mouse subcutaneous xenograft model of human melanoma A375 mixed with human PBMCs were shown.

[0247] Figures 10A-10B The effects of anti-PVRIG / TIGIT bispecific antibodies on mouse body weight and tumor volume in a mouse subcutaneous xenograft model of human melanoma A375 mixed with human PBMCs were shown. Detailed Implementation

[0248] I. Terminology

[0249] To facilitate understanding of this disclosure, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0250] The three-letter and single-letter codes for amino acids used in this disclosure are as described in J. Biol. Chem, 243, p3558 (1968).

[0251] The terms “PVRIG,” “PVRIG protein,” or “PVRIG polypeptide” may optionally include any such protein or its variants, conjugates, or fragments, including (but not limited to) known or wild-type PVRIG as described herein, as well as any naturally occurring splice variants, amino acid variants, or isoforms, and especially the soluble extracellular domain (ECD) fragment of PVRIG. The definition of ECD herein is as in patent WO2016134333. The complete human PVRIG sequence can be found in GenBank accession number AAH73861.1.

[0252] "PVRIG-binding protein" means any protein capable of specifically binding to PVRIG or any molecule containing said protein. PVRIG-binding proteins may include antibodies against PVRIG as defined in this disclosure, their antigen-binding fragments, or conjugates thereof. PVRIG-binding proteins also encompass immunoglobulin superfamily antibodies (IgSF) or CDR transplantation molecules. The "PVRIG-binding protein" of this disclosure may contain at least one PVRIG-binding immunoglobulin single variable domain (e.g., VHH). In some embodiments, the "PVRIG-binding protein" may contain 2, 3, 4, or more PVRIG-binding immunoglobulin single variable domains (e.g., VHH). In addition to containing the PVRIG-binding immunoglobulin single variable domain, the PVRIG-binding protein of this disclosure may also contain a linker and / or an effector-functional portion, such as a half-life-extending portion (e.g., an immunoglobulin single variable domain binding serum albumin), and / or a fusion partner (e.g., serum albumin) and / or a conjugated polymer (e.g., PEG) and / or an Fc region. In some embodiments, the “PVRIG binding protein” of this disclosure also encompasses bispecific / multispecific antibodies containing immunoglobulins that bind different antigens (such as a first antibody binding a first antigen (such as PVRIG) and a second antibody binding a second antigen (such as TIGIT), optionally including a third antibody binding a third antigen, and further optionally including a fourth antibody binding a fourth antigen).

[0253] The terms “TIGIT”, “TIGIT protein”, or “TIGIT polypeptide” may optionally include any such protein or its variants, conjugates, or fragments, including (but not limited to) known or wild-type TIGIT as described herein, and any naturally occurring splice variants, amino acid variants, or isoforms. The complete TIGIT sequence can be found in GenBank accession number AAI01289.1.

[0254] "Binds to PVRIG" means that the antigen can interact with a PVRIG or its epitope, which may be of human origin. "Binds to TIGIT" means that the antigen can interact with a TIGIT or its epitope, which may be of human origin. "Antigen binding site" refers to a discontinuous three-dimensional spatial site on the antigen that is recognized by the antibody or antigen-binding fragment of this disclosure.

[0255] The term "antibody" or "immunoglobulin" broadly encompasses conventional antibodies (tetrapeptide chains consisting of two identical heavy chains and two identical light chains linked by interchain disulfide bonds), as well as Fab, Fv, sFv, F(ab')2, linear antibodies, single-chain antibodies, scFv, sdAb, sdFv, nanobodies, peptide antibodies, domain antibodies (heavy chain (VH) antibodies, light chain (VL) antibodies), and multispecific antibodies (bispecific antibodies, diabody, triabody, and tetrabody, tandem di-scFv, tandem tri-scFv). Therefore, the term "antibody" as used in this disclosure includes full-length antibodies, their individual chains and any portion, domain, or fragment having antigen-binding activity, as well as multispecific antibodies (including, but not limited to, antigen-binding domains or fragments, such as VHH domains or VH / VL domains, respectively) comprising their individual chains and any portion, domain, or fragment having antigen-binding activity. Traditional antibodies or immunoglobulins are typically tetrapeptide chains composed of two identical heavy chains and two identical light chains linked by interchain disulfide bonds. The amino acid composition and sequence of the constant region of the heavy chain differ, thus their antigenicity varies. Based on this, immunoglobulins can be classified into five classes, or isotypes of immunoglobulins: IgM, IgD, IgG, IgA, and IgE, with corresponding heavy chains of μ, δ, γ, α, and ε chains, respectively. Within the same class of Ig, differences in the amino acid composition of its hinge region and the number and position of disulfide bonds in the heavy chain can further lead to different subclasses; for example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. Light chains are classified as κ (kappa) or λ (lambda) chains based on differences in their constant regions. Each of the five classes of Ig can have either a κ (kappa) chain or a λ (lambda) chain. In some embodiments, the antibodies of this disclosure specifically or substantially specifically bind to PVRIG and / or TIGIT.

[0256] The “antibody” disclosed herein includes, but is not limited to: (i) Fab fragments consisting of VL, VH, CL, and CH1 domains; (ii) Fd fragments consisting of VH and CH1 domains; (iii) F(ab')2 fragments, a bivalent fragment comprising two linked Fab fragments; (vii) single-chain Fv molecules (scFv) wherein the VH and VL domains are linked by a peptide linker that allows the two domains to bind to form an antigen-binding site; (Bird et al., 1988, Science 242: 423-426; Huston et al., 1988, Proceedings of the National Academy of Sciences 85: 5879-5883) 242, which are incorporated herein by reference in their entirety; (iv) “bifunctional antibodies” or “trifunctional antibodies”, multivalent or multispecific fragments constructed by gene fusion (Tomlinson et al., 2000, Methods of Enzymology). (Enzymol.) 326: 461-479; WO94 / 13804; Holliger et al., 1993, Proceedings of the National Academy of Sciences of the United States of America 90: 6444-6448, all of which are incorporated herein by reference in their entirety); (v) “domain antibodies” or “dAbs” (sometimes referred to as “immunoglobulin single variable domains”), including immunoglobulin single variable domains from other species, such as rodents (e.g., as disclosed in WO00 / 29004), nurse sharks and camelid V-HH dAbs; (vi) SMIPs (small molecule immunotherapeutic agents), camel antibodies, nanobodies and IgNARs; (vii) humanized antibodies of (i)-(vi) above.

[0257] Unless otherwise specified, the antibodies disclosed herein generally use the Kabat numbering system. EU numbers in Kabat are also generally used for constant domains and / or Fc domains.

[0258] The antibodies disclosed herein can be polyclonal, monoclonal, xenogeneic, allogeneic, syngeneic, or modified forms thereof, with monoclonal antibodies being particularly suitable in many embodiments. Generally, the antibodies disclosed herein are recombinant antibodies. As used herein, “recombinant” refers to products such as cells or nucleic acids, proteins, or vectors, indicating that said cells, nucleic acids, proteins, or vectors have been modified by introducing heterologous nucleic acids or proteins or by altering native nucleic acids or proteins, or that said cells are derived from such modified cells. For example, recombinant cells express genes not present in native (non-recombinant) cell forms or express native genes that are abnormally expressed, poorly expressed, or not expressed at all.

[0259] "Monoclonal antibody" and "monoclonal antibody composition" refer to a population of antibody molecules containing only one species with an antigen-binding site capable of responding to an immune response to a specific antigenic epitope, while "polyclonal antibody" and "polyclonal antibody composition" refer to a population of antibody molecules containing multiple species with antigen-binding sites capable of interacting with a specific antigen. Monoclonal antibody compositions typically exhibit a single binding affinity to the specific antigen with which they respond to an immune response.

[0260] "Antigen" refers to a vertebrate molecule used for immunization to generate antibodies that recognize the antigen, or for screening expression libraries (e.g., phage, yeast, or ribosome display libraries, in particular). In this document, antigen is defined more broadly and is generally expected to include target molecules that are specifically recognized by antibodies, and therefore include a portion or mimic of molecules used in immunization processes to generate antibodies or in library screening to select antibodies.

[0261] The term “sequence” (as used in terms such as “immunoglobulin sequence,” “antibody sequence,” “single variable domain sequence,” “VHH sequence,” or “protein sequence”) should generally be understood to include both the relevant amino acid sequence and the nucleic acid or nucleotide sequence encoding the sequence, unless further definition is required by this disclosure.

[0262] "Polynucleotide" or "nucleic acid" refers to a nucleotide chain of any length, including DNA and RNA. Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases and / or their analogs, or any substrate that can be incorporated into the chain by DNA or RNA polymerases. Polynucleotides may contain modified nucleotides, such as methylated nucleotides and their analogs. If present, modifications to the nucleotide structure may be imparted before or after chain assembly. Polynucleotides may also contain similar forms of ribose or deoxyribose known in the art, including, for example, 2′-O-methyl-, 2′-O-allyl, 2′-fluoro-, or 2′-azido-ribose, carbocyclic sugar analogs, α- or β-anomeric sugars, epimeric sugars (such as arabinose, xylose or lythose, pyranose, furanose, sedoheptulose), acyclic analogs, and non-base nucleoside analogs such as methylriboside.

[0263] "Homology" or "identity" refers to the sequence similarity between two polynucleotide sequences or two polypeptides. When positions in two compared sequences are occupied by the same base or amino acid monomer subunit—for example, if every position in two DNA molecules is occupied by adenine—then the molecules are homologous at that position. The percentage of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared, multiplied by 100%. For example, at optimal sequence alignment, if six out of ten positions in two sequences match or are homologous, then the two sequences are 60% homologous. Generally, comparisons are made when the highest percentage of homology is obtained by aligning the two sequences.

[0264] A "domain" of a polypeptide or protein refers to a folded protein structure that can maintain its tertiary structure independently of the rest of the protein. Generally, a domain is responsible for a single functional property of a protein and, in many cases, can be added to, removed from, or transferred to other proteins without losing the function of the rest of the protein and / or the domain itself.

[0265] An "immunoglobulin domain" refers to a globular region of an antibody chain (such as the chain of a conventional tetrapeptide chain antibody or a heavy chain antibody), or a polypeptide that is essentially composed of such globular regions. An immunoglobulin domain is characterized by its ability to maintain the immunoglobulin folding properties of the antibody molecule, consisting of two interlayers of approximately seven antiparallel β-sheet strands arranged in two β-sheets, optionally stabilized by conserved disulfide bonds.

[0266] An immunoglobulin variable domain refers to an immunoglobulin domain essentially composed of four "frame regions," referred to in the art and hereinafter as "frame region 1" or "FR1," "frame region 2" or "FR2," "frame region 3" or "FR3," and "frame region 4" or "FR4," respectively. These frame regions are separated by three "complementarity-determining regions" or "CDRs," referred to in the art and hereinafter as "complementarity-determining region 1" or "CDR1," "complementarity-determining region 2" or "CDR2," and "complementarity-determining region 3" or "CDR3," respectively. Therefore, the general structure or sequence of an immunoglobulin variable domain can be represented as: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The immunoglobulin variable domain confers antigen specificity due to the presence of antigen-binding sites.

[0267] "Antibody framework (FR)" refers to a portion of a variable domain that serves as a scaffold for the antigen-binding loop (CDR) of that variable domain.

[0268] The determination or definition of a “CDR” can be accomplished by resolving the structure of the antibody and / or the structure of the antibody-ligand complex, thereby enabling the definitive depiction of the CDR and the identification of residues containing the antibody binding site. This can be achieved using any of the various techniques known to those skilled in the art, such as X-ray crystallography. A variety of analytical methods can be used to identify CDRs, including but not limited to the Kabat numbering system, the Chothia numbering system, the AbM numbering system, the IMGT numbering system, contact definition, and conformation definition. The Kabat numbering system is the standard for numbering residues in antibodies and is commonly used to identify CDR regions (see, for example, Johnson & Wu, 2000, Nucleic Acids Res., 28: 214-8). The Chothia numbering system is similar to the Kabat numbering system, but it takes into account the location of certain structural loop regions (see, for example, Chothia et al., 1986, J. Mol. Biol., 196: 901-17; Chothia et al., 1989, Nature, 342: 877-83). The AbM numbering system uses a computer program integration suite produced by the Oxford Molecular Group to model antibody structures (see, for example, Martin et al., 1989, ProcNatl Acad Sci (USA), 86: 9268-9272; "AbMTM, A Computer Program for Modeling Variable Regions of Antibodies," Oxford, UK; Oxford Molecular, Ltd). The AbM numbering system uses a combination of knowledge databases and a de novo approach to model the tertiary structure of antibodies from basic sequences (see those described in Samudrala et al., 1999, "Ab Initio Protein Structure Prediction Using a Combined Hierarchical Approach" in PROTEINS, Structural, Function and Genetics Suppl., 3: 194-198). Contact definitions are based on the analysis of available complex crystal structures (see, for example, MacCallum et al., 1996, J. Mol. Biol., 5: 732-45). In the conformational definition, the position of CDR can be identified as a residue that contributes enthalpy to antigen binding (see, for example, Makabe et al., 2008, Journal of Biological Chemistry, 283: 1156-1166).Other CDR boundary definitions may not strictly follow one of the methods described above, but still overlap with at least a portion of the Kabat CDR, although they may be shortened or lengthened depending on whether a particular residue or group of residues significantly affects the predicted or experimental results of antigen binding. As used herein, a CDR may refer to a CDR defined by any method (including combinations of methods) known in the art. The methods used herein may utilize a CDR defined according to any of these methods. For any given embodiment containing more than one CDR, the CDR may be defined according to any of the Kabat, Chothia, extended, AbM, IMGT, contact, and / or conformational definitions.

[0269] "Immunoglobulin single variable domain" is generally used to refer to an immunoglobulin variable domain (which can be a heavy chain or light chain domain, including VH, VHH, or VL domains) that can form a functional antigen-binding site without interacting with other variable domains (e.g., without the VH / VL interaction required between the VH and VL domains of a conventional four-chain monoclonal antibody). Examples of "immunoglobulin single variable domain" include nanobodies (including VHH, humanized VHH, and / or camelified VH, such as camelified human VH), IgNAR, domains, and (single-domain) antibodies (such as dAbs) that are VH domains or derived from VH domains. TM ) and antibodies that are VL domains or derived from VL domains (such as dAbs) TM Immunoglobulin single variable domains based on and / or derived from heavy chain variable domains (such as VH or VHH domains) are generally preferred. A specific example of an immunoglobulin single variable domain is the “VHH domain” (or simply “VHH”) as defined below.

[0270] The “VHH domain,” also known as a heavy chain single-domain antibody, VHH, VHH antibody fragment, VHH antibody, or nanobody, is a variable domain of an antigen-binding immunoglobulin called a “heavy chain antibody” (i.e., an antibody lacking a light chain) (Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hamers C, Songa EB, Bendahman N, Hamers R.: “Naturally occurring antibodies devoid of light chains”; Nature 363, 446-448 (1993)). The term “VHH domain” is used to distinguish the variable domain from the heavy chain variable domain (referred to herein as the “VH domain”) and the light chain variable domain (referred to herein as the “VL domain”) present in conventional tetrapeptide chain antibody structures. The VHH domain specifically binds to an epitope without the need for other antigen-binding domains (unlike the VH or VL domains in conventional tetrapeptide chain antibodies, where the epitope is recognized by both the VL and VH domains). The VHH domain is a small, stable, and highly efficient antigen-recognition unit formed by a single immunoglobulin domain. The terms “heavy chain single-domain antibody,” “VHH domain,” “VHH,” “VHH domain,” “VHH antibody fragment,” “VHH antibody,” and “domain” (“Nanobody” is a trademark of Ablynx NV, Ghent, Belgium) are used interchangeably. “VHH domain” includes, but is not limited to, naturally occurring antibodies produced by camelids, antibodies produced by camelids that have been humanized, or antibodies obtained through phage display technology.

[0271] As is known in the art regarding VH and VHH domains, the total number of amino acid residues in each CDR may differ and may not correspond to the total number of amino acid residues indicated by the Kabat number (i.e., one or more positions according to the Kabat number may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than allowed by the Kabat number). This means that, in general, the Kabat number may or may not correspond to the actual number of amino acid residues in the actual sequence. Other numbering systems or encoding rules include Chothia, IMGT, and AbM.

[0272] The total number of amino acid residues in the VHH domain will typically be in the range of 110 to 120, often between 112 and 115. However, it should be noted that smaller and longer sequences may also be suitable for the purposes described in this disclosure.

[0273] The VHH domain (alone or as part of a larger polypeptide) offers many significant advantages over using conventional VH and VL domains, scFv, or conventional antibody fragments (such as Fab- or F(ab')2- fragments):

[0274] - Only a single domain is needed to bind to the antigen with high affinity and high selectivity, so that there is no need for two separate domains, nor is it necessary to ensure that the two domains exist in the appropriate spatial conformation and configuration (for example, scFv generally requires the use of specially designed adapters).

[0275] The -VHH domain can be expressed by a single gene and does not require post-translational folding or modification;

[0276] -VHH domains can be easily modified into multivalent and multispecific formats;

[0277] - The VHH domain is highly soluble and has no tendency to aggregate;

[0278] The -VHH domain is highly stable to heat, pH, proteases, and other denaturants or conditions, and therefore can be prepared, stored, or transported without the use of refrigeration equipment, thus saving costs, time, and the environment;

[0279] -VHH domains are easy to prepare and relatively inexpensive, even at the scale required for production;

[0280] The -VHH domain is relatively small compared to conventional tetrapeptide chain antibodies and their antigen-binding fragments (approximately 15 kDa or 1 / 10 the size of conventional IgG), thus exhibiting higher tissue penetration and allowing for higher dose administration compared to conventional tetrapeptide chain antibodies and their antigen-binding fragments.

[0281] The -VHH domain can exhibit so-called cavity-binding properties (especially due to its elongated CDR3 loop compared to the conventional VH domain), thereby reaching targets and epitopes that are inaccessible to conventional tetrapeptide chain antibody and its antigen-binding fragments.

[0282] Methods for obtaining VHHs that bind to specific antigens or epitopes have been previously disclosed in the following literature: R. van der Linden et al., Journal of Immunological Methods, 240(2000)185-195; Li et al., JBiol Chem., 287(2012)13713-13721; Deffar et al., African Journal of Biotechnology Vol.8(12), pp.2645-2652, 17June, 2009 and WO94 / 04678.

