Combination therapy involving anti-HLA-G antibodies and anti-EGFR antibodies, anti-PD1 or anti-PD-L1 antibodies and / or anti-CD47

Abstract

Provided herein are combination therapies involving an antibody having a binding specificity for HLA-G and an antibody having a binding specificity for EGFR, PD-1 and / or PD-L1 and / or CD47 or SIRP [alpha].

Description

[0001] Related Applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 329,065, filed on April 8, 2022, the contents of which are incorporated herein by reference in their entirety. Technical Field

[0003] Provided herein are combination therapies involving an antibody having binding specificity for HLA-G and an antibody having binding specificity for EGFR, PD-1 and / or PD-L1 and / or CD47 or SIRPα.

[0004] Sequence Listing

[0005] This application contains a sequence listing that has been submitted electronically in .XML format and is hereby incorporated by reference in its entirety. The .XML copy created on April 6, 2023 is named "1107368.00128.xml" and is 161,005 bytes in size. The sequence listing contained in this .XML file is part of the specification and is hereby incorporated by reference in its entirety. Background Art

[0006] HLA-G histocompatibility antigen class I G, also known as human leukocyte antigen G (HLA-G), is a protein encoded by the HLA-G gene in humans. HLA-G belongs to the HLA nonclassical class I heavy chain paralog. HLA-G is a heterodimer composed of a heavy chain and a light chain (beta-2 microglobulin). HLA-G has membrane-bound and soluble forms.

[0007] HLA-G is usually expressed at the maternal-fetal interface and other immune privileged sites. HLA-G plays a role in pregnancy immune tolerance and is expressed in the placenta by extravillous trophoblast cells, while the classical MHC class I genes (HLA-A and HLA-B) do not. Since HLA-G was first identified in placental samples, many studies have evaluated its role in pregnancy conditions such as preeclampsia and recurrent miscarriage. See Michita, Rafael Tomoya et al., Human Immunology 2016, 77 (10): 892-897, which is incorporated herein by reference in its entirety, including any drawings.

[0008] HLA-G has been shown to be immunosuppressive. By binding to receptors expressed on a variety of myeloid and lymphoid cells, HLA-G can directly inhibit the function of NK cells, cytotoxic T lymphocytes, B cells, neutrophils, monocytes, macrophages, and dendritic cells. HLA-G also inhibits T and NK cell proliferation and cytolytic activity. HLA-G inhibits phagocytosis and induces the generation or expansion of regulatory T cells.

[0009] HLA-G mediates immune function through at least three ITIM-containing inhibitory receptors: ILT2, ILT4, and KIR2DL4. For example, on lymphoid and myeloid cells, HLA-G mediates function through ILT2. On myeloid cells, HLA-G mediates function through ILT4. On decidual NK cells, HLA-G mediates immune function through KIR2DL4 and ILT2.

[0010] HLA-G is an immune checkpoint target. HLA-G can directly inhibit immune cell function by receptor binding and / or trogocytosis and impair chemotaxis. HLA-G can give tumor cells higher invasion and metastasis potential. HLA-G promotes escape from tumor immune surveillance and enhances metastasis and progression of malignant tumors. During tumor progression, HLA-G has other effects, such as inhibiting immune cell lysis, inducing immune cell apoptosis, and / or producing regulatory cells by receptor binding and / or trogocytosis.

[0011] HLA-G expression is upregulated in a wide variety of tumors and is associated with poor prognosis and disease progression. Serum HLA-G levels are elevated in breast cancer, lung cancer, colorectal cancer (CRC), gastric cancer, esophageal cancer, neuroblastoma, cervical cancer, and hematological cancers. HLA-G has also been found to be associated with clinical parameters of advanced disease, such as tumor metastasis, poor prognosis, immune escape, and tumor aggressiveness.