[0283] The term "Fc variant" or "variant Fc" refers to a protein containing amino acid modifications in its Fc domain. Fc variants of this disclosure are defined according to the amino acid modifications constituting them. Thus, for example, S228P or 228P is an Fc variant with a proline substitution at position 228 relative to the parental Fc polypeptide, where the designation is based on the EU index. The identity of the WT amino acid may be left unspecified; in this case, the aforementioned variant is referred to as 228P.

[0284] Examples of "humanization" include the ability to "humanize" a VHH domain derived from camelids by replacing one or more amino acid residues in the original VHH sequence with one or more amino acid residues present at the corresponding position in the VH domain of a conventional human tetrapeptide antibody (also referred to herein as "sequence optimization," which, in addition to humanization, may also encompass other modifications to the sequence by providing one or more mutations that enhance the properties of the VHH, such as removing potential post-translational modification sites). The humanized VHH domain may contain one or more fully human frame region sequences, and in some embodiments, may contain the human frame region sequence of IGHV3. Another example of "humanization" includes transplanting a mouse CDR sequence into a human antibody variable region frame, i.e., antibodies generated from different types of human germline antibody framework sequences. This can overcome the strong antibody-variable antibody response induced by chimeric antibodies due to carrying a large amount of mouse protein components. Humanization methods include protein surface amino acid resurfacing and CDR grafting to a universal framework, which involves "grafting" the CDR onto other "scaffolds" (including but not limited to human scaffolds or non-immunoglobulin scaffolds). Suitable scaffolds and techniques for CDR grafting are known in the art. Germline DNA sequences of human heavy and light chain variable region genes, for example, can be found in the VBase human germline sequence database (available at www.mrccpe.com.ac.uk / vbase) and in Kabat, E.A. et al., 1991, Sequences of Proteins of Immunological Interest, 5th edition. The humanized antibodies disclosed herein also include humanized antibodies further matured by phage display with affinity for the CDR. Furthermore, to avoid a decrease in activity along with a decrease in immunogenicity, minimal reverse or reversion mutations can be performed on the human antibody variable region framework sequence to maintain activity.

[0285] An antibody that has reached “affinity maturity” has one or more variations in one or more CDRs that result in an increased affinity for the antigen compared to its respective parent antibody. Affinity-matured antibodies can be prepared, for example, by methods known in the art as described below: Marks et al., 1992, Biotechnology 10: 779-783 or Barbas et al., 1994, Proc. Nat. Acad. Sci, USA 91: 3809-3813; Shier et al., 1995, Gene 169: 147-155; Yelton et al., 1995, Immunol. 155: 1994-2004; Jackson et al., 1995, J. Immunol. 154(7): 3310-9; and Hawkins et al., 1992, J. MoI. Biol. 226(3): 889896; KS Johnson and RE Hawkins, “Affinity maturation of antibodies using phage display”, Oxford University Press 1996.

[0286] Typically, the antibodies disclosed herein will be measured in a preferred 10 as in Biacore, KinExA, or Fortibio assays. -7 Up to 10 -10 mol / L (M), more preferably 10 -8 Up to 10 -10 moles per liter, or even more preferably 10 -9 Up to 10 -10 Or a lower dissociation constant (KD), and / or at least 10 -7 M, preferably at least 10 -8 M, more preferably at least 10 -9 M, more preferably at least 10 -10 The association constant (KA) of M binds to the antigen it wishes to bind to (i.e., PVRIG). Any value greater than 10... -4 The KD value of M is generally considered to indicate nonspecific binding. The specific binding of antigen-binding proteins to antigens or epitopes can be determined in any suitable manner known, including, for example, surface plasmon resonance (SPR) assays, Scatchard assays, and / or competitive binding assays (e.g., radioimmunoassay (RIA), enzyme immunoassay (EIA), and sandwich competitive assays) as described in this disclosure.

[0287] The term “epitope” or the interchangeable term “antigenic determinant” refers to any antigenic determinant on an antigen to which the complementary site of an antibody binds. Antigenic determinants typically contain chemically active surface groups of a molecule, such as amino acid or sugar side chains, and usually possess specific three-dimensional structural features and specific charge characteristics. For example, an epitope typically comprises at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive or discontinuous amino acids in a unique spatial conformation, and can be a “linear” epitope or a “conformal” epitope. In a linear epitope, all points of interaction between the protein and the interacting molecule (e.g., an antibody) are present linearly along the primary amino acid sequence of the protein. In a conformal epitope, points of interaction are present across protein amino acid residues that are separated from each other. Epitopes of a given antigen can be identified using many epitope localization techniques well known in the art (e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, GEMorris, Ed. (1996), US4708871). Antibodies can be competitively screened for binding to the same epitope using conventional techniques known to those skilled in the art. For example, competitive and cross-competitive studies can be performed to obtain antibodies that compete or cross-competitively bind to the antigen (see WO03 / 48731 for high-throughput screening methods). Therefore, antibodies and their antigen-binding fragments that compete with the antibody molecules of this disclosure for binding to the same epitope on PVRIG can be obtained using conventional techniques known to those skilled in the art.

[0288] "Specific binding" or "selective binding" refers to the binding of an antibody to a predetermined epitope on an antigen. Typically, when recombinant human PVRIG, TIGIT, or their epitopes are used as analytes and antibodies are used as ligands, the antibody binds at approximately less than 10⁻⁶ ppm when measured in an instrument using surface plasmon resonance (SPR) technology. -7 M or even smaller equilibrium dissociation constant (K) D Antibodies that recognize antigens bind to a predetermined antigen or its epitope, and the affinity of the antibody for the predetermined antigen or its epitope is at least twice that for the antibody for binding to a nonspecific antigen other than the predetermined antigen (or its epitope) or a closely related antigen (such as BSA). The term "antibody that recognizes an antigen" may be used interchangeably with "antibody that specifically binds" in this document.

[0289] "Binding affinity" is used in this paper as a measure of the strength of the non-covalent interaction between two molecules (e.g., an antibody or a portion thereof with an antigen), used to describe monovalent interactions (intrinsic activity). The binding affinity between two molecules can be determined by determining the dissociation constant (K). D Quantification can be achieved by using methods such as surface plasmon resonance (SPR) (Biacore) to measure the kinetics of complex formation and dissociation.D The rate constants corresponding to the binding and dissociation of monovalent complexes are called the binding rate constant ka (or kon) and the dissociation rate constant kd (or koff), respectively. D Through equation K D =kd / ka is related to ka and kd. The value of the dissociation constant can be determined directly by well-known methods (see Caceci et al., 1984, Byte 9: 340-362; Wong & Lohman, 1993, PNAS 90: 5428-5432). Other standard assays for assessing the binding ability of antibodies to target antigens are known in the art, including, for example, ELISA, Western blotting, RIA and flow cytometry, as well as other assays exemplified elsewhere in this document. Similarly, the specificity of an interaction can be determined and compared by Ka of the target interaction (e.g., the specific interaction between antibody and antigen). D Values ​​of K for non-target interactions (e.g., control antibodies known not to bind to PVRIG) D The value is evaluated. In some embodiments, the anti-PVRIG antibody of this disclosure has an affinity for its target that is at least 2, 10, 50, 100, 200, 500, 1,000, or 10,000 times greater than its affinity for another non-PVRIG molecule, which is not a limiting definition herein.

[0290] "Conservative modification" applies to both amino acid and nucleotide sequences. For a specific nucleotide sequence, a conservative modification refers to the substitution of one another between nucleic acids that encode the same or substantially the same amino acid sequence, or, where the nucleotide does not encode an amino acid sequence, substantially the same nucleotide sequence. For an amino acid sequence, a conservative modification refers to the substitution of other amino acids in a protein with similar characteristics (e.g., charge, side chain size, hydrophobicity / hydrophilicity, main chain conformation, and rigidity) such that changes can be made frequently without altering the protein's biological activity. Those skilled in the art will recognize that, in general, a single amino acid substitution in a non-essential region of a polypeptide does not substantially alter its biological activity (see, for example, Watson et al. (1987) Molecule Lar Biology of the Gene, The Benjamin / Cummings Pub. Co., p. 224, (4th edition)).

[0291] "Amino acid mutation" includes amino acid substitution, deletion, insertion, modification, and any combination thereof to achieve the final construct, giving it desired properties such as enhanced stability or increased activity. Amino acid sequence deletions and insertions include amino and / or carboxyl terminus deletions and amino acid insertions. Preferred amino acid mutations are amino acid substitutions. To alter the binding properties of, for example, anti-PVRIG antibodies, non-conserved amino acids can be substituted, i.e., replacing one amino acid with another amino acid with different structure and / or chemical properties. Preferred amino acid substitutions include replacing hydrophobic amino acids with hydrophilic amino acids. Amino acid substitutions include substitution with non-naturally occurring amino acids or with naturally occurring amino acid derivatives of 20 standard amino acids (e.g., 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5-hydroxylysine). Amino acid mutations can be generated using genetic or chemical methods known in the art, including site-directed mutagenesis, PCR, gene synthesis, chemical modification, etc. Amino acid mutations can occur in the CDR, FR, or Fc regions of the antibody.

[0292] For amino acid mutations in the Fc region, mutations can be introduced into the Fc sequence of the wild-type antibody disclosed herein to alter Fc-mediated related activities. These mutations include, but are not limited to: a) mutations altering Fc-mediated CDC activity; b) mutations altering Fc-mediated ADCC activity; or c) mutations altering FcRn-mediated in vivo half-life (see Leonard G. Presta, Current Opinion in Immunology 2008, 20: 460-470; Esohe E. Idusogie et al., J Immunol 2000, 164: 4178-4184; RAPHAEL A. CLYNES et al., Nature Medicine, 2000, Volume 6, Number 4: 443-446; Paul R. Hinton et al., J Immunol, 2006, 176: 346-356). Specifically, this includes: modifying the hinge region of CH1 to alter the number of cysteine ​​residues in the hinge region, such as by increasing or decreasing the number (see US5,677,425, incorporated herein by reference in its entirety). Introducing mutations that enhance binding to FcγRIIIa (to induce enhanced ADCC) and mutations that weaken binding to FcγRIIb, such as 236A, 239D, 239E, 332E, 332D, 239D / 332E, 267D, 267E, 328F, 267E / 328F, 236A / 332E, 239D / 332E / 330Y, 239D, 332E / 330L, 299T, and 297N (see US11 / 124,620, US6,737,056, incorporated herein by reference in its entirety). Fc modification can be performed to increase its biological half-life, for example, by introducing one or more of the following mutations: T252L, T254S, T256F (see US6,277,375); to increase the biological half-life, the antibody can be modified in the CH1 or CL region to contain a rescue receptor binding epitope obtained from two loops of the CH2 domain of the Fc region of IgG (see US5,869,046, US6,121,022); additional mutations for increasing serum half-life include 428L, 434A, 434S, and 428L / 434S (see US8,883,973, US6,737,056, US7,371,826, which are incorporated herein by reference in their entirety). The effector function of an antibody can be altered by replacing at least one amino acid residue in the Fc region. For example, one or more amino acids selected from amino acid residues 234, 235, 236, 237, 297, 318, 320, and 322 can be replaced to change the antibody’s affinity for the effector ligand, but retain the antigen-binding ability of the parent antibody.Effector ligands with altered affinity can be, for example, the Fc receptor or C1 component of complement (see US5,624,821, US5,648,260, incorporated herein by reference). Altering one or more amino acid residues within amino acid positions 231 and 239 alters the antibody's ability to fix complement (see WO 94 / 29351, incorporated herein by reference). Modifying the Fc region by modifying one or more amino acids at the following positions can enhance the antibody's ability to mediate antibody-dependent cytotoxicity (ADCC) and / or increase antibody affinity for the Fcγ receptor: 238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296. 6, 298, 301, 303, 305, 307, 309, 312, 315, 320, 322, 324, 326, 327, 329, 330, 331, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378, 382, ​​388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438 or 439 (see WO00 / 42072, cited in full). Furthermore, binding sites for FcγRI, FcγRII, FcγRIII, and FcRn on human IgG1 have been mapped, and variants with modified binding have been described (see Shields, RL et al. (2001), *Journal of Biochemistry*, 276: 6591-6604). Specific mutations at positions 256, 290, 298, 333, 334, and 339 have been shown to modify binding to FcyRIII. Additionally, the following combination mutants have shown modified FcγRIII binding: T256A / S298A, S298A / E333A, S298A / K224A, and S298A / E333A / K334A. In addition, mutations such as M252Y / S254T / T256E or M428L / N434S improve binding to FcRn and increase the antibody's circulating half-life (see Chan CA and Carter PJ (2010) Nature Rev Immunol 10:301-316).

[0293] The antibodies disclosed herein are modified by PEGylation or the addition of other water-soluble moieties, for example, to enhance half-life. "PEGylation" refers to the linkage of at least one PEG molecule to another molecule (e.g., a therapeutic protein). For example, PEG is a linear or branched polyether with a hydroxyl group at one end and has the following conventional structure: HO-(CH2CH2O). n -CH2CH2-OH. To couple PEG to molecules (peptides, polysaccharides, polynucleotides, and small organic molecules), PEG can be activated by preparing derivatives of PEG with one or two terminal functional groups. A common route for PEG conjugation of proteins is to activate PEG with functional groups suitable for reaction with lysine and N-terminal amino acid groups. In particular, common reactive groups involved in conjugation are the α- or ε-amino groups of lysine. The reaction of the PEGylation linker with the protein can lead to the attachment of the PEG moiety primarily at the following sites: the α-amino group at the N-terminus of the protein, the ε-amino group on the side chain of a lysine residue, or the imidazole group on the side chain of a histidine residue. Since most recombinant proteins have a single α-amino group and many ε-amino and imidazole groups, many positional isomers can be generated depending on the chemical nature of the linker.

[0294] The engineered antibodies or antigen-binding fragments disclosed herein can be prepared and purified using conventional methods. For example, cDNA sequences encoding heavy and light chains can be cloned and recombined into expression vectors. Recombinant immunoglobulin expression vectors can stably transfect CHO cells. Mammalian expression systems lead to glycosylation of the antibody, particularly at the highly conserved N-terminus of the Fc region. Stable clones are obtained by expressing antibodies that specifically bind to human antigens. Positive clones are scaled up in serum-free medium in a bioreactor to produce antibodies. Cultures secreting antibodies can be purified and collected using conventional techniques. Antibodies can be concentrated by filtration using conventional methods. Soluble mixtures and polymers can also be removed using conventional methods, such as molecular sieving or ion exchange. The resulting product must be immediately frozen, e.g., at -70°C, or lyophilized.

[0295] "Giving," "applying," and "treatment," when applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, refer to the contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid, such as in therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Cellular treatment includes contact between a reagent and a cell, as well as contact between a reagent and a fluid, wherein the fluid is in contact with the cell. "Giving," "applying," and "treatment" also mean treatment, such as of cells, by means of a reagent, diagnostic agent, conjugate composition, or by means of another cell in vitro and ex vivo. When applied to humans, veterinary, or research subjects, it refers to therapeutic treatment, preventative or prophylactic measures, research, and diagnostic applications.

[0296] "Treatment" means administering an oral or topical therapeutic agent, such as an antibody or pharmaceutical composition thereof comprising any of the present disclosure, to a subject who has, is suspected of having, or is predisposed to having one or more proliferative diseases or their symptoms, and the therapeutic agent is known to have a therapeutic effect on these symptoms. Typically, the therapeutic agent is administered in a treated subject or population in an amount that effectively relieves symptoms of one or more diseases, whether by inducing the regression of such symptoms or inhibiting their development to any clinically measurable degree. The amount of therapeutic agent that effectively relieves any specific disease symptom (also referred to as the "therapeuticly effective amount") can vary depending on a variety of factors, such as the subject's disease state, age, and weight, and the drug's ability to produce the desired therapeutic effect in the subject. Whether the disease symptoms have been relieved can be evaluated using any clinical test that a physician or other healthcare professional typically uses to assess the severity or progression of the symptoms. Although the embodiments of this disclosure (e.g., treatment methods or products) may be ineffective in alleviating the symptoms of the target disease in a particular subject, they should reduce the symptoms of the target disease in a statistically significant number of subjects, as determined by any statistical test known in the art, such as the Student t-test, chi-square test, U-test according to Mann and Whitney, Kruskal-Wallis test (H-test), Jonckheere-Terpstra test, and Wilcoxon test.

[0297] "Effective amount" includes an amount sufficient to improve or prevent the symptoms or condition of a medical condition. Effective amount also means an amount sufficient to allow or facilitate diagnosis. The effective amount used on a subject may vary depending on factors such as the condition to be treated, the subject's overall health, the route and dosage of administration, and the severity of side effects. Effective amount may be the maximum dose or administration regimen that avoids significant side effects or toxicity. Subjects in this disclosure may be animal or human subjects.

[0298] "Host cell" includes individual cells or cell cultures that may be, or have been, recipients of vectors for incorporating polynucleotide inserts. Host cells include progeny of a single host cell, and progeny may not necessarily be identical to the original parent cell (in morphology or genomic DNA complementation) due to natural, accidental, or intentional mutations. Host cells include cells transfected and / or transformed in vivo with the polynucleotides of this disclosure. "Cell," "cell line," and "cell culture" are used interchangeably, and all such names include their progeny. It should also be understood that, due to intentional or unintentional mutations, all progeny may not be exactly identical in DNA content. This includes mutant progeny with the same function or biological activity as those screened from the originally transformed cells.

[0299] "Vector" refers to a construct capable of being delivered into a host cell and, in some embodiments, expressing one or more target genes or sequences. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, granules or phage vectors, DNA or RNA expression vectors bound to cationic condensers, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells such as production cells.

[0300] "Optional" or "optionally" means that the event or circumstances described below may, but do not have to, occur, including the circumstances in which the event or circumstances may or may not occur. For example, "optionally contains 1-3 antibody heavy chain variable regions" means that the antibody heavy chain variable regions of a particular sequence may, but do not have to, be present.

[0301] "Pharmaceutical composition" means a mixture containing one or more antibodies or antigen-binding fragments described herein, or their physiologically / pharmacologically acceptable salts or prodrugs, along with other chemical components, such as physiologically / pharmacologically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to a living organism, thereby promoting the absorption of the active ingredient and the exertment of its biological activity.

[0302] "Pharmaceutically acceptable carriers" or "pharmaceutically acceptable excipients" include any material that, when combined with an active ingredient, allows that ingredient to retain its biological activity and does not react with the subject's immune system. Examples include, but are not limited to, any standard pharmaceutical carrier, such as phosphate-buffered saline solutions, water, emulsions such as oil / water emulsions, and various types of wetting agents. In some embodiments, the diluent for aerosol or parenteral administration is phosphate-buffered saline (PBS) or physiological (0.9%) saline. Compositions containing such carriers are formulated using well-known conventional methods (see, for example, Remington's Pharmaceutical Sciences, 18th edition, A. Gennaro, editor, Mack Publishing Co., Easton, PA, 1990; and R. Remington, The Science and Practice of Pharmacy, 20th edition, Mack Publishing, 2000).

[0303] The "PVRIG binding protein" or "PVRIG antibody" disclosed herein may, for example, comprise one or more effector molecules in a conjugated manner. The "effector molecules" include, for example, antitumor agents, drugs, toxins, bioactive proteins (e.g., enzymes), other antibodies or antibody fragments, synthetic or naturally occurring polymers, nucleic acids and fragments thereof (e.g., DNA, RNA and fragments thereof), radionuclides, particularly radioiodides, radioisotopes, chelated metals, nanoparticles, and reporter groups such as fluorescent compounds or compounds detectable by NMR or ESR spectroscopy. When the effector molecule is a polymer, it can typically be a synthetic or naturally occurring polymer, such as optionally substituted linear or branched polyalkylene, polyolefin, or polyoxyalkylene polymers, or branched or unbranched polysaccharides, such as homopolymers or heteropolymers. Specific optional substituents that may be present on the aforementioned synthetic polymers include one or more hydroxyl, methyl, or methoxy groups. Specific examples of synthetic polymers include optionally substituted linear or branched poly(ethylene glycol), poly(propylene glycol), poly(vinyl alcohol), or derivatives thereof, particularly optionally substituted poly(ethylene glycol) such as methoxylated poly(ethylene glycol) or derivatives thereof. Specific naturally occurring polymers include lactose, amylose, dextran, glycogen, or derivatives thereof. In one embodiment, the polymer is albumin or a fragment thereof, such as human serum albumin or a fragment thereof. The conjugation of the polymer with a PVRIG-binding protein or a PVRIG antibody can be achieved using conventional methods.

[0304] The following examples are used for further description, but these examples are not intended to limit the scope.

[0305] Experimental methods not specifically described in the examples or test cases are generally performed under standard conditions or as recommended by the raw material or product manufacturer. See Sambrook et al., Molecular Cloning, Laboratory Handbook, Cold Spring Harbor Laboratory; Methods in Modern Molecular Biology, Ausubel et al., Greene Publishing Association, Wiley Interscience, NY. Reagents not specifically named are commercially available, standard reagents.

[0306] Example 1. PVRIG protein sequence and preparation

[0307] The recombinant human PVRIG (h-PVRIG-his) protein with his tag, the recombinant human PVRIG (h-PVRIG-mIgG2a Fc) protein with mouse IgG2a Fc tag, and the mouse PVRIG (m-PVRIG-hIgG1 Fc) protein with human IgG1 Fc tag were purified commercial protein reagents purchased from Acrobiosystems, and their sequences are shown in Table 1.

[0308] Table 1. Amino acid sequence of recombinant proteins

[0309] name amino acid sequence start and end Genbank login ID h-PVRIG-his Thr41-Asp171 Q6DKI7-1 h-PVRIG-mIgG2a Fc Thr41-Asp171 Q6DKI7-1 m-PVRIG-hIgG1 Fc Ser35-Asp165 A0A1B0GS01-1

[0310] The recombinant protein sequence of cynomolgus monkey PVRIG (cyno-PVRIG-his) with the his tag is as follows:

[0311]

[0312] The recombinant protein was transiently transfected into HEK293 cells using standard methods. The supernatant was collected and purified using Ni-NTA. The resulting product was cyno-PVRIG-his.

[0313] Example 2. Generation of anti-human PVRIG single-domain antibody

[0314] Anti-human PVRIG monoclonal single-domain antibodies were produced by immunizing camels. The immunoantigen was the his-tagged recombinant human PVRIG protein (h-PVRIG-his). Emulsification was performed using Freund's adjuvant (Sigma, Lot No.: F5881 / F5506): initial immunizations used Freund's complete adjuvant (CFA), with subsequent booster immunizations using Freund's incomplete adjuvant (IFA). Immunization was administered on days 0, 14, 28, and 42. Blood samples were collected on day 56 for serum analysis, and the antibody titer in the camel serum was determined using ELISA.

[0315] 200 mL of camel peripheral blood was collected, and PBMCs were isolated. RNA was extracted from the cells using Trizol and reverse transcribed into cDNA. The gene of the variable region of the single-domain antibody was amplified by PCR and cloned into a phage vector to establish a phage library of anti-human PVRIG single-domain antibody.

[0316] Phage libraries were diluted and blocked with BSA and co-incubated with Dynabeads (M-280, Invitrogen). Phages after negative screening incubation were collected. Dynabeads were blocked with biotin-labeled, his-tagged human PVRIGs. The phage suspension collected after negative screening was incubated with the Dynabeads, and the phages were eluted with trypsin. After three rounds of screening, 400 clones obtained in the third round were sequenced. The heavy chain sequences of five single-domain antibodies are shown in Table 2, and the CDRs for different coding rules are shown in Table 3.

[0317] Table 2. Heavy chain variable region (HCVR) sequence of anti-PVRIG antibody

[0318]

[0319] Table 3. Heavy chain complementarity-determining region (CDR) sequence of anti-PVRIG antibody

[0320]

[0321]

[0322] Example 3. Preparation of full-length anti-PVRIG antibody

[0323] The heavy chain variable regions of the five antibodies in Example 2 were linked to the Fc region of the human IgG4 heavy chain to construct a full-length anti-PVRIG antibody. The heavy chain Fc region includes a hinge region and carries S228P, F234A, L235A, and K447A mutations (Eu nomenclature system). The anti-PVRIG antibody CPA.7.021 shown in WO2016134333 was screened from an antibody phage library; its subtype is IgG1, which binds well to human PVRIG but not to cynomolgus monkey PVRIG. The heavy and light chain variable regions of CPA.7.021 were linked to the constant region of the human IgG4 heavy chain (carrying S228P, F234A, L235A, and K447A mutations) and the constant region of the human Kappa light chain, respectively, to construct the positive antibody Tab5.

[0324] The full-length sequences of the five antibodies and the positive antibody are shown in Table 4.

[0325] Table 4. Full-length heavy and light chain sequences of fully human anti-PVRIG antibodies

[0326]

[0327]

[0328] (Note: The underlined part represents the Fc domain of the heavy chain)

[0329] The above sequence was synthesized, digested with BamHI and XhoI, and then inserted into the pcDNA3.1 expression vector (Life Technologies Cat. No. V790-20) via the BamHI / XhoI restriction site. The expression vector and transfection reagent PEI (Polysciences, Inc. Cat. No. 23966) were transfected into HEK293 cells (Life Technologies Cat. No. 11625019) at a 1:2 ratio and incubated in a CO2 incubator for 4-5 days. The expressed antibody was recovered by centrifugation, purified using standard methods, and the target antibody was obtained upon detection.

[0330] Example 4. Binding experiment of anti-PVRIG antibody to recombinant PVRIG protein

[0331] ELISA assays are used to detect the binding properties of anti-PVRIG antibodies. The antibody is directly coated with a his-tagged recombinant PVRIG protein. After antibody addition, the antibody-antigen binding activity is detected by adding a secondary antibody (HRP-conjugated anti-primary Fc antibody) and the HRP substrate TMB.

[0332] Human, cynomolgus monkey, or mouse PVRIG protein was coated onto 96-well microplates at a concentration of 1 μg / mL (100 μL per well) and incubated overnight at 4°C. Wash three times with 250 μL of wash buffer per well, vortexing for 10 seconds each time to ensure thorough washing. Add 300 μL / well blocking buffer (PBS + 0.05% Tween 20 + 1% BSA) and incubate at room temperature for 1 hour. Wash three times with 250 μL of wash buffer per well, vortexing for 10 seconds each time to ensure thorough washing. Add 100 μL of diluted anti-PVRIG antibody to each well. Incubate at 37°C for 1 hour. Wash three times with 250 μL of wash buffer per well. Add 100 μL of HRP-labeled anti-human IgG secondary antibody (Sigma, A8667) to each well. Incubate at 37°C for 1 hour. Wash three times with 250 μL of wash buffer per well. Add 100 μL of TMB to each well and incubate in the dark for 15 minutes. Add 50 μL of 0.16M sulfuric acid to each well. Use a Thermo MμLtiSkanFc microplate reader to read the OD value at 450 nm and calculate the EC50 of the anti-PVRIG antibody binding to PVRIG. 50 Values. All antibodies showed strong binding ability to human or cynomolgus monkey PVRIG recombinant protein, but not to mouse PVRIG recombinant protein.

[0333] Table 5. Results of binding experiments of anti-PVRIG antibodies to recombinant PVRIG proteins from different strains.

[0334]

[0335] Example 5. Assay of anti-PVRIG antibody binding to cells expressing PVRIG

[0336] The binding properties of anti-PVRIG antibodies were detected using flow cytometry (FACS). Cell lines overexpressing human or cynomolgus monkey PVRIG were constructed, and the activity of antibody-antigen binding was detected by adding secondary antibody after antibody addition.

[0337] Expression plasmids carrying human or cynomolgus monkey PVRIG gene sequences were transfected into HEK293 cells, and stable overexpression monoclonal cell lines were obtained by antibiotic selection and indefinite dilution. 2 x 10⁶ cells were seeded per well in 96-well plates. 5Overexpressing cells were centrifuged at 300g for 5 minutes, the supernatant was removed, and 100 μL of the test antibody was added. The cells were incubated at 4°C for 1 hour. After centrifugation to remove the supernatant, the cells were washed three times with 200 μL of washing buffer (PBS + 2% FBS). 100 μL of 1:500 diluted anti-human IgG secondary antibody (Invitrogen, A-11013) labeled with Alexa Fluor 488 was added, and the cells were incubated at 4°C for 1 hour. After centrifugation to remove the supernatant, the cells were washed three times with 200 μL of washing buffer (PBS + 2% FBS). The cells were resuspended in 100 μL of PBS and analyzed by flow cytometry (BD FACS Calibur or BD FACS Canto_II). All antibodies showed strong binding ability to human or cynomolgus monkey PVRIG expressed on the cell surface, significantly stronger than the positive antibody Tab5, while Tab5 did not bind to cynomolgus monkey PVRIG at all.

[0338] Table 6. Results of cell binding assays of anti-PVRIG antibodies against different strains of PVRIG.

[0339]

[0340] (Note: NA, not available, indicates that the binding is too strong, and the antibody does not dissociate even under low concentration conditions, making it impossible to obtain an accurate EC.) 50 。 )

[0341] Example 6. Experiment on the blocking of PVRIG and PVRL2 binding by anti-PVRIG antibody

[0342] In this experiment, the ability of the screened anti-PVRIG antibodies to block the binding of human PVRIG and its ligand human PVRL2 was detected through in vitro blocking experiments. Specifically, recombinant human PVRIG protein (h-PVRIG-mIgG2a Fc) tagged with mouse IgG2a Fc was coated onto a 96-well ELISA plate. After the anti-PVRIG antibody was added and fully bound to occupy the epitope, PVRL2 (PV2-H52E2, AcroBiosystem) tagged with his was added. The binding amount of PVRIG to PVRL2 was calculated by detecting the his tag, and the IC50 of the anti-PVRIG antibody blocking the PVRIG active site was calculated. 50 value.

[0343] Coat 96-well microplates with h-PVRIG-mIgG2a Fc protein at a concentration of 1 μg / mL (100 μL per well) and incubate overnight at 4°C. Wash three times with 250 μL of washing buffer per well, vortexing for 10 seconds each time to ensure thorough washing. Add 300 μL / well blocking buffer and incubate at room temperature for 1 hour. Wash three times with 250 μL of washing buffer per well, vortexing for 10 seconds each time to ensure thorough washing. Add 50 μL of diluted anti-PVRIG test antibody and 50 μL of his-tagged ligand PVRL2 to each well and incubate at 37°C for 1 hour. Wash three times with 250 μL of washing buffer per well. Add 100 μL of HRP-labeled anti-his-tagged secondary antibody (Genscrpit) diluted 1:2000 to each well. Incubate at 37°C for 1 hour. Wash three times with 250 μL of washing buffer per well. Add 100 μL of TMB to each well and incubate in the dark for 15 minutes. Add 50 μL of 0.16 M sulfuric acid to each well. Read the OD value at 450 nm using a Thermo Microplate reader (MμLtiSkanFc) and calculate the IC50 of the anti-PVRIG antibody against the binding of PVRIG to PVRL2. 50 value.

[0344] The results showed that all the detected antibodies could strongly inhibit the binding of human PVRIG to human PVRL2.

[0345] Table 7. Antibody blocking assays for human PVRIG / PVRL2 binding

[0346] Antibody number <![CDATA[ELISA IC 50 (nM)]]> 20 1.18 30 1.11 38 0.93 39 0.76 151 0.37 Tab5 1.16 IgG4 Unblocked

[0347] Example 7. Affinity determination of anti-PVRIG antibody with PVRIG

[0348] The Protein A biosensor (Fortebio, #18-5010) was wetted by immersing it in 200 μL of KB buffer (PBS, pH 7.4, 0.02% Tween-20, 0.1% BSA) for 60 seconds. Then, the anti-PVRIG antibody was diluted to 10 μg / mL with KB buffer, and the sensor was placed in 200 μL of this solution until a reading of 1.2 nm was obtained. The sensor was then immersed in KB buffer for 100 seconds to elute excess antibody. Human PVRIG with the his tag was serially diluted with KB buffer in a 2-fold gradient to between 64 nM and 4 nM. The sensor was placed in this solution for 300 seconds to bind. The sensor was then placed in KB buffer for 600 seconds to dissociate. The affinity of the anti-PVRIG antibody for human PVRIG was fitted using a dynamic 1:1 binding modality, as shown in Table 8.

[0349] The results showed that all the detected antibodies had high affinity for human PVRIG.

[0350] Table 8. Affinity of anti-PVRIG antibodies to human PVRIG

[0351] Antibody number Kon(1 / Ms) Koff(1 / s) KD(M) 20 3.43E+05 8.07E-05 2.36E-10 30 2.84E+05 2.05E-04 7.23E-10 38 1.32E+05 2.87E-04 2.17E-09 39 2.42E+05 1.69E-04 6.96E-10 151 2.61E+05 5.22E-05 2.00E-10 Tab5 7.37E+05 1.61E-05 2.19E-10

[0352] Example 8. Anti-PVRIG antibody reporter gene cell activity assay

[0353] First, the plvx-OS8 (G418 resistant) plasmid was constructed and transfected into 293F cells. G418 selection was performed, and the expression of OS8 in cloned cells was detected by flow cytometry. At the same time, the activation of Jurkat cells by OS8 was detected. Clones with moderate activation were selected to obtain the 293F-OS8 cell line. The plvx-PVRL2 plasmid was constructed and used to infect 293F-OS8 cells. Clones with the highest PVRL2 expression level were selected by flow cytometry to obtain the 293F-OS8-PVRL2 cell line.

[0354] Secondly, plvx-NFAT-Luc (Hygromycin resistant) was constructed, packaged into a lentivirus, and used to infect Jurkat E6.1 cells. Hygromycin was added to screen for resistant clones, and OKT3 was used to stimulate clones to screen for clones with moderate Luciferase signal, thus obtaining the Jurkat-NFAT-Luc cell line. The plvx-PVRIG (Puromycin resistant) vector was constructed, packaged into a lentivirus, and used to infect Jurkat-NFAT-Luc cells. Flow cytometry was used to screen for clones with the highest PVRIG expression, thus obtaining the Jurkat-NFAT-Luc-PVRIG cell line.

[0355] 1E4 Jurkat-NFAT-Luc-PVRIG cells were incubated with the test antibody at 37°C for 20 minutes. 1E5 293F-OS8-PVRL2 cells were then added and incubated at 37°C for 5 hours. After centrifugation to remove the supernatant, cells were lysed with Luciferase buffer (Promega, E6130) and fluorescence values ​​were detected. EC50 was calculated. 50 The in vitro cell activity of the anti-PVRIG antibody was evaluated. Experimental results are as follows: Figure 1 As shown in Table 9.

[0356] The results showed that all the detected antibodies had a strong ability to activate Luciferase in Jurkat cells, with an activity 3.7-18.5 times that of the positive antibodies, proving that these antibodies can bind to PVRIG and block the binding of PVRL2 to PVRIG.

[0357] Table 9. Results of anti-PVRIG antibody reporter gene cell activity assay

[0358] Antibody strain number <![CDATA[PVRIG reporter gene cell viability assay EC 50 (nM)]]> 20 0.04 30 0.06 38 0.20 39 0.06 151 0.04 Tab5 0.74 IgG4 No combination

[0359] Example 9. NK cell killing assay using anti-PVRIG antibody

[0360] PVRIG is expressed on NK cells, while PVRL2 is expressed in many tumor cells (including K562 cells). Anti-PVRIG antibodies can relieve the inhibitory effect of tumor cells on NK cell activity by blocking the binding of PVRL2 to PVRIG.

[0361] The cultured NK92 cell line (NK cells from human patients with malignant non-Hodgkin lymphoma) was washed twice with washing buffer (including RPMI 1640, 5% FBS, 10 ng / mL IL-2) and resuspended to 2 × 10⁻⁶. 6 A density of cells / mL was used. 50 μL was added to each well of a 96-well plate (total 1 × 10⁻⁶). 5 NK92 cells (number of cells). Add 50 μL of 20 nM or 100 nM of the test antibody and incubate at 37°C for 30 minutes. Wash twice with washing buffer and resuspend to 2 × 10⁻⁶. 5 Density of cells / mL. Add 50 μL (total 1×10⁻⁶). 4 (Number) human chronic myeloid leukemia K562 cells were used to achieve a NK92 cell to K562 cell ratio of 10:1. Cells were incubated at 37°C for 4 hours. Cytotoxic activity was measured using the CytoTox-Glo cytotoxicity system (Promega, G9292). First, 50 μL of AAF-Glo reagent was added, and the cells were incubated at room temperature for 15 minutes. The fluorescence of K562 cells killed by NK92 cells was measured. Then, 50 μL of lysis buffer was added, and the cells were incubated at room temperature for 15 minutes. All cells in the lysed wells were then lysed, and the fluorescence of all cells was measured. Three control groups were prepared: a sample containing only culture medium (control group 1), a sample containing only NK92 cells (control group 2), and a 150 μL sample containing only K562 cells (control group 3). The same procedure was performed on these control groups.