[0012] PD-1 is a membrane protein with 268 amino acids. PD-1 contains an extracellular IgV domain, a transmembrane region, and an intracellular tail. The tail contains two phosphorylation sites, located in an immunoreceptor tyrosine-based inhibitory motif and an immunoreceptor tyrosine-based switch motif. It has been shown that PD-1 negatively regulates T cell receptor signaling.

[0013] PD-1 is moderately expressed on naive T cells, B cells, and NK cells, and is upregulated by T / B cell receptor signaling on lymphocytes, monocytes, and myeloid cells. PD-1 plays a role in modulating the immune system's response by downregulating the immune system and promoting self-tolerance by inhibiting T cell inflammatory activity. It prevents autoimmune diseases, but may also prevent the immune system from killing cancer cells.

[0014] PD-1 is considered an important player in immune regulation and maintenance of peripheral tolerance. PD-1 can be considered an immune checkpoint and operates through a variety of different mechanisms. For example, PD-1 promotes apoptosis of antigen-specific T cells in lymph nodes. Furthermore, PD-1 reduces apoptosis of regulatory T cells (anti-inflammatory, suppressive T cells).

[0015] PD-1 binds to two ligands, PD-L1 and PD-L2. Both PD-1 ligands are members of the CD28-B7 family of co-signaling molecules, which play important roles in all stages of T cell function and other cell functions. The interaction of PD-1 with its ligands sends a signal into the T cell and essentially shuts down or inhibits the T cell.

[0016] Cancer cells exploit this system by driving high levels of PD-L1 expression. This allows cancer cells to gain control of the PD-1 pathway and shut down PD-1-expressing T cells, thereby suppressing anti-cancer immune responses. PD-L1 is associated with poor prognosis in ovarian cancer, renal cancer, colorectal cancer, pancreatic cancer, liver cancer, and melanoma. Similarly, PD-1 expression on tumor-infiltrating lymphocytes indicates T cell dysfunction in breast cancer and melanoma and is associated with poor prognosis in renal cancer.

[0017] PD-1 therapies that “unblock” an existing immune response or unblock the onset of an immune response are effective, but sometimes only a subset of subjects respond. In addition, even among those who respond, the response is not always complete or optimal.

[0018] Epidermal growth factor receptor (EGFR; ErbB-1; HER1 in humans) is a transmembrane protein that is a receptor for members of the epidermal growth factor family (EGF family) of extracellular protein ligands. The epidermal growth factor receptor is a member of the ErbB receptor family, which is a subfamily of the following four closely related receptor tyrosine kinases: EGFR (ErbB-1), HER2 / neu (ErbB-2), Her 3 (ErbB-3) and Her 4 (ErbB-4). In many cancer types, mutations that affect EGFR expression or activity may lead to more progressive cancers. Insufficient signaling of EGFR and other receptor tyrosine kinases in humans is associated with diseases such as Alzheimer's disease, while overexpression is associated with the development of a variety of tumors. Interrupting EGFR signaling by blocking the EGFR binding site on the extracellular domain of the receptor or by inhibiting intracellular tyrosine kinase activity can prevent the growth of tumors expressing EGFR and improve the patient's condition.

[0019] EGFR is a transmembrane protein that is activated by binding to its specific ligands, including epidermal growth factor and transforming growth factor α (TGFα). ErbB2 has no known direct activating ligands and may be constitutively activated or become active after heterodimerization with other family members such as EGFR. Upon activation by its growth factor ligands, EGFR switches from an inactive monomeric form to an active homodimer.

[0020] EGFR dimerization stimulates its intrinsic intracellular protein-tyrosine kinase activity. As a result, several tyrosine (Y) residues in the C-terminal domain of EGFR have undergone autophosphorylation. These residues include Y992, Y1045, Y1068, Y1148 and Y1173. Autophosphorylation triggers downstream activation and signal transduction of several other proteins, which are related to phosphorylated tyrosine by their own phosphotyrosine binding SH2 domains. These downstream signal transduction proteins start several signal transduction cascades, mainly MAPK, Akt and JNK pathways, thereby causing DNA synthesis and cell proliferation. Such proteins regulate phenotypes such as cell migration, adhesion and proliferation. The kinase domain of EGFR can also cross-phosphorylate the tyrosine residues of other receptors with which it is polymerized, and itself can be activated in this way.