[0362] The lethality is calculated using the following formula:

[0363] Killing activity (%) = {[(R–BG)-(T–BG)-(E–BG)] / [(TL–BGL)–(T–BG)]}×100

[0364] Wherein, R is the fluorescence value after adding AAF-Glo, BG is the fluorescence value of control group 1 after adding AAF-Glo, E is the fluorescence value of control group 2 after adding AAF-Glo, T is the fluorescence value of control group 3 after adding AAF-Glo, TL is the fluorescence value of control group 3 after adding lysis buffer, and BGL is the fluorescence value of control group 1 after adding lysis buffer.

[0365] Experimental results are as follows Figure 2As shown in Table 10, all detected anti-PVRIG antibodies can significantly activate NK92 cells and kill K562 cells.

[0366] Table 10. NK cell killing assay with anti-PVRIG antibody

[0367]

[0368] Example 10. Mixed Lymphocyte Response (MLR) Assay with Anti-PVRIG Antibody

[0369] PVRIG is expressed on T cells, while PVRL2 is expressed in dendritic cells (DCs). Anti-PVRIG antibodies can activate T cells by blocking the binding of PVRL2 to PVRIG, thereby relieving the inhibitory effect of dendritic cells on T cells.

[0370] Mixed lymphocyte reaction refers to the phenomenon where lymphocytes from two unrelated individuals, with normal function, can mutually stimulate T cell proliferation when cultured together in vitro due to differences in major histocompatibility antigens. PBMCs were isolated from peripheral blood of the first individual and cultured in RPMI 1640 medium containing 10% FBS. Cytokines were added to a final concentration of 50 ng / mL GM-CSF (Peprotech, 300-03-100UG) and 50 ng / mL IL-4 (Peprotech, 200-04-100UG), with fresh medium containing cytokines added every 2-3 days. After 6 days of culture, 1 μg / mL LPS (Sigma, L2880-25MG) was added and incubated for 24 hours, and the differentiated dendritic cells (DCs) were collected. PBMCs were then isolated from peripheral blood of the second individual and cultured using EasySep human CD3... + T-cell isolation kit (Stemcell, 17952) for isolating CD3 + T cells. Adjusting CD3 + The density of T cells and DC cells was such that 1×10⁻⁶ cells were added to each well. 5 CD3 + T cells and 2×10 4 Add the test antibody to each DC cell, incubate at 37°C for 120 h, collect the supernatant, and detect the IFNγ content in the supernatant using an ELISA kit (R&D, DY202).

[0371] Experimental results are as follows Figure 3 As shown in Table 11, all detected anti-PVRIG antibodies significantly activated T cells to secrete IFNγ compared to the control antibody IgG4. Furthermore, at low doses (e.g., 4 nM, 20 nM), the antibodies disclosed herein were more effective than the positive control Tab5.

[0372] Table 11. IFNγ secretion levels in mixed lymphocyte responses to anti-PVRIG antibodies

[0373]

[0374] Example 11. Humanization of anti-PVRIG antibody

[0375] Based on the typical VH structures of the obtained camel single-domain antibodies 20, 30, 38, 39, and 151, the heavy chain variable region sequences were compared with the antibody GermLine database to obtain highly homologous human genotype templates. The frame region of the camel single-domain antibody was replaced with the heavy chain frame region of the human genotype template, while the CDR (according to the Kabat numbering system) was retained. This was then recombined with the Fc region of human IgG (IgG4Fc with S228P, F234A, L235A, and K447A mutations). Based on the three-dimensional structure of the camel single-domain antibody, reverse mutations were performed on embedded residues, residues that directly interact with the CDR region, and residues that significantly affect the conformation of the variable region. The chemically unstable amino acid residues in the CDR region were also optimized to generate a series of humanized single-domain antibodies. The human genotype templates for each single-domain antibody and the heavy chain variable region sequences of the humanized antibodies are shown in Tables 12-16.

[0376] Table 12. Human lineage template and humanized antibody sequence for antibody 20

[0377]

[0378]

[0379] According to Table 12, antibodies 20H1-20H5 contain CDR1 as shown in TDCMG (SEQ ID NO: 7), CDR2 as shown in HIDSDGIPRYVDSVKG (SEQ ID NO: 8), and CDR3 as shown in GFKFDEDYCAPND (SEQ ID NO: 150).

[0380] Table 13. Human lineage template and humanized antibody sequence of antibody 30

[0381]

[0382] According to Table 13, antibodies 30H1-30H5 contain CDR1 as shown in GDCMG (SEQ ID NO: 10), CDR2 as shown in TIDNAGRIKYADSVKG (SEQ ID NO: 11), and CDR3 as shown in GWTFGGQCSPAD (SEQ ID NO: 151).

[0383] Table 14. Human lineage template and humanized antibody sequence of antibody 38

[0384]

[0385]

[0386] Table 15. Human lineage template and humanized antibody sequence of antibody 39

[0387]

[0388] Table 16. Human lineage template and humanized antibody sequence of antibody 151

[0389]

[0390]

[0391] The variable region of the humanized antibody heavy chain was linked to the Fc region of the human IgG4 heavy chain to construct a full-length anti-PVRIG antibody. The Fc region of the heavy chain includes a hinge region and carries S228P, F234A, L235A, and K447A mutations.

[0392] >Human IgG4 heavy chain Fc region (S228P / F234A / L235A / K447A)

[0393]

[0394] >Human IgG4 heavy chain Fc region (S228P / K447A)

[0395]

[0396] The antibody was expressed and purified using conventional methods, and the target antibody was obtained after testing.

[0397] Example 12. Binding assay of humanized anti-PVRIG antibody to cells expressing PVRIG

[0398] Following the method in Example 5, the binding of the humanized anti-PVRIG antibody to human or cynomolgus monkey PVRIG was detected by flow cytometry. The experimental results are shown in Table 17.

[0399] Table 17. FACS binding assay results of humanized anti-PVRIG single-domain antibodies against different strains of PVRIG.

[0400]

[0401]

[0402] (Note: NT stands for not tested.)

[0403] Example 13. Determination of the affinity between humanized anti-PVRIG antibody and PVRIG.

[0404] The affinity of the humanized anti-PVRIG antibody for human PVRIG was tested according to the method in Example 7. The results are shown in Table 18. All antibodies listed in the table have high affinity for human PVRIG.

[0405] Table 18. Affinity of humanized anti-PVRIG antibodies to human PVRIG

[0406] Antibody number Kon(1 / Ms) Koff(1 / s) KD(M) 20H5 1.93E+05 1.35E-05 6.98E-11 30H2 1.69E+05 3.25E-04 1.92E-09 30H3 1.48E+05 3.58E-04 2.41E-09 39H1 2.64E+05 8.65E-04 3.28E-09 39H2 1.80E+05 1.24E-04 6.92E-10 39H4 1.89E+05 9.11E-05 4.82E-10 151H7 1.57E+05 1.88E-04 1.20E-09

[0407] Example 14. Humanized anti-PVRIG antibody reporter gene cell activity assay

[0408] The activity of the humanized anti-PVRIG antibody in reporter cells was detected according to the method in Example 8. The experimental results are as follows: Figures 4A-4B As shown in Table 19. All antibodies listed in the table have the ability to activate Jurkat cells.

[0409] Table 19. Reporter gene activity assay of humanized anti-PVRIG antibody

[0410]

[0411] Example 15. Experiment on the NK cell killing ability of humanized anti-PVRIG antibody

[0412] The activation ability of the humanized anti-PVRIG antibody on NK cells was detected according to the method in Example 9. The experimental results are as follows: Figures 5A-5B As shown in Tables 20 and 21. The results show that the humanized anti-PVRIG antibodies of this disclosure have a significant ability to activate NK cells and promote the killing of target cells K562 by NK cells.

[0413] Table 20. NK cell killing assay of humanized anti-PVRIG antibody

[0414]

[0415] Table 21. NK cell killing assay of humanized anti-PVRIG antibody

[0416]

[0417]

[0418] Example 16. Preparation of anti-PVRIG / TIGIT bispecific antibody

[0419] To explore the effects of different anti-PVRIG / TIGIT bispecific antibodies on antibody function, anti-PVRIG single-domain antibody 151 was linked to the N-terminus or C-terminus of the heavy or light chain of anti-TIGIT antibody 1708 via the GGGGSGGGGS (SEQ ID NO: 152) linker. This resulted in four anti-PVRIG / TIGIT bispecific antibodies, named 1708-151-1, 1708-151-2, 1708-151-3, and 1708-151-4, corresponding to 151 being linked to the N-terminus of the heavy chain, C-terminus of the heavy chain, N-terminus of the light chain, and C-terminus of the light chain of 1708, respectively. Anti-TIGIT antibody 1708 was of human IgG4 subtype and contained the S228P (Eu nomenclature system) mutation. The sequences of anti-TIGIT antibody 1708 and the bispecific antibody formed with 151 are shown in Table 22 below. The sequence information of the anti-TIGIT antibody is shown in Tables 23-24. The TIGIT antibody in WO2019062832A is cited in full here.

[0420] Table 22. First and second polypeptide chain sequences of anti-PVRIG / TIGIT bispecific antibodies

[0421]

[0422]

[0423]

[0424] Table 23. CDR region sequences of anti-TIGIT antibody heavy and light chains (Kabat numbering rules)

[0425]

[0426]

[0427] Table 24. Heavy chain VH and light chain VL sequences of anti-TIGIT antibody

[0428]

[0429] The antibody was transiently transfected, expressed, and purified using conventional methods, and the full-length anti-PVRIG / TIGIT bispecific antibody of this disclosure was obtained after identification. The expression level and purity of the bispecific antibody are shown in Table 25. When nanobodies were conjugated to ordinary monoclonal antibodies, good expression levels and purity were observed at both the N-terminus and C-terminus of the heavy and light chains.

[0430] Table 25. Expression level and purity of anti-PVRIG / TIGIT bispecific antibody

[0431] Antibody number Expression level (mg / L) SEC purity (%) 1708-151-1 140 97.6 1708-151-2 108 95.7 1708-151-3 160 94.7 1708-151-4 158 96.2

[0432] Example 17. Binding of anti-PVRIG / TIGIT bispecific antibody to PVRIG and TIGIT and its blocking effect on the corresponding ligands.

[0433] A) Binding of different configurations of bispecific antibodies to human PVRIG and their blocking of ligand PVRL2

[0434] The experiments were conducted according to the methods of Examples 4, 5, and 6, and the results are shown in Table 26. The results show that the binding of different configurations of bispecific antibodies to recombinant human PVRIG protein and cells overexpressing human PVRIG, as well as their blocking of PVRL2 binding to PVRIG, are basically consistent and without difference.

[0435] Table 26. Binding and ligand blocking of PVRIG by bispecific antibodies

[0436]

[0437] B) Binding of different configurations of bispecific antibodies to human TIGIT and their blocking of ligand PVR

[0438] Following the methods of Examples 4, 5, and 6 (with the corresponding receptors and ligands replaced by human TIGIT and human PVR), experiments were conducted, and the results are shown in Table 27. The results indicate that the binding of different conformations of the bispecific antibody and the anti-TIGIT antibody to recombinant human TIGIT protein and cells overexpressing human TIGIT, as well as their blocking of TIGIT binding to its ligand PVR, were essentially consistent and without difference. The conjugation mode of the anti-PVRIG antibody 151 had virtually no effect on the binding of the anti-TIGIT antibody to TIGIT.

[0439] Table 27. Binding and ligand blocking of TIGIT by bispecific antibodies

[0440]

[0441] Based on the data in Tables 24-25, we found that anti-PVRIG antibodies, whether linked to the N-terminus or C-terminus of the heavy or light chain of anti-TIGIT antibodies, maintained the binding of PVRIG and TIGIT and the blocking of ligands, and all showed good expression levels and purity.

[0442] Example 18. Preparation of humanized anti-PVRIG / TIGIT bispecific antibody

[0443] Different humanized anti-PVRIG antibodies (20H5, 30H2, 39H2, 151H7, 151H8) were linked to the N-terminus of the heavy chain of anti-TIGIT antibody 1708, i.e., a bispecific antibody structure similar to 1708-151-1 was constructed to create bispecific antibodies, the sequences of which are shown in Table 28.

[0444] Table 28. Full-length sequences of the first and second polypeptide chains of humanized anti-PVRIG / TIGIT bispecific antibodies

[0445]

[0446]

[0447]

[0448] The antibody was transiently transfected, expressed, and purified using conventional methods, and the target bispecific antibody was obtained after identification.

[0449] Example 19. Binding of humanized anti-PVRIG / TIGIT bispecific antibody to PVRIG and TIGIT and its blocking of corresponding ligands

[0450] Following the methods described in Examples 4, 5, and 6, the binding of the humanized anti-PVRIG / TIGIT bispecific antibody to human and cynomolgus monkey PVRIG and its ligand blocking effect on human PVRIG were detected. The results are shown in Table 29. Figures 6A-6E As shown in the figure. The results indicate that all humanized bispecific antibodies can bind to human PVRIG and block PVRIG from binding to PVRL2. 1708-151H8 showed weaker binding to cynomolgus monkey PVRIG.

[0451] Table 29. Binding and ligand blocking of PVRIG by humanized bispecific antibodies

[0452]

[0453] Similar to Examples 4, 5, and 6, the binding of the humanized anti-PVRIG / TIGIT bispecific antibody to human and cynomolgus monkey TIGIT, and its blocking effect on the binding of human TIGIT to its ligand, were tested. In this case, the PVRIG protein was replaced with TIGIT, and PVRL2 was replaced with PVR. The results are shown in Table 30 and... Figures 7A-7E As shown in the figure. The results indicate that all the bispecific antibodies can bind to human and cynomolgus monkey TIGIT and block TIGIT binding to PVR.

[0454] Table 30. Binding and ligand blocking of TIGIT by humanized bispecific antibodies

[0455]

[0456]

[0457] The affinity of humanized bispecific antibodies for human PVRIG, cynomolgus monkey PVRIG, and human TIGIT was detected using Biacore. The humanized bispecific antibodies were captured on the Protein A biosensor chip (GE lifesciences, 29127557) of a Biacore instrument (Biacore X100, GE), and then a series of concentration gradients of human PVRIG antigen (AcroBiosystem, PVG-H52H4), cynomolgus monkey PVRIG antigen (SEQ ID NO:1), or human TIGIT antigen (AcroBiosystem, TIT-H52H3) were flowed over the chip surface. The reaction signals were detected in real time using the Biacore instrument (Biacore X100, GE) to obtain binding and dissociation curves. The experimental data were fitted using a (1:1) binding model with Biacore X100 evaluation software 2.0 GE software to obtain affinity values, as shown in Table 31.

[0458] Table 31. Affinity of humanized bispecific antibodies to human PVRIG, cynomolgus monkey PVRIG, and human TIGIT

[0459]

[0460] Example 20. Mixed lymphocyte reaction (MLR) assay with humanized anti-PVRIG / TIGIT bispecific antibody

[0461] The activation ability of the humanized anti-PVRIG / TIGIT bispecific antibody on T cells was detected according to the method in Example 10. The experimental results are as follows: Figure 8 As shown in Table 32, the results indicated that the humanized anti-PVRIG / TIGIT bispecific antibody 1708-151H8 significantly activated T cells and promoted T cell secretion of IFNγ. Importantly, the activity of the bispecific antibody was stronger than that of anti-PVRIG antibody 151H8 alone and anti-TIGIT antibody 1708 alone.

[0462] Table 32. IFNγ secretion levels in mixed lymphocyte responses to humanized bispecific antibodies

[0463]

[0464] Example 21. Evaluation of the antitumor effect of anti-PVRIG / TIGIT bispecific antibody in a mouse subcutaneous xenograft model of human melanoma A375 mixed with human PBMCs

[0465] To further investigate the role of bispecific antibody subtypes in animal efficacy, in addition to the aforementioned IgG4 subtype bispecific antibody, we also synthesized the corresponding IgG1 subtype antibody for animal efficacy studies. Other previously undescribed antibody sequences used in this experiment are shown in Table 33.

[0466] Table 33. Full-length sequences of the first and second polypeptide chains of IgG1-type humanized anti-PVRIG / TIGIT bispecific antibodies

[0467]

[0468]

[0469] Female NCG mice, 4-8 weeks old, weighing approximately 18-22g, were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. All NCG mice were cultured under SPF-grade IVC (Indoor Temperature and Pressure) system conditions.

[0470] A375 cells were cultured in DMEM medium containing 10% fetal bovine serum (FBS). A375 cells in the exponential growth phase were collected and resuspended in HBSS to a suitable concentration for subcutaneous tumor inoculation in NCG mice. A375 cells used for co-culture were treated with Mitomycin C for 2 hours and then washed three times with PBS. Peripheral blood from healthy individuals was collected, and human PBMCs were isolated and counted using density gradient centrifugation. The PBMCs were then resuspended in RPMI 1640 medium (containing IL-2 and 10% FBS) to a concentration of 3 × 10⁻⁶. 6 A375 cells treated with Mitomycin C were co-cultured at a concentration of [number] cells / mL. After 6 days of co-culture, PBMCs were harvested, along with freshly digested A375 cells. Each mouse was inoculated with 5 × 10⁶ PBMCs. 5 4 × 10⁴ A375 cells 6 Inoculation volume: 0.2 mL / mouse (containing 50% Matrigel); subcutaneous inoculation on the right side of female NCG mice. Mice were randomly assigned to groups based on body weight. Detailed administration methods, dosages, and routes are shown in Table 34. Day 0 was the day of group administration. Due to the different molecular weights of anti-PVRIG and anti-TIGIT antibodies, this dosage ensured that anti-PVRIG and anti-TIGIT antibodies had the same initial molar concentration.

[0471] Table 34. Dosing Regimen

[0472]

[0473]

[0474] (Note: N: Number of animals used; ip: Intraperitoneal injection; Q2D: Once every two days; Dosage volume: Adjust the dosage volume according to the weight of tumor-bearing mice (0.1 mL / 10 g).)

[0475] After drug administration began, mouse body weight and tumor volume were measured twice weekly. The experimental results are shown in Tables 35-36 and 36, respectively. Figures 9A-9B .

[0476] Table 35. Effects of anti-PVRIG / TIGIT bispecific antibody on mouse body weight

[0477]

[0478] Table 36. Antitumor effect of anti-PVRIG / TIGIT bispecific antibody in mouse human A375 tumor model

[0479]

[0480] (Note: Compared with the control group (hIgG1), a statistically significant difference is considered to be ×P<0.05, ××P<0.01, or ×××P<0.001.)

[0481] At the end of the experiment (day 26 post-drug administration), there was no significant difference in the anti-PVRIG antibody 151 monotherapy group compared to the control group. Tumor volume decreased in the anti-TIGIT antibody 1708-IgG1 monotherapy group, the combination of anti-PVRIG antibody 151 and anti-TIGIT antibody 1708-IgG1 group, and the 1708-151-IgG1 bispecific antibody group. The 1708-151-IgG4 bispecific antibody group even completely inhibited tumor growth, showing a significant difference compared to other groups (see...). Figure 9B ).

[0482] Mice were randomly grouped according to their body weight and given medication. Detailed administration methods, dosages and routes are shown in Table 37. The day of administration was designated as day 0.

[0483] Table 37. Dosing Regimen

[0484]

[0485]

[0486] (Note: N: Number of animals used; ip: Intraperitoneal injection; Q2D: Once every two days; Dosage volume: Adjust the dosage volume according to the weight of tumor-bearing mice (0.1 mL / 10 g).)

[0487] After drug administration began, mouse body weight and tumor volume were measured twice weekly. The experimental results are shown in Tables 38-39 and 39, respectively. Figures 10A-10B .

[0488] Table 38. Effects of anti-P RIG / TIGIT bispecific antibody on mouse body weight

[0489]

[0490] Table 39. Antitumor effect of anti-PVRIG / TIGIT bispecific antibody in mouse human A375 tumor model

[0491]

[0492] (Note: Compared with the control group (hIgG1), a statistically significant difference is considered to be ×P<0.05, ××P<0.01, or ×××P<0.001.)

[0493] At the end of the experiment (day 28 after administration), compared with the control group, both the 1708-30H2IgG4 and 1708-151H7IgG4 double antibody groups effectively inhibited tumor growth at low doses, showing a significant difference compared with the control group (see...). Figure 10A and Figure 10B ).

[0494] While specific embodiments of this disclosure have been described above, those skilled in the art should understand that these are merely illustrative examples, and various changes or modifications can be made to these embodiments without departing from the principles and essence of the invention. Therefore, the scope of protection of this disclosure is defined by the appended claims. sequence list <110> Jiangsu Hengrui Medicine Co., Ltd. Shanghai Shengdi Pharmaceutical Co., Ltd. <120> PVRIG-binding protein and its medicinal uses <130> 721016CPCT <150> CN202010174835.4 <151> 2020-03-13 <160> 153 <170> PatentIn version 3.5 <210> 1 <211> 137 <212> PRT <213> Artificial sequence <220> <223> his1PVRIG (cyno‑PVRIG‑his) <400> 1 Thr Pro Glu Val Trp Val Gln Val Gln Met Glu Ala Thr Glu Leu Ser 1 5 10 15 Ser Phe Thr Val His Cys Gly Phe Leu Gly Pro Gly Ser Ile Ser Leu 20 25 30 Val Thr Val Ser Trp Gly Gly Pro Asp Gly Ala Gly Gly Thr Lys Leu 35 40 45 Ala Val Leu His Pro Glu Leu Gly Thr Arg Gln Trp Ala Pro Ala Arg 50 55 60 Gln Ala Arg Trp Glu Thr Gln Ser Ser Ile Ser Leu Ala Leu Glu Asp 65 70 75 80 Ser Gly Ala Ser Ser Pro Phe Ala Asn Thr Thr Phe Cys Cys Lys Phe 85 90 95 Ala Ser Phe Pro Glu Gly Ser Trp Glu Ser Cys Gly Ser Leu Pro Pro 100 105 110 Ser Ser Asp Pro Gly Leu Ser Ala Pro Pro Thr Pro Val Pro Ile Leu 115 120 125 Arg Ala Asp His His His His His His 130 135 <210> 2 <211> 121 <212> PRT <213> Synthetic Sequence <220> <223> 20‑HCVR <400> 2 Asp Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Val Ala Ser Arg Tyr Thr Ser Arg Thr Asp 20 25 30 Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu His Glu Gly Val 35 40 45 Ala His Ile Asp Ser Asp Gly Ile Pro Arg Tyr Val Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Gln Asp His Ala Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Ser Ala Thr Tyr Tyr Cys Val 85 90 95 Val Gly Phe Lys Phe Asp Asp Asp Tyr Cys Ala Pro Asn Asp Trp Gly 100 105 110[[ID=2:8]] Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 3 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> 30‑HCVR <400> 3 His Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Glu Ala Ser Gly Tyr Ser Tyr Ser Gly Asp 20 25 30 Cys Met Gly Trp Phe Arg Arg Ala Pro Gly Lys Glu Arg Asp Glu Gly 35 40 45 Val Ala Thr Ile Asp Asn Ala Gly Arg Ile Lys Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser His Gly Asn Gly Lys Tyr Ile Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Asp Met Tyr Tyr Cys 85 90 95 Ala Ala Gly Trp Thr Phe Gly Gly Asn Cys Ser Pro Ala Asp Trp Gly 100 105 110 Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 4 <211> 123 <212> PRT <213> Artificial Sequence <220> <223> 38 - HCVR <400> 4 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Pro Ser Thr Tyr Gly Pro Ser 20 25 30 Asp Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Gln Arg Glu Gly Val 35 40 45 Ala Thr Ile Ser Ala Ala Gly Arg Leu Thr Tyr Tyr Thr Asp Ser Val 50 55 60 Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Met Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Ala Asp Phe Ala Gly Gly Ser Ser Leu Phe Ala Asp Tyr Lys Tyr 100 105 110 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 5 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> 39‑HCVR <400> 5 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Arg Tyr Thr Ser Arg Thr Asp 20 25 30 Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Ala His Ile Asp Ser Asp Gly Ile Pro Arg Tyr Val Glu Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Gln Asp His Ala Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Ser Ala Thr Tyr Tyr Cys Val 85 90 95 Val Gly Phe Lys Phe Gly Asp Tyr Cys Ala Pro Asn Asp Trp Gly Gln 100 105 110 Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 6 <211> 127 <212> PRT <213> Artificial Sequence <220> <223> 151 - HCVR <400> 6 His Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Val Ala Ser Ala Ser Gly Phe Thr Tyr Arg 20 25 30 Pro Tyr Cys Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu 35 40 45 Ala Val Ala Gly Ile Asp Ile Phe Gly Gly Thr Thr Tyr Ala Asp Ser 50 55 60 Val Lys Gly Arg Phe Thr Ala Ser Arg Asp Asn Ala Gly Phe Ser Leu 65 70 75 80 Phe Leu Gln Met Asn Asp Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr 85 90 95 Cys Ala Ala Gly Asp Ser Pro Asp Gly Arg Cys Pro Pro Leu Gly Gln 100 105 110 Gly Leu Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 125 <210> 7 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> 20 HCDR1 <400> 7 Thr Asp Cys Met Gly 1 5 <210> 8 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> 20 HCDR2 <400> 8 His Ile Asp Ser Asp Gly Ile Pro Arg Tyr Val Asp Ser Val Lys Gly 1 5 10 15 <210> 9 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> 20 HCDR3 <400> 9 Gly Phe Lys Phe Asp Asp Asp Tyr Cys Ala Pro Asn Asp 1 5 10 <210> 10 <211> 5 <212> PRT <213> Artificial sequence <220> <223> 30 HCDR1 <400> 10 Gly Asp Cys Met Gly 1 5 <210> 11 <211> 16 <212> PRT <213> Artificial sequence <220> <223> 30 HCDR2 <400> 11 Thr Ile Asp Asn Ala Gly Arg Ile Lys Tyr Ala Asp Ser Val Lys Gly 1 5 10 15 <210> 12 <211> 12 <212> PRT <213> Artificial sequence <220> <223> 30 HCDR3 <400> 12 Gly Trp Thr Phe Gly Gly Asn Cys Ser Pro Ala Asp 1 5 10 <210> 13 <211> 5 <212> PRT <213> Artificial sequence <220> <223> 38 HCDR1 <400> 13 Pro Ser Asp Met Ala 1 5 <210> 14 <211> 17 <212> PRT <213> Artificial sequence <220> <223> 38 HCDR2 <400> 14 Thr Ile Ser Ala Ala Gly Arg Leu Thr Tyr Tyr Thr Asp Ser Val Arg 1 5 10 15 Gly <210> 15 <211> 14 <212> PRT <213> Artificial sequence <220> <223> 38 HCDR3 <400> 15 Asp Phe Ala Gly Gly Ser Ser Leu Phe Ala Asp Tyr Lys Tyr 1 5 10 <210> 16 <211> 5 <212> PRT <213> Artificial sequence <220> <223> 39 HCDR1 <400> 16 Thr Asp Cys Met Gly 1 5 <210> 17 <211> 16 <212> PRT <213> Artificial sequence <220> <223> 39 HCDR2 <400> 17 His Ile Asp Ser Asp Gly Ile Pro Arg Tyr Val Glu Ser Val Lys Gly 1 5 10 15 <210> 18 <211> 12 <212> PRT <213> Artificial sequence <220> <223> 39 HCDR3 <400> 18 Gly Phe Lys Phe Gly Asp Tyr Cys Ala Pro Asn Asp 1 5 10 <210> 19 <211> 7 <212> PRT <213> Artificial sequence <220> <223> 151 HCDR1 <400> 19 Tyr Arg Pro Tyr Cys Met Ala 1 5 <210> 20 <211> 16 <212> PRT <213> Artificial sequence <220> <223> 151 HCDR2 <400> 20 Gly Ile Asp Ile Phe Gly Gly Thr Thr Tyr Ala Asp Ser Val Lys Gly 1 5 10 15 <210> twenty one <211> 17 <212> PRT <213> Artificial sequence <220> <223> 151 HCDR3 <400> twenty one Gly Asp Ser Pro Asp Gly Arg Cys Pro Pro Leu Gly Gln Gly Leu Asn 1 5 10 15 Tyr <210> twenty two <211> 7 <212> PRT <213> Artificial sequence <220> <223> 20 HCDR1 CHO <400> twenty two Arg Tyr Thr Ser Arg Thr Asp 1 5 <210> twenty three <211> 5 <212> PRT <213> Artificial sequence <220> <223> 20 HCDR2 CHO <400> twenty three Asp Ser Asp Gly Ile 1 5 <210> twenty four <211> 13 <212> PRT <213> Artificial sequence <220> <223> 20 HCDR3 CHO <400> twenty four Gly Phe Lys Phe Asp Asp Asp Tyr Cys Ala Pro Asn Asp 1 5 10 <210> 25 <211> 7 <212> PRT <213> Artificial sequence <220> <223> 30 HCDR1 CHO <400> 25 Gly Tyr Ser Tyr Ser Gly Asp 1 5 <210> 26 <211> 5 <212> PRT <213> Artificial sequence <220> <223> 30 HCDR2 CHO <400> 26 Asp Asn Ala Gly Arg 1 5 <210> 27 <211> 12 <212> PRT <213> Artificial sequence <220> <223> 30 HCDR3 CHO <400> 27 Gly Trp Thr Phe Gly Gly Asn Cys Ser Pro Ala Asp 1 5 10 <210> 28 <211> 7 <212> PRT <213> Artificial sequence <220> <223> 38 HCDR1 CHO <400> 28 Pro Ser Thr Tyr Gly Pro Ser 1 5 <210> 29 <211> 6 <212> PRT <213> Artificial sequence <220> <223> 38 HCDR2 CHO <400> 29 Ser Ala Ala Gly Arg Leu 1 5 <210> 30 <211> 14 <212> PRT <213> Artificial sequence <220> <223> 38 HCDR3 CHO <400> 30 Asp Phe Ala Gly Gly Ser Ser Leu Phe Ala Asp Tyr Lys Tyr 1 5 10 <210> 31 <211> 7 <212> PRT <213> Artificial sequence <220> <223> 39 HCDR1 CHO <400> 31 Arg Tyr Thr Ser Arg Thr Asp 1 5 <210> 32 <211> 5 <212> PRT <213> Artificial sequence <220> <223> 39 HCDR2 CHO <400> 32 Asp Ser Asp Gly Ile 1 5 <210> 33 <211> 12 <212> PRT <213> Artificial sequence <220> <223> 39 HCDR3 CHO <400> 33 Gly Phe Lys Phe Gly Asp Tyr Cys Ala Pro Asn Asp 1 5 10 <210> 34 <211> 9 <212> PRT <213> Artificial sequence <220> <223> 151 HCDR1 CHO <400> 34 Ala Ser Gly Phe Thr Tyr Arg Pro Tyr 1 5 <210> 35 <211> 5 <212> PRT <213> Artificial sequence <220> <223> 151 HCDR2 CHO <400> 35 Asp Ile Phe Gly Gly 1 5 <210> 36 <211> 17 <212> PRT <213> Artificial sequence <220> <223> 151 HCDR3 CHO <400> 36 Gly Asp Ser Pro Asp Gly Arg Cys Pro Pro Leu Gly Gln Gly Leu Asn 1 5 10 15 Tyr <210> 37 <211> 8 <212> PRT <213> Artificial sequence <220> <223> 20 HCDR1 IMGT <400> 37 Arg Tyr Thr Ser Arg Thr Asp Cys 1 5 <210> 38 <211> 7 <212> PRT <213> Artificial sequence <220> <223> 20 HCDR2 IMGT <400> 38 Ile Asp Ser Asp Gly Ile Pro 1 5 <210> 39 <211> 15 <212> PRT <213> Artificial sequence <220> <223> 20 HCDR3 IMGT <400> 39 Val Val Gly Phe Lys Phe Asp Asp Asp Tyr Cys Ala Pro Asn Asp 1 5 10 15 <210> 40 <211> 8 <212> PRT <213> Artificial sequence <220> <223> 30 HCDR1 IMGT <400> 40 Gly Tyr Ser Tyr Ser Gly Asp Cys 1 5 <210> 41 <211> 7 <212> PRT <213> Artificial sequence <220> <223> 30 HCDR2 IMGT <400> 41 Ile Asp Asn Ala Gly Arg Ile 1 5 <210> 42 <211> 14 <212> PRT <213> Artificial sequence <220> <223> 30 HCDR3 IMGT <400> 42 Ala Ala Gly Trp Thr Phe Gly Gly Asn Cys Ser Pro Ala Asp 1 5 10 <210> 43 <211> 8 <212> PRT <213> Artificial sequence <220> <223> 38 HCDR1 IMGT <400> 43 Pro Ser Thr Tyr Gly Pro Ser Asp 1 5 <210> 44 <211> 8 <212> PRT <213> Artificial sequence <220> <223> 38 HCDR2 IMGT <400> 44 Ile Ser Ala Ala Gly Arg Leu Thr 1 5 <210> 45 <211> 16 <212> PRT <213> Artificial sequence <220> <223> 38 HCDR3 IMGT <400> 45 Ala Ala Asp Phe Ala Gly Gly Ser Ser Leu Phe Ala Asp Tyr Lys Tyr 1 5 10 15 <210> 46 <211> 8 <212> PRT <213> Artificial sequence <220> <223> 39 HCDR1 IMGT <400> 46 Arg Tyr Thr Ser Arg Thr Asp Cys 1 5 <210> 47 <211> 7 <212> PRT <213> Artificial sequence <220> <223> 39 HCDR2 IMGT <400> 47 Ile Asp Ser Asp Gly Ile Pro 1 5 <210> 48 <211> 14 <212> PRT <213> Artificial sequence <220> <223> 39 HCDR3 IMGT <400> 48 Val Val Gly Phe Lys Phe Gly Asp Tyr Cys Ala Pro Asn Asp 1 5 10 <210> 49 <211> 10 <212> PRT <213> Artificial sequence <220> <223> 151 HCDR1 IMGT <400> 49 Ala Ser Gly Phe Thr Tyr Arg Pro Tyr Cys 1 5 10 <210> 50 <211> 7 <212> PRT <213> Artificial sequence <220> <223> 151 HCDR2 IMGT <400> 50 Ile Asp Ile Phe Gly Gly Thr 1 5 <210> 51 <211> 19 <212> PRT <213> Artificial sequence <220> <223> 151 HCDR3 IMGT <400> 51 Ala Ala Gly Asp Ser Pro Asp Gly Arg Cys Pro Pro Leu Gly Gln Gly 1 5 10 15 Leu Asn Tyr <210> 52 <211> 10 <212> PRT <213> Artificial sequence <220> <223> 20 HCDR1 ABM <400> 52 Arg Tyr Thr Ser Arg Thr Asp Cys Met Gly 1 5 10 <210> 53 <211> 9 <212> PRT <213> Artificial sequence <220> <223> 20 HCDR2 ABM <400> 53 His Ile Asp Ser Asp Gly Ile Pro Arg 1 5 <210> 54 <211> 13 <212> PRT <213> Artificial sequence <220> <223> 20 HCDR3 ABM <400> 54 Gly Phe Lys Phe Asp Asp Asp Tyr Cys Ala Pro Asn Asp 1 5 10 <210> 55 <211> 10 <212> PRT <213> Artificial sequence <220> <223> 30 HCDR1 ABM <400> 55 Gly Tyr Ser Tyr Ser Gly Asp Cys Met Gly 1 5 10 <210> 56 <211> 9 <212> PRT <213> Artificial sequence <220> <223> 30 HCDR2 ABM <400> 56 Thr Ile Asp Asn Ala Gly Arg Ile Lys 1 5 <210> 57 <211> 12 <212> PRT <213> Artificial sequence <220> <223> 30 HCDR3 ABM <400> 57 Gly Trp Thr Phe Gly Gly Asn Cys Ser Pro Ala Asp 1 5 10 <210> 58 <211> 10 <212> PRT <213> Artificial sequence <220> <223> 38 HCDR1 ABM <400> 58 Pro Ser Thr Tyr Gly Pro Ser Asp Met Ala 1 5 10 <210> 59 <211> 10 <212> PRT <213> Artificial sequence <220> <223> 38 HCDR2 ABM <400> 59 Thr Ile Ser Ala Ala Gly Arg Leu Thr Tyr 1 5 10 <210> 60 <211> 14 <212> PRT <213> Artificial sequence <220> <223> 38 HCDR3 ABM <400> 60 Asp Phe Ala Gly Gly Ser Ser Leu Phe Ala Asp Tyr Lys Tyr 1 5 10 <210> 61 <211> 10 <212> PRT <213> Artificial sequence <220> <223> 39 HCDR1 ABM <400> 61 Arg Tyr Thr Ser Arg Thr Asp Cys Met Gly 1 5 10 <210> 62 <211> 9 <212> PRT <213> Artificial sequence <220> <223> 39 HCDR2 ABM <400> 62 His Ile Asp Ser Asp Gly Ile Pro Arg 1 5 <210> 63 <211> 12 <212> PRT <213> Artificial sequence <220> <223> 39 HCDR3 ABM <400> 63 Gly Phe Lys Phe Gly Asp Tyr Cys Ala Pro Asn Asp 1 5 10 <210> 64 <211> 12 <212> PRT <213> Artificial sequence <220> <223> 151 HCDR1 ABM <400> 64 Ala Ser Gly Phe Thr Tyr Arg Pro Tyr Cys Met Ala 1 5 10 <210> 65 <211> 9 <212> PRT <213> Artificial sequence <220> <223> 151 HCDR2 ABM <400> 65 Gly Ile Asp Ile Phe Gly Gly Thr Thr 1 5 <210> 66 <211> 17 <212> PRT <213> Artificial sequence <220> <223> 151 HCDR3 ABM <400> 66 Gly Asp Ser Pro Asp Gly Arg Cys Pro Pro Leu Gly Gln Gly Leu Asn 1 5 10 15 Tyr <210> 67 <211> 350 <212> PRT <213> Artificial sequence <220> <223> 20 HC <400> 67 Asp Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Val Ala Ser Arg Tyr Thr Ser Arg Thr Asp 20 25 30 Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu His Glu Gly Val 35 40 45 Ala His Ile Asp Ser Asp Gly Ile Pro Arg Tyr Val Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Gln Asp His Ala Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Ser Ala Thr Tyr Tyr Cys Val 85 90 95 Val Gly Phe Lys Phe Asp Asp Asp Tyr Cys Ala Pro Asn Asp Trp Gly 100 105 110 Gln Gly Thr Gln Val Thr Val Ser Ser Glu Ser Lys Tyr Gly Pro Pro 115 120 125 Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe 130 135 140 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 145 150 155 160 Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val 165 170 175 Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 180 185 190 Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val 195 200 205 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 210 215 220 Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser 225 230 235 240 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 245 250 255 Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 260 265 270 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 275 280 285 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 290 295 300 Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp 305 310 315 320 Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 325 330 335 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Ala 340 345 350 <210> 68 <211> 350 <212> PRT <213> Artificial Sequence <220> <223> 30 HC <400> 68 His Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Glu Ala Ser Gly Tyr Ser Tyr Ser Gly Asp 20 25 30 Cys Met Gly Trp Phe Arg Arg Ala Pro Gly Lys Glu Arg Asp Glu Gly 35 40 45 Val Ala Thr Ile Asp Asn Ala Gly Arg Ile Lys Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser His Gly Asn Gly Lys Tyr Ile Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Asp Met Tyr Tyr Cys 85 90 95 Ala Ala Gly Trp Thr Phe Gly Gly Asn Cys Ser Pro Ala Asp Trp Gly 100 105 110 Gln Gly Thr Gln Val Thr Val Ser Ser Glu Ser Lys Tyr Gly Pro Pro 115 120 125 Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe 130 135 140 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 145 150 155 160 Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val 165 170 175 Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 180 185 190 Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val 195 200 205 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 210 215 220 Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser 225 230 235 240 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 245 250 255 Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 260 265 270 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 275 280 285 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 290 295 300 Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp 305 310 315 320 Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 325 330 335 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Ala 340 345 350 <210> 69 <211> 352 <212> PRT <213> Artificial sequence <220> <223> 38 HC <400> 69 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Pro Ser Thr Tyr Gly Pro Ser 20 25 30 Asp Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Gln Arg Glu Gly Val 35 40 45 Ala Thr Ile Ser Ala Ala Gly Arg Leu Thr Tyr Tyr Thr Asp Ser Val 50 55 60 Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Met Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Ala Asp Phe Ala Gly Gly Ser Ser Leu Phe Ala Asp Tyr Lys Tyr 100 105 110 Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Glu Ser Lys Tyr Gly 115 120 125 Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser 130 135 140 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 145 150 155 160 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro 165 170 175 Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 180 185 190 Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val 195 200 205 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 210 215 220 Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr 225 230 235 240 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 245 250 255 Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys 260 265 270 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 275 280 285 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 290 295 300 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser 305 310 315 320 Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 325 330 335 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Ala 340 345 350 <210> 70 <211> 349 <212> PRT <213> Artificial Sequence <220> <223> 39 HC <400> 70 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Arg Tyr Thr Ser Arg Thr Asp 20 25 30 Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Ala His Ile Asp Ser Asp Gly Ile Pro Arg Tyr Val Glu Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Gln Asp His Ala Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Ser Ala Thr Tyr Tyr Cys Val 85 90 95 Val Gly Phe Lys Phe Gly Asp Tyr Cys Ala Pro Asn Asp Trp Gly Gln 100 105 110 Gly Thr Gln Val Thr Val Ser Ser Glu Ser Lys Tyr Gly Pro Pro Cys 115 120 125 Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu 130 135 140 Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 145 150 155 160 Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln 165 170 175 Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 180 185 190 Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu 195 200 205 Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 210 215 220 Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys 225 230 235 240 Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 245 250 255 Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys 260 265 270 Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 275 280 285 Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly 290 295 300 Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln 305 310 315 320 Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 325 330 335 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Ala 340 345 <210> 71 <211> 356 <212> PRT <213> Artificial Sequence <220> <223> 151 HC <400> 71 His Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Val Ala Ser Ala Ser Gly Phe Thr Tyr Arg 20 25 30 Pro Tyr Cys Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu 35 40 45 Ala Val Ala Gly Ile Asp Ile Phe Gly Gly Thr Thr Tyr Ala Asp Ser 50 55 60 Val Lys Gly Arg Phe Thr Ala Ser Arg Asp Asn Ala Gly Phe Ser Leu 65 70 75 80 Phe Leu Gln Met Asn Asp Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr 85 90 95 Cys Ala Ala Gly Asp Ser Pro Asp Gly Arg Cys Pro Pro Leu Gly Gln 100 105 110 Gly Leu Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Glu 115 120 125 Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala 130 135 140 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 145 150 155 160 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 165 170 175 Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu 180 185 190 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr 195 200 205 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 210 215 220 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser 225 230 235 240 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 245 250 255 Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val 260 265 270 Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 275 280 285 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 290 295 300 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr 305 310 315 320 Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val 325 330 335 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 340 345 350 Ser Leu Gly Ala 355 <210> 72 <211> 449 <212> PRT <213> Artificial Sequence <220> <223> tab5 HC[[ID=2�]] <400> 72 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Gly Thr Ser 20 25 30 Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Ser Phe Asp Gly Thr Glu Ile His Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Ser Thr Val Phe 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro Asp Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Ala Lys Gly Ser Gly Asn Ile Tyr Phe Tyr Ser Gly Met Asp Val Trp 100 105 110 Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125 Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr 130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190 Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp 195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr 210 215 220 Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp 260 265 270 Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly 435 440 445 Ala <210> 73 <211> 214 <212> PRT <213> Artificial sequence <220> <223> tab5 LC <400> 73 Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Gly Gln Ser Ile Ser Gly Trp 20 25 30 Leu Ala Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro Asn Leu Leu Ile 35 40 45 Tyr Glu Thr Ser Thr Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Ser Tyr Pro Leu 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 74 <211> 98 <212> PRT <213> Artificial Sequence <220> <223> IGHV3-7 *01 HCVR <400> 74 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Asn Ile Lys Gln Asp Gly Ser Glu Lys Tyr Tyr Val Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg <210> 75 <211> 121 <212> PRT <213> artificial sequence <220> <223> 20H1 HCVR <400> 75 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Asp 20 25 30 Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Gly Val 35 40 45 Ala His Ile Asp Ser Asp Gly Ile Pro Arg Tyr Val Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Val 85 90 95 Val Gly Phe Lys Phe Asp Glu Asp Tyr Cys Ala Pro Asn Asp Trp Gly 100 105 110 Gln Gly Thr Met Val Thr Val Ser Ser 115 120 <210> 76 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> 20H2 HCVR <400> 76 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Ser Arg Thr Asp 20 25 30 Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Gly Val 35 40 45 Ala His Ile Asp Ser Asp Gly Ile Pro Arg Tyr Val Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Val 85 90 95 Val Gly Phe Lys Phe Asp Glu Asp Tyr Cys Ala Pro Asn Asp Trp Gly 100 105 110 Gln Gly Thr Met Val Thr Val Ser Ser 115 120 <210> 77 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> 20H3 HCVR <400> 77 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Ser Thr Asp 20 25 30 Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Gly Val 35 40 45 Ala His Ile Asp Ser Asp Gly Ile Pro Arg Tyr Val Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Gln Asp His Ala Lys Asn Ser Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Val 85 90 95 Val Gly Phe Lys Phe Asp Glu Asp Tyr Cys Ala Pro Asn Asp Trp Gly 100 105 110 Gln Gly Thr Met Val Thr Val Ser Ser 115 120 <210> 78 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> 20H4 HCVR <400> 78 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Ser Arg Thr Asp 20 25 30 Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Gly Val 35 40 45 Ala His Ile Asp Ser Asp Gly Ile Pro Arg Tyr Val Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Gln Asp His Ala Lys Asn Ser Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Val 85 90 95 Val Gly Phe Lys Phe Asp Glu Asp Tyr Cys Ala Pro Asn Asp Trp Gly 100 105 110 Gln Gly Thr Met Val Thr Val Ser Ser 115 120 <210> 79 <211> 121 <212> PRT <213> Synthetic Sequence <220> <223> 20H5 HCVR <400> 79 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Ser Arg Thr Asp 20 25 30 Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu His Glu Gly Val 35 40 45 Ala His Ile Asp Ser Asp Gly Ile Pro Arg Tyr Val Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Gln Asp His Ala Lys Asn Ser Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Val 85 90 95 Val Gly Phe Lys Phe Asp Glu Asp Tyr Cys Ala Pro Asn Asp Trp Gly 100 105 110 Gln Gly Thr Met Val Thr Val Ser Ser 115 120 <210> 80 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> 30H1 HCVR <400> 80 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Gly Asp 20 25 30 Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Gly Val 35 40 45 Ala Thr Ile Asp Asn Ala Gly Arg Ile Lys Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Ala Gly Trp Thr Phe Gly Gly Gln Cys Ser Pro Ala Asp Trp Gly Gln ​ Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 81 <211> 121 <212> PRT <213> Synthetic Sequence <220> <223> 30H2 HCVR <400> 81 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Gly Asp 20 25 30 Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Asp Glu Gly 35 40 45 Val Ala Thr Ile Asp Asn Ala Gly Arg Ile Lys Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Gly Trp Thr Phe Gly Gly Gln Cys Ser Pro Ala Asp Trp Gly 100 105 110 Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 82 <211> 121 <212> PRT <213> Artificial sequence <220> <223> 30H3 HCVR <400> 82 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ser Tyr Ser Gly Asp 20 25 30 Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Asp Glu Gly 35 40 45 Val Ala Thr Ile Asp Asn Ala Gly Arg Ile Lys Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Gly Trp Thr Phe Gly Gly Gln Cys Ser Pro Ala Asp Trp Gly 100 105 110 Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 83 <211> 121 <212> PRT <213> Artificial sequence <220> <223> 30H4 HCVR <400> 83 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Gly Asp 20 25 30 Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Asp Glu Gly 35 40 45 Val Ala Thr Ile Asp Asn Ala Gly Arg Ile Lys Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser His Gly Asn Ala Lys Tyr Ile Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Gly Trp Thr Phe Gly Gly Gln Cys Ser Pro Ala Asp Trp Gly 100 105 110 Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 84 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> 30H5 HCVR <400> 84 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ser Tyr Ser Gly Asp 20 25 30 Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Asp Glu Gly 35 40 45 Val Ala Thr Ile Asp Asn Ala Gly Arg Ile Lys Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser His Gly Asn Ala Lys Tyr Ile Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Gly Trp Thr Phe Gly Gly Gln Cys Ser Pro Ala Asp Trp Gly 100 105 110 Gln Gly Thr Gln Val Thr Val Ser Ser 115 120 <210> 85 <211> 98 <212> PRT <213> Artificial Sequence <220> <223> IGHV3-30 *02 <400> 85 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Phe Ile Arg Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys <210> 86 <211> 123 <212> PRT <213> Synthetic Sequence <220> <223> 38H2 HCVR <400> 86 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Ser 20 25 30 Asp Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Gly Val 35 40 45 Ala Thr Ile Ser Ala Ala Gly Arg Leu Thr Tyr Tyr Thr Asp Ser Val 50 55 60 Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Asp Phe Ala Gly Gly Ser Ser Leu Phe Ala Asp Tyr Lys Tyr 100 105 110 Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser 115 120 <210> 87 <211> 123 <212> PRT <213> artificial sequence <220> <223> 38H4 HCVR <400> 87 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Pro Phe Thr Tyr Gly Pro Ser 20 25 30 Asp Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Gly Val 35 40 45 Ala Thr Ile Ser Ala Ala Gly Arg Leu Thr Tyr Tyr Thr Asp Ser Val 50 55 60 Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Asp Phe Ala Gly Gly Ser Ser Leu Phe Ala Asp Tyr Lys Tyr 100 105 110 Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser 115 120 <210> 88 <211> 123 <212> PRT <213> artificial sequence <220> <223> 38H7 HCVR <400> 88 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Pro Ser Thr Tyr Gly Pro Ser 20 25 30 Asp Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Gln Arg Glu Gly Val 35 40 45 Ala Thr Ile Ser Ala Ala Gly Arg Leu Thr Tyr Tyr Thr Asp Ser Val 50 55 60 Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Met Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Asp Phe Ala Gly Gly Ser Ser Leu Phe Ala Asp Tyr Lys Tyr 100 105 110 Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser 115 120 <210> 89 <211> 123 <212> PRT <213> artificial sequence <220> <223> 38H8 HCVR <400> 89 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Pro Ser Thr Tyr Gly Pro Ser 20 25 30 Asp Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Gly Val 35 40 45 Ala Thr Ile Ser Ala Ala Gly Arg Leu Thr Tyr Tyr Thr Asp Ser Val 50 55 60 Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Asp Phe Ala Gly Gly Ser Ser Leu Phe Ala Asp Tyr Lys Tyr 100 105 110 Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser 115 120 <210> 90 <211> 123 <212> PRT <213> artificial sequence <220> <223> 38H9 HCVR <400> 90 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Pro Ser Thr Tyr Gly Pro Ser 20 25 30 Asp Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Gly Val 35 40 45 Ala Thr Ile Ser Ala Ala Gly Arg Leu Thr Tyr Tyr Thr Asp Ser Val 50 55 60 Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Met Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Asp Phe Ala Gly Gly Ser Ser Leu Phe Ala Asp Tyr Lys Tyr 100 105 110 Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser 115 120 <210> 91 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> 39H1 HCVR <400> 91 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Asp 20 25 30 Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Gly Val 35 40 45 Ala His Ile Asp Ser Asp Gly Ile Pro Arg Tyr Val Glu Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Val 85 90 95 Val Gly Phe Lys Phe Gly Asp Tyr Cys Ala Pro Asn Asp Trp Gly Gln 100 105 110 Gly Thr Met Val Thr Val Ser Ser 115 120 <210> 92 <211> 120 <212> PRT <213> Artificial sequence <220> <223> 39H2 HCVR <400> 92 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Ser Arg Thr Asp 20 25 30 Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Gly Val 35 40 45 Ala His Ile Asp Ser Asp Gly Ile Pro Arg Tyr Val Glu Ser Val Lys<舍 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Val 85 90 95 Val Gly Phe Lys Phe Gly Asp Tyr Cys Ala Pro Asn Asp Trp Gly Gln 100 105 110 Gly Thr Met Val Thr Val Ser Ser 115 120 <210> 93 <211> 120 <212> PRT <213> Artificial sequence <220> <223> 39H3 HCVR <400> 93 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Ser Thr Asp 20 25 30 Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Gly Val 35 40 45 Ala His Ile Asp Ser Asp Gly Ile Pro Arg Tyr Val Glu Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Gln Asp His Ala Lys Asn Ser Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Val 85 90 95 Val Gly Phe Lys Phe Gly Asp Tyr Cys Ala Pro Asn Asp Trp Gly Gln 100 105 110 Gly Thr Met Val Thr Val Ser Ser 115 120 <210> 94 <211> 120 <212> PRT <213> Artificial sequence <220> <223> 39H4 HCVR <400> 94 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Ser Arg Thr Asp 20 25 30 Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Gly Val 35 40 45 Ala His Ile Asp Ser Asp Gly Ile Pro Arg Tyr Val Glu Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Gln Asp His Ala Lys Asn Ser Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Val 85 90 95 Val Gly Phe Lys Phe Gly Asp Tyr Cys Ala Pro Asn Asp Trp Gly Gln 100 105 110 Gly Thr Met Val Thr Val Ser Ser 115 120 <210> 95 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> 39H5 HCVR <400> 95 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Ser Arg Thr Asp 20 25 30 Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Ala His Ile Asp Ser Asp Gly Ile Pro Arg Tyr Val Glu Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Gln Asp His Ala Lys Asn Ser Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Val 85 90 95 Val Gly Phe Lys Phe Gly Asp Tyr Cys Ala Pro Asn Asp Trp Gly Gln 100 105 110 Gly Thr Met Val Thr Val Ser Ser 115 120 <210> 96 <211> 125 <212> PRT <213> Synthetic Sequence <220> <223> 151H2 HCVR <400> 96 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Tyr Arg Pro Tyr 20 25 30 Cys Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Ala Val 35 40 45 Ala Gly Ile Asp Ile Phe Gly Gly Thr Thr Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Ala Gly Asp Ser Pro Asp Gly Arg Cys Pro Pro Leu Gly Gln Gly Leu 100 105 110 Asn Tyr Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser 115 120 125 <210> 97 <211> 127 <212> PRT <213> Artificial sequence <220> <223> 151H4 HCVR <400> 97 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Val Ala Ser Ala Ser Gly Phe Thr Tyr Arg 20 25 30 Pro Tyr Cys Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu 35 40 45 Ala Val Ala Gly Ile Asp Ile Phe Gly Gly Thr Thr Tyr Ala Asp Ser 50 55 60 Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu 65 70 75 80 Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Ala Gly Asp Ser Pro Asp Gly Arg Cys Pro Pro Leu Gly Gln 100 105 110 Gly Leu Asn Tyr Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser 115 120 125 <210> 98 <211> 127 <212> PRT <213> Artificial Sequence <220> <223> 151H7 HCVR <400> 98 His Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Val Ala Ser Ala Ser Gly Phe Thr Tyr Arg 20 25 30 Pro Tyr Cys Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu 35 40 45 Ala Val Ala Gly Ile Asp Ile Phe Gly Gly Thr Thr Tyr Ala Asp Ser 50 55 60 Val Lys Gly Arg Phe Thr Ala Ser Arg Asp Asn Ala Gly Phe Ser Leu 65 70 75 80 Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Ala Gly Asp Ser Pro Asp Gly Arg Cys Pro Pro Leu Gly Gln 100 105 110 Gly Leu Asn Tyr Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser 115 120 125 <210> 99 <211> 127 <212> PRT <213> Synthetic Sequence <220> <223> 151H8 HCVR <400> 99 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Val Ala Ser Ala Ser Gly Phe Thr Tyr Arg 20 25 30 Pro Tyr Cys Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu 35 40 45 Ala Val Ala Gly Ile Asp Ile Phe Gly Gly Thr Thr Tyr Ala Asp Ser 50 55 60 Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Gly Phe Ser Leu 65 70 75 80 Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Ala Gly Asp Ser Pro Asp Gly Arg Cys Pro Pro Leu Gly Gln 100 105 110 Gly Leu Asn Tyr Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser 115 120 125 <210> 100 <211> 127 <212> PRT <213> Synthetic Sequence <220> <223> 151H9 HCVR[[ID=3V]] <400> 100 His Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Val Ala Ser Ala Ser Gly Phe Thr Tyr Arg 20 25 30 Pro Tyr Cys Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu 35 40 45 Ala Val Ala Gly Ile Asp Ile Phe Gly Gly Thr Thr Tyr Ala Asp Ser 50 55 60 Val Lys Gly Arg Phe Thr Ala Ser Arg Asp Asn Ala Gly Phe Ser Leu 65 70 75 80 Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Ala Gly Asp Ser Pro Asp Gly Arg Cys Pro Pro Leu Gly Gln 100 105 110 Gly Leu Asn Tyr Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser 115 120 125 <210> 101 <211> 229 <212> PRT <213> Artificial Sequence <220> <223> Human IgG4 heavy chain Fc region (S228P / F234A / L235A / K447A) <400> 101 Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala 1 5 10 15 Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 20 25 30 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 35 40 45 Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val 50 55 60 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser 65 70 75 80 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 85 90 95 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser 100 105 110 Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 115 120 125 Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln 130 135 140 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 145 150 155 160 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 165 170 175 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu 180 185 190 Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser 195 200 205 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 210 215 220 Leu Ser Leu Gly Ala 225 <210> 102 <211> 446 <212> PRT <213> Artificial Sequence <220> <223> 1708TIGITHC <400> 102 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Asp Pro Asp Ser Thr Gly Ser Lys Tyr Asn Glu Lys Phe 50 55 60 Lys Thr Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Gly Ala Tyr Gly Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro 210 215 220 Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe 225 230 235 240 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 245 250 255 Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val 260 265 270 Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280 285 Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val 290 295 300 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 305 310 315 320 Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser 325 330 335 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 340 345 350 Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 355 360 365 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 385 390 395 400 Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp 405 410 415 Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 420 425 430 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 435 440 445 <210> 103 <211> 214 <212> PRT <213> Artificial sequence <220> <223> 1708TIGIT LC <400> 103 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Ile Tyr Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ser Pro Lys Leu Leu Ile 35 40 45 Tyr Asn Ala Arg Thr Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Tyr His Ser Gly Ser Pro Leu 85 90 95 Pro Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 104 <211> 583 <212> PRT <213> Synthetic Sequence <220> <223> 1708-151-11511708N <400> 104 His Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Val Ala Ser Ala Ser Gly Phe Thr Tyr Arg 20 25 30 Pro Tyr Cys Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu 35 40 45 Ala Val Ala Gly Ile Asp Ile Phe Gly Gly Thr Thr Tyr Ala Asp Ser 50 55 60 Val Lys Gly Arg Phe Thr Ala Ser Arg Asp Asn Ala Gly Phe Ser Leu 65 70 75 80 Phe Leu Gln Met Asn Asp Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr 85 90 95 Cys Ala Ala Gly Asp Ser Pro Asp Gly Arg Cys Pro Pro Leu Gly Gln 100 105 110 Gly Leu Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly 115 120 125 Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Gln Ser 130 135 140 Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys 145 150 155 160 Ala Ser Gly Tyr Thr Phe Thr Asn Tyr Trp Met His Trp Val Arg Gln 165 170 175 Ala Pro Gly Gln Gly Leu Glu Trp Met Gly Arg Ile Asp Pro Asp Ser 180 185 190 Thr Gly Ser Lys Tyr Asn Glu Lys Phe Lys Thr Arg Val Thr Met Thr 195 200 205 Arg Asp Thr Ser Thr Ser Thr Val Tyr Met Glu Leu Ser Ser Leu Arg 210 215 220 Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Glu Gly Ala Tyr Gly 225 230 235 240 Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 245 250 255 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 260 265 270 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 275 280 285 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 290 295 300 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 305 310 315 320 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr 325 330 335 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 340 345 350 Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro 355 360 365 Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 370 375 380 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 385 390 395 400 Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 405 410 415 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 420 425 430 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 435 440 445 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 450 455 460 Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 465 470 475 480 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys 485 490 495 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 500 505 510 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 515 520 525 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 530 535 540 Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 545 550 555 560 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 565 570 575 Leu Ser Leu Ser Leu Gly Lys 580 <210> 105 <211> 583 <212> PRT <213> Artificial Sequence <220> <223> 1708-151-21511708C <400> 105 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Asp Pro Asp Ser Thr Gly Ser Lys Tyr Asn Glu Lys Phe 50 55 60 Lys Thr Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Gly Ala Tyr Gly Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro 210 215 220 Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe 225 230 235 240 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 245 250 255 Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val 260 265 270 Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280 285 Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val 290 295 300 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 305 310 315 320 Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser 325 330 335 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 340 345 350 Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 355 360 365 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 385 390 395 400 Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp 405 410 415 Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 420 425 430 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys Gly Gly 435 440 445 Gly Gly Ser Gly Gly Gly Gly Ser His Val Gln Leu Val Glu Ser Gly 450 455 460 Gly Gly Ser Val Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Val Ala 465 470 475 480 Ser Ala Ser Gly Phe Thr Tyr Arg Pro Tyr Cys Met Ala Trp Phe Arg 485 490 495 Gln Ala Pro Gly Lys Glu Arg Glu Ala Val Ala Gly Ile Asp Ile Phe 500 505 510 Gly Gly Thr Thr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ala Ser 515 520 525 Arg Asp Asn Ala Gly Phe Ser Leu Phe Leu Gln Met Asn Asp Leu Lys 530 535 540 Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala Ala Gly Asp Ser Pro Asp 545 550 555 560 Gly Arg Cys Pro Pro Leu Gly Gln Gly Leu Asn Tyr Trp Gly Gln Gly 565 570 575 Thr Gln Val Thr Val Ser Ser 580 <210> 106 <211> 351 <212> PRT <213> Artificial sequence <220> <223> 1708‑151‑31511708N <400> 106 His Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Val Ala Ser Ala Ser Gly Phe Thr Tyr Arg 20 25 30 Pro Tyr Cys Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu 35 40 45 Ala Val Ala Gly Ile Asp Ile Phe Gly Gly Thr Thr Tyr Ala Asp Ser 50 55 60 Val Lys Gly Arg Phe Thr Ala Ser Arg Asp Asn Ala Gly Phe Ser Leu 65 70 75 80 Phe Leu Gln Met Asn Asp Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr 85 90 95 Cys Ala Ala Gly Asp Ser Pro Asp Gly Arg Cys Pro Pro Leu Gly Gln 100 105 110 Gly Leu Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly 115 120 125 Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser 130 135 140 Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys 145 150 155 160 Arg Ala Ser Glu Asn Ile Tyr Ser Tyr Leu Ala Trp Tyr Gln Gln Lys 165 170 175 Pro Gly Lys Ser Pro Lys Leu Leu Ile Tyr Asn Ala Arg Thr Leu Ala 180 185 190 Glu Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe 195 200 205 Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr 210 215 220 Cys Gln Tyr His Ser Gly Ser Pro Leu Pro Phe Gly Gly Gly Thr Lys 225 230 235 240 Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro 245 250 255 Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu 260 265 270 Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp 275 280 285 Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp 290 295 300 Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys 305 310 315 320 Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln 325 330 335 Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 340 345 350 <210> 107 <211> 351 <212> PRT <213> Artificial Sequence <220> <223> 1708‑151‑41511708C <400> 107 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Ile Tyr Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ser Pro Lys Leu Leu Ile 35 40 45 Tyr Asn Ala Arg Thr Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Tyr His Ser Gly Ser Pro Leu 85 90 95 Pro Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 210 215 220 His Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly 225 230 235 240 Ser Leu Arg Leu Ser Cys Val Ala Ser Ala Ser Gly Phe Thr Tyr Arg 245 250 255 Pro Tyr Cys Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu 260 265 270 Ala Val Ala Gly Ile Asp Ile Phe Gly Gly Thr Thr Tyr Ala Asp Ser 275 280 285 Val Lys Gly Arg Phe Thr Ala Ser Arg Asp Asn Ala Gly Phe Ser Leu 290 295 300 Phe Leu Gln Met Asn Asp Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr 305 310 315 320 Cys Ala Ala Gly Asp Ser Pro Asp Gly Arg Cys Pro Pro Leu Gly Gln 325 330 335 Gly Leu Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser 340 345 350 <210> 108 <211> 577 <212> PRT <213> Artificial sequence <220> <223> 1708-20H5 <400> 108 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Ser Arg Thr Asp 20 25 30 Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu His Glu Gly Val 35 40 45 Ala His Ile Asp Ser Asp Gly Ile Pro Arg Tyr Val Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Gln Asp His Ala Lys Asn Ser Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Val 85 90 95 Val Gly Phe Lys Phe Asp Glu Asp Tyr Cys Ala Pro Asn Asp Trp Gly 100 105 110 Gln Gly Thr Met Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly 115 120 125 Gly Gly Ser Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 130 135 140 Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe 145 150 155 160 Thr Asn Tyr Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu 165 170 175 Glu Trp Met Gly Arg Ile Asp Pro Asp Ser Thr Gly Ser Lys Tyr Asn 180 185 190 Glu Lys Phe Lys Thr Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser 195 200 205 Thr Val Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val 210 215 220 Tyr Tyr Cys Ala Arg Glu Gly Ala Tyr Gly Tyr Tyr Phe Asp Tyr Trp 225 230 235 240 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 245 250 255 Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr 260 265 270 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 275 280 285 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 290 295 300 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 305 310 315 320 Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp 325 330 335 His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr 340 345 350 Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro 355 360 365 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 370 375 380 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp 385 390 395 400 Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 405 410 415 Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val 420 425 430 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 435 440 445 Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys 450 455 460 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 465 470 475 480 Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 485 490 495 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 500 505 510 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 515 520 525 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys 530 535 540 Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu 545 550 555 560 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly 565 570 575 Lys <210> 109 <211> 577 <212> PRT <213> Artificial Sequence <220> <223> 1708-30H2 <400> 109 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Gly Asp 20 25 30 Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Asp Glu Gly 35 40 45 Val Ala Thr Ile Asp Asn Ala Gly Arg Ile Lys Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Gly Trp Thr Phe Gly Gly Gln Cys Ser Pro Ala Asp Trp Gly 100 105 110 Gln Gly Thr Gln Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly 115 120 125 Gly Gly Ser Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 130 135 140 Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe 145 150 155 160 Thr Asn Tyr Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu 165 170 175 Glu Trp Met Gly Arg Ile Asp Pro Asp Ser Thr Gly Ser Lys Tyr Asn 180 185 190 Glu Lys Phe Lys Thr Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser 195 200 205 Thr Val Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val 210 215 220 Tyr Tyr Cys Ala Arg Glu Gly Ala Tyr Gly Tyr Tyr Phe Asp Tyr Trp 225 230 235 240 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 245 250 255 Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr 260 265 270 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 275 280 285 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 290 295 300 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 305 310 315 320 Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp 325 330 335 His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr 340 345 350 Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro 355 360 365 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 370 375 380 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp 385 390 395 400 Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 405 410 415 Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val 420 425 430 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 435 440 445 Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys 450 455 460 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 465 470 475 480 Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 485 490 495 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 500 505 510 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 515 520 525 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys 530 535 540 Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu 545 550 555 560 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly 565 570 575 Lys <210> 110 <211> 576 <212> PRT <213> Artificial sequence <220> <223> 1708-39H2 <400> 110 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Ser Arg Thr Asp 20 25 30 Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Gly Val 35 40 45 Ala His Ile Asp Ser Asp Gly Ile Pro Arg Tyr Val Glu Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Val 85 90 95 Val Gly Phe Lys Phe Gly Asp Tyr Cys Ala Pro Asn Asp Trp Gly Gln 100 105 110 Gly Thr Met Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly 115 120 125 Gly Ser Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro 130 135 140 Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr 145 150 155 160 Asn Tyr Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu 165 170 175 Trp Met Gly Arg Ile Asp Pro Asp Ser Thr Gly Ser Lys Tyr Asn Glu 180 185 190 Lys Phe Lys Thr Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr 195 200 205 Val Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr 210 215 220 Tyr Cys Ala Arg Glu Gly Ala Tyr Gly Tyr Tyr Phe Asp Tyr Trp Gly 225 230 235 240 Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 245 250 255 Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala 260 265 270 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 275 280 285 Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 290 295 300 Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 305 310 315 320 Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His 325 330 335 Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly 340 345 350 Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser 355 360 365 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 370 375 380 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro 385 390 395 400 Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 405 410 415 Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val 420 425 430 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 435 440 445 Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr 450 455 460 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 465 470 475 480 Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys 485 490 495 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 500 505 510 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 515 520 525 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser 530 535 540 Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 545 550 555 560 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 565 570 575 <210> 111 <211> 583 <212> PRT <213> Artificial Sequence <220> <223> 1708-151H7 <400> 111 His Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Val Ala Ser Ala Ser Gly Phe Thr Tyr Arg 20 25 30 Pro Tyr Cys Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu 35 40 45 Ala Val Ala Gly Ile Asp Ile Phe Gly Gly Thr Thr Tyr Ala Asp Ser 50 55 60 Val Lys Gly Arg Phe Thr Ala Ser Arg Asp Asn Ala Gly Phe Ser Leu 65 70 75 80 Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Ala Gly Asp Ser Pro Asp Gly Arg Cys Pro Pro Leu Gly Gln 100 105 110 Gly Leu Asn Tyr Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Gly 115 120 125 Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Gln Ser 130 135 140 Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys 145 150 155 160 Ala Ser Gly Tyr Thr Phe Thr Asn Tyr Trp Met His Trp Val Arg Gln 165 170 175 Ala Pro Gly Gln Gly Leu Glu Trp Met Gly Arg Ile Asp Pro Asp Ser 180 185 190 Thr Gly Ser Lys Tyr Asn Glu Lys Phe Lys Thr Arg Val Thr Met Thr 195 200 205 Arg Asp Thr Ser Thr Ser Thr Val Tyr Met Glu Leu Ser Ser Leu Arg 210 215 220 Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Glu Gly Ala Tyr Gly 225 230 235 240 Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 245 250 255 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 260 265 270 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 275 280 285 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 290 295 300 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 305 310 315 320 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr 325 330 335 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 340 345 350 Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro 355 360 365 Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 370 375 380 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 385 390 395 400 Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 405 410 415 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 420 425 430 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 435 440 445 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 450 455 460 Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 465 470 475 480 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys 485 490 495 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 500 505 510 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 515 520 525 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 530 535 540 Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 545 550 555 560 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 565 570 575 Leu Ser Leu Ser Leu Gly Lys 580 <210> 112 <211> 583 <212> PRT <213> Artificial sequence <220> <223> 1708-151H8 <400> 112 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Val Ala Ser Ala Ser Gly Phe Thr Tyr Arg 20 25 30 Pro Tyr Cys Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu 35 40 45 Ala Val Ala Gly Ile Asp Ile Phe Gly Gly Thr Thr Tyr Ala Asp Ser 50 55 60 Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Gly Phe Ser Leu 65 70 75 80 Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Ala Gly Asp Ser Pro Asp Gly Arg Cys Pro Pro Leu Gly Gln 100 105 110 Gly Leu Asn Tyr Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Gly 115 120 125 Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Gln Ser 130 135 140 Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys 145 150 155 160 Ala Ser Gly Tyr Thr Phe Thr Asn Tyr Trp Met His Trp Val Arg Gln 165 170 175 Ala Pro Gly Gln Gly Leu Glu Trp Met Gly Arg Ile Asp Pro Asp Ser 180 185 190 Thr Gly Ser Lys Tyr Asn Glu Lys Phe Lys Thr Arg Val Thr Met Thr 195 200 205 Arg Asp Thr Ser Thr Ser Thr Val Tyr Met Glu Leu Ser Ser Leu Arg 210 215 220 Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Glu Gly Ala Tyr Gly 225 230 235 240 Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 245 250 255 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 260 265 270 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 275 280 285 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 290 295 300 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 305 310 315 320 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr 325 330 335 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 340 345 350 Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro 355 360 365 Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 370 375 380 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 385 390 395 400 Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 405 410 415 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 420 425 430 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 435 440 445 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 450 455 460 Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 465 470 475 480 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys 485 490 495 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 500 505 510 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 515 520 525 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 530 535 540 Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 545 550 555 560 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 565 570 575 Leu Ser Leu Ser Leu Gly Lys 580 <210> 113 <211> 449 <212> PRT <213> Artificial Sequence <220> <223> 1708-IgG1 (1708IgG1) HC <400> 113 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Asp Pro Asp Ser Thr Gly Ser Lys Tyr Asn Glu Lys Phe 50 55 60 Lys Thr Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Gly Ala Tyr Gly Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 Lys <210> 114 <211> 586 <212> PRT <213> Synthetic sequence <220> <223> 1708-151-IgG1 (1708-151IgG1) <400> 114 His Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Val Ala Ser Ala Ser Gly Phe Thr Tyr Arg 20 25 30 Pro Tyr Cys Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu 35 40 45 Ala Val Ala Gly Ile Asp Ile Phe Gly Gly Thr Thr Tyr Ala Asp Ser 50 55 60 Val Lys Gly Arg Phe Thr Ala Ser Arg Asp Asn Ala Gly Phe Ser Leu 65 70 75 80 Phe Leu Gln Met Asn Asp Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr 85 90 95 Cys Ala Ala Gly Asp Ser Pro Asp Gly Arg Cys Pro Pro Leu Gly Gln 100 105 110 Gly Leu Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly 115 120 125 Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Gln Ser 130 135 140 Gly Ala Glu Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys 145 150 155 160 Ala Ser Gly Tyr Thr Phe Thr Asn Tyr Trp Met His Trp Val Arg Gln 165 170 175 Ala Pro Gly Gln Gly Leu Glu Trp Met Gly Arg Ile Asp Pro Asp Ser 180 185 190 Thr Gly Ser Lys Tyr Asn Glu Lys Phe Lys Thr Arg Val Thr Met Thr 195 200 205 Arg Asp Thr Ser Thr Ser Thr Val Tyr Met Glu Leu Ser Ser Leu Arg 210 215 220 Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Glu Gly Ala Tyr Gly 225 230 235 240 Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 245 250 255 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 260 265 270 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 275 280 285 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 290 295 300 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 305 310 315 320 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 325 330 335 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 340 345 350 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 355 360 365 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 370 375 380 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 385 390 395 400 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 405 410 415 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 420 425 430 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 435 440 445 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 450 455 460 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 465 470 475 480 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 485 490 495 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 500 505 510 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 515 520 525 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 530 535 540 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 545 550 555 560 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 565 570 575 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 580 585 <210> 115 <211> 5 <212> PRT <213> artificial sequence <220> <223> 1707 HCDR1 <400> 115 Asp Tyr His Met Tyr 1 5 <210> 116 <211> 17 <212> PRT <213> artificial sequence <220> <223> 1707 HCDR2 <400> 116 Tyr Ile Ser Lys Gly Gly Ile Ser Thr Tyr Tyr Pro Asp Thr Val Lys 1 5 10 15 Gly <210> 117 <211> 10 <212> PRT <213> artificial sequence <220> <223> 1707 HCDR3 <400> 117 Gln Ser Ser Tyr Asp Phe Ala Met Asp Tyr 1 5 10 <210> 118 <211> 11 <212> PRT <213> Artificial sequence <220> <223> 1707 LCDR1 <400> 118 Lys Ala Ser Gln Asp Val Gly Thr Ser Val Ala 1 5 10 <210> 119 <211> 7 <212> PRT <213> Artificial sequence <220> <223> 1707 LCDR2 <400> 119 Trp Ala Ser Ala Arg His Thr 1 5 <210> 120 <211> 9 <212> PRT <213> Artificial sequence <220> <223> 1707 LCDR3 <400> 120 Gln Gln Tyr Ser Ser Tyr Pro Leu Thr 1 5 <210> 121 <211> 5 <212> PRT <213> Artificial sequence <220> <223> 1708 HCDR1 <400> 121 Asn Tyr Trp Met His 1 5 <210> 122 <211> 17 <212> PRT <213> Artificial sequence <220> <223> 1708 HCDR2 <400> 122 Arg Ile Asp Pro Asp Ser Thr Gly Ser Lys Tyr Asn Glu Lys Phe Lys 1 5 10 15 Thr <210> 123 <211> 10 <212> PRT <213> Artificial sequence <220> <223> 1708 HCDR3 <400> 123 Glu Gly Ala Tyr Gly Tyr Tyr Tyr Phe Asp Tyr 1 5 10 <210> 124 <211> 11 <212> PRT <213> Artificial sequence <220> <223> 1708 LCDR1 <400> 124 Arg Ala Ser Glu Asn Ile Tyr Ser Tyr Leu Ala 1 5 10 <210> 125 <211> 7 <212> PRT <213> Artificial sequence <220> <223> 1708 LCDR2 <400> 125 Asn Ala Arg Thr Leu Ala Glu 1 5 <210> 126 <211> 9 <212> PRT <213> Artificial sequence <220> <223> 1708 LCDR3 <400> 126 Gln Tyr His Ser Gly Ser Pro Leu Pro 1 5 <210> 127 <211> 5 <212> PRT <213> Artificial sequence <220> <223> 1709 HCDR1 <400> 127 Asp Tyr Tyr Met His 1 5 <210> 128 <211> 17 <212> PRT <213> Artificial sequence <220> <223> 1709 HCDR2 <400> 128 Leu Val Tyr Pro Tyr Asn Asp Asn Thr Gly Tyr Asn Arg Lys Phe Lys 1 5 10 15 Gly <210> 129 <211> 11 <212> PRT <213> Artificial sequence <220> <223> 1709 HCDR3 <400> 129 Gly Gly Pro Ser Asn Trp Asn Tyr Phe Asp Tyr 1 5 10 <210> 130 <211> 11 <212> PRT <213> Artificial sequence <220> <223> 1709 LCDR1 <400> 130 Lys Ala Ser Gln Asn Val Val Thr Ala Val Ala 1 5 10 <210> 131 <211> 7 <212> PRT <213> Artificial sequence <220> <223> 1709 LCDR2 <400> 131 Ser Ala Ser Asn Arg Tyr Thr 1 5 <210> 132 <211> 9 <212> PRT <213> Artificial sequence <220> <223> 1709 HCDR3 <400> 132 Gln Gln Tyr Thr Leu Tyr Pro Leu Thr 1 5 <210> 133 <211> 5 <212> PRT <213> Artificial sequence <220> <223> 1710 HCDR1 <400> 133 Asn Tyr Tyr Met His 1 5 <210> 134 <211> 17 <212> PRT <213> Artificial sequence <220> <223> 1710 HCDR2 <400> 134 Arg Ile Asp Pro Thr Ser Gly Ala Thr Lys Tyr Asn Asp Asn Phe Lys 1 5 10 15 Gly <210> 135 <211> 10 <212> PRT <213> Artificial sequence <220> <223> 1710 HCDR3 <400> 135 Glu Gly Gly Phe Gly Tyr Tyr Tyr Phe Asp Tyr 1 5 10 <210> 136 <211> 11 <212> PRT <213> Artificial sequence <220> <223> 1710 LCDR1 <400> 136 Arg Thr Ser Glu Asn Ile Phe Thr Tyr Leu Ala 1 5 10 <210> 137 <211> 7 <212> PRT <213> Artificial sequence <220> <223> 1710 LCDR2 <400> 137 Asn Ala Lys Thr Phe Ala Glu 1 5 <210> 138 <211> 9 <212> PRT <213> Artificial sequence <220> <223> 1710 LCDR3 <400> 138 Gln His His Tyr Gly Ile Pro Leu Pro 1 5 <210> 139 <211> 5 <212> PRT <213> Artificial sequence <220> <223> 1711 HCDR1 <400> 139 Asn Tyr Trp Ile Gly 1 5 <210> 140 <211> 17 <212> PRT <213> Artificial sequence <220> <223> 1711 HCDR2 <400> 140 Asp Ile Tyr Pro Gly Gly Ala Tyr Thr Asn Tyr Asn Glu Lys Phe Lys 1 5 10 15 Asp <210> 141 <211> 13 <212> PRT <213> Artificial sequence <220> <223> 1711 HCDR3 <400> 141 Gly Asp Tyr Tyr Asp Ser Ser Gly Arg Ala Met Asp Tyr 1 5 10 <210> 142 <211> 17 <212> PRT <213> Artificial sequence <220> <223> 1711 LCDR1 <400> 142 Lys Ser Ser Gln Ser Leu Leu Tyr Ser Arg Asn Gln Met Asn Tyr Leu 1 5 10 15 Ala <210> 143 <211> 7 <212> PRT <213> Artificial sequence <220> <223> 1711 LCDR2 <400> 143 Trp Thr Ser Thr Arg Glu Ser 1 5 <210> 144 <211> 9 <212> PRT <213> Synthetic Sequence <220> <223> 1711 LCDR3 <400> 144 Gln Gln Tyr Tyr Ser Tyr Pro Tyr Thr 1 5 <210> 145 <211> 119 <212> PRT <213> Synthetic Sequence <220> <223> 1708-VH1 <400> 145 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Asp Pro Asp Ser Thr Gly Ser Lys Tyr Asn Glu Lys Phe 50 55 60 Lys Thr Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Gly Ala Tyr Gly Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 146 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> 1708-VH2 <400> 146 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Asp Pro Asp Ser Thr Gly Ser Lys Tyr Asn Glu Lys Phe 50 55 60 Lys Thr Arg Val Thr Met Thr Val Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Gly Ala Tyr Gly Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 147 <211> 119 <212> PRT <213> Artificial sequence <220> <223> 1708-VH3 <400> 147 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Arg Ile Asp Pro Asp Ser Thr Gly Ser Lys Tyr Asn Glu Lys Phe 50 55 60 Lys Thr Arg Val Thr Met Thr Val Asp Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Gly Ala Tyr Gly Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 148 <211> 107 <212> PRT <213> artificial sequence <220> <223> 1708‑VL1 <400> 148 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Ile Tyr Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Asn Ala Arg Thr Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Tyr His Ser Gly Ser Pro Leu 85 90 95 Pro Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 149 <211> 107 <212> PRT <213> artificial sequence <220> <223> 1708‑VL2 <400> 149 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Ile Tyr Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ser Pro Lys Leu Leu Ile 35 40 45 Tyr Asn Ala Arg Thr Leu Ala Glu Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Tyr His Ser Gly Ser Pro Leu 85 90 95 Pro Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 150 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> 20 HCDR3 <400> 150 Gly Phe Lys Phe Asp Glu Asp Tyr Cys Ala Pro Asn Asp 1 5 10 <210> 151 <211> 12 <212> PRT <213> Artificial sequence <220> <223> 30 HCDR3 <400> 151 Gly Trp Thr Phe Gly Gly Gln Cys Ser Pro Ala Asp 1 5 10 <210> 152 <211> 10 <212> PRT <213> Artificial sequence <220> <223> connector <400> 152 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 <210> 153 <211> 229 <212> PRT <213> Artificial sequence <220> <223> Human IgG4 heavy chain Fc region (S228P / K447A) <400> 153 Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe 1 5 10 15 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 20 25 30 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 35 40 45 Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val 50 55 60 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser 65 70 75 80 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 85 90 95 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser 100 105 110 Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 115 120 125 Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln 130 135 140 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 145 150 155 160 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 165 170 175 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu 180 185 190 Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser 195 200 205 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 210 215 220 Leu Ser Leu Gly Lys 225

Claims

1. A PVRIG binding protein comprising at least one immunoglobulin single variable domain, the amino acid sequences of CDR1, CDR2 and CDR3 of said immunoglobulin single variable domain are selected from the following combinations: CDR1 as set forth in SEQ ID NO: 10, CDR2 as set forth in SEQ ID NO: 11 and CDR3 as set forth in SEQ ID NO: 12 or 151; CDR1 as set forth in SEQ ID NO: 7, CDR2 as set forth in SEQ ID NO: 8 and CDR3 as set forth in SEQ ID NO: 9 or 150; CDR1 as set forth in SEQ ID NO: 13, CDR2 as set forth in SEQ ID NO: 14 and CDR3 as set forth in SEQ ID NO: 15; CDR1 as set forth in SEQ ID NO: 16, CDR2 as set forth in SEQ ID NO: 17 and CDR3 as set forth in SEQ ID NO: 18; or CDR1 as set forth in SEQ ID NO: 19, CDR2 as set forth in SEQ ID NO: 20 and CDR3 as set forth in SEQ ID NO:

21.

2. The PVRIG binding protein of claim 1, wherein the immunoglobulin single variable domain of said PVRIG binding protein is a humanized and / or affinity matured VHH; said humanized and / or affinity matured VHH comprises a heavy chain framework region of human germline template IGHV3-7*01 or IGHV3-30*02.

3. The PVRIG binding protein of claim 1, wherein the amino acid sequence of said immunoglobulin single variable domain is set forth in any one of SEQ ID NO: 3, 80-84; or SEQ ID NO: 2, 75-79; or SEQ ID NO: 4, 86-90; or SEQ ID NO: 5, 91-95; or SEQ ID NO: 6, 96-100; or has at least 80% sequence identity to any of the foregoing sequences.

4. The PVRIG binding protein of any one of claims 1-3, further comprising a human immunoglobulin Fc region.

5. A PVRIG / TIGIT binding protein comprising a first antigen binding domain that specifically binds PVRIG and a second antigen binding domain that specifically binds TIGIT, wherein 1) said first antigen binding domain that specifically binds PVRIG comprises at least one immunoglobulin single variable domain, the amino acid sequences of CDR1, CDR2 and CDR3 of said immunoglobulin single variable domain are selected from the following combinations: CDR1 as set forth in SEQ ID NO: 10, CDR2 as set forth in SEQ ID NO: 11 and CDR3 as set forth in SEQ ID NO: 12 or 151; a CDR1 as set forth in SEQ ID NO: 7, a CDR2 as set forth in SEQ ID NO: 8, and a CDR3 as set forth in SEQ ID NO: 9 or 150; a CDR1 as set forth in SEQ ID NO: 13, a CDR2 as set forth in SEQ ID NO: 14, and a CDR3 as set forth in SEQ ID NO: 15; a CDR1 as set forth in SEQ ID NO: 16, a CDR2 as set forth in SEQ ID NO: 17, and a CDR3 as set forth in SEQ ID NO: 18; or a CDR1 as set forth in SEQ ID NO: 19, a CDR2 as set forth in SEQ ID NO: 20, and a CDR3 as set forth in SEQ ID NO: 21; and 2) the second antigen binding domain that specifically binds to TIGIT comprises a heavy chain variable region (VH) and a light chain variable region (VL), the HCDR1, HCDR2, and HCDR3 of the heavy chain variable region are respectively an amino acid sequence as set forth in SEQ ID NOs: 121, 122, and 123, and the LCDR1, LCDR2, and LCDR3 of the light chain variable region are respectively an amino acid sequence as set forth in SEQ ID NOs: 124, 125, and 126.

6. The PVRIG / TIGIT binding protein of claim 5, wherein the amino acid sequence of the immunoglobulin single variable domain in the first antigen binding domain is respectively as set forth in any one of SEQ ID NO: 3, 80-84; or SEQ ID NO: 2, 75-79; or SEQ ID NO: 4, 86-90; or SEQ ID NO: 5, 91-95; or SEQ ID NO: 6, 96-100; or has at least 80% sequence identity to any of the foregoing sequences.

7. The PVRIG / TIGIT binding protein of claim 5, wherein, the heavy chain variable region of the second antigen binding domain that specifically binds to TIGIT comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 145-147 or has at least 90% sequence identity thereto, and the light chain variable region comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 148-149 or has at least 90% sequence identity thereto.

8. The PVRIG / TIGIT binding protein of claim 5, wherein, the second antigen binding domain that specifically binds to TIGIT comprises a full length heavy chain (HC) and a full length light chain (LC); the heavy chain sequence comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 145-147 or has at least 90% sequence identity thereto; and the light chain sequence comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 148-149 or has at least 90% sequence identity thereto.

9. The PVRIG / TIGIT binding protein of claim 7, wherein, the second antigen binding domain that specifically binds to TIGIT comprises a full length heavy chain (HC) and a full length light chain (LC); the heavy chain sequence comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 145-147 or has at least 90% sequence identity thereto; and the light chain sequence comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 148-149 or has at least 90% sequence identity thereto. the heavy chain sequence is set forth in SEQ ID NO: 102 or has at least 90% sequence identity thereto and the light chain sequence is set forth in SEQ ID NO: 103 or has at least 90% sequence identity thereto.

10. The PVRIG / TIGIT binding protein of any one of claims 5-9, the second antigen binding domain that specifically binds to TIGIT comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein: the immunoglobulin single variable domain that specifically binds to PVRIG is N-terminal to the heavy chain variable region of the second antigen binding domain that specifically binds to TIGIT; the immunoglobulin single variable domain that specifically binds to PVRIG is C-terminal to the heavy chain variable region of the second antigen binding domain that specifically binds to TIGIT; the immunoglobulin single variable domain that specifically binds to PVRIG is N-terminal to the light chain variable region of the second antigen binding domain that specifically binds to TIGIT; and / or the immunoglobulin single variable domain that specifically binds to PVRIG is C-terminal to the light chain variable region of the second antigen binding domain that specifically binds to TIGIT.

11. The PVRIG / TIGIT binding protein of claim 10, wherein, the immunoglobulin single variable domain that specifically binds to PVRIG is directly or via a linker connected to the second antigen binding domain that specifically binds to TIGIT; the linker is an amino acid sequence set forth as (G4S)2.

12. The PVRIG / TIGIT binding protein of any one of claim 11, comprising a first polypeptide chain and a second polypeptide chain, wherein: the first polypeptide chain comprises an amino acid sequence set forth in any one of SEQ ID NOs: 108-112 and 114 and the second polypeptide chain comprises an amino acid sequence set forth in SEQ ID NO: 103; or the first polypeptide chain comprises an amino acid sequence set forth in SEQ ID NO: 104 or 105 and the second polypeptide chain comprises an amino acid sequence set forth in SEQ ID NO: 103; or the first polypeptide chain comprises an amino acid sequence set forth in SEQ ID NO: 102 and the second polypeptide chain comprises an amino acid sequence set forth in SEQ ID NO: 106 or 107.

13. An anti-PVRIG antibody or antigen-binding fragment thereof, comprising the immunoglobulin single variable domain of any one of claims 1-3 or 5-6.

14. The anti-PVRIG antibody or antigen-binding fragment thereof of claim 13, further comprising the second antigen binding domain that specifically binds to TIGIT of claim 5.

15. A polynucleotide encoding the PVRIG binding protein of any one of claims 1-4, the PVRIG / TIGIT binding protein of any one of claims 5-12, or the anti-PVRIG antibody or antigen-binding fragment thereof of claim 13 or 14.

16. A host cell comprising the polynucleotide of claim 15.

17. A method of making a PVRIG binding protein, a PVRIG / TIGIT binding protein, or an anti-PVRIG antibody or antigen-binding fragment thereof, comprising: expressing the polynucleotide of claim 15 in the host cell of claim 16, and isolating the expressed PVRIG binding protein, PVRIG / TIGIT binding protein, or anti-PVRIG antibody or antigen-binding fragment thereof from the host cell.

18. A pharmaceutical composition comprising the PVRIG binding protein of any one of claims 1-4, the PVRIG / TIGIT binding protein of any one of claims 5-12, or the anti-PVRIG antibody or antigen-binding fragment thereof of claim 13 or 14, and a pharmaceutically acceptable excipient, diluent, or carrier.

19. Use of the PVRIG binding protein of any one of claims 1-4, the PVRIG / TIGIT binding protein of any one of claims 5-12, or the anti-PVRIG antibody or antigen-binding fragment thereof of claim 13 or 14, the polynucleotide of claim 15, or the pharmaceutical composition of claim 18 in the manufacture of a medicament for treating cancer, the cancer being melanoma.

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