[0021] Mutations that cause EGFR overexpression (called upregulation or amplification) are associated with many cancers, including lung adenocarcinoma (40% of cases), anal cancer, glioblastoma (50%), and head and neck epithelial tumors (80%-100%). These somatic mutations involving EGFR cause it to be constantly activated, thereby producing uncontrolled cell division. In glioblastoma, specific mutations of EGFR are often observed, and the specific mutation is referred to as EGFRvIII. About 30% of all epithelial cancers are associated with mutations, amplifications, or disorders of EGFR or its family members.

[0022] CD47 (cluster of differentiation 47), also known as integrin-associated protein (IAP), is a transmembrane protein encoded by the CD47 gene in humans. CD47 belongs to the immunoglobulin superfamily and binds to membrane integrins, and also binds to the ligands thrombospondin-1 (TSP-1) and signal regulatory protein α (SIRPα). CD47 is involved in a range of cellular processes, including apoptosis, proliferation, adhesion and migration. In addition, CD47 plays a key role in immune and angiogenic responses. CD47 is ubiquitously expressed in human cells, and has been found to be overexpressed in many different tumor cells.

[0023] CD47 is a 50 kDa membrane receptor with an extracellular N-terminal IgV domain, five transmembrane domains, and a short C-terminal intracellular tail. There are four alternatively spliced ​​isoforms of CD47 that differ only in the length of their cytoplasmic tails.

[0024] Type 2 is the most widely expressed form found in all circulating and immune cells. The second most abundant isoform is type 4, which is primarily expressed in the brain and peripheral nervous system. Only keratinocytes express large amounts of type 1.

[0025] CD47 is a high affinity receptor for thrombospondin-1 (TSP-1), a secreted glycoprotein that plays a role in vascular development and angiogenesis, and in the latter capacity, the TSP1 / CD47 interaction inhibits nitric oxide signaling at multiple levels in vascular cells. Binding of TSP-1 to CD47 affects several fundamental cellular functions, including cell migration and adhesion, cell proliferation or apoptosis, and plays a role in the regulation of angiogenesis and inflammation.

[0026] CD47 interacts with signal regulatory protein α (SIRPα), an inhibitory transmembrane receptor present on myeloid cells. The CD47 / SIRPα interaction results in bidirectional signaling, leading to different intercellular responses, including inhibition of phagocytosis, stimulation of intercellular fusion, and T cell activation. CD47 interacts with several membrane integrins, the most common of which is integrin α V β3. These interactions generate the CD47 / integrin complex, which influences a range of cellular functions including adhesion, spreading, and migration.

[0027] CD47 was first identified as a tumor antigen on human ovarian cancer in the 1980s. Since then, CD47 has been found to be expressed on a variety of human tumor types, including acute myeloid leukemia (AML), chronic myeloid leukemia, acute lymphocytic leukemia (ALL), non-Hodgkin's lymphoma (NHL), multiple myeloma (MM), bladder cancer, and other solid tumors. CD47 is also highly expressed in brain tumors in children and adults.

[0028] High levels of CD47 prevent cancer cells from being phagocytosed. This is due to the binding of CD47 to SIRP-α on macrophages. Binding of SIRP-α leads to inhibition of phagocytosis.

[0029] Anti-CD47 antibody-mediated phagocytosis of cancer by macrophages can trigger an anti-tumor T cell immune response. Anti-CD47 antibody treatment not only enables macrophages to engulf cancer cells, but also promotes the activation of cancer-specific lymphocytes: cancer cells display mutant proteins that the immune system can now respond to.

[0030] There is a need for combination therapies involving an antibody having binding specificity for HLA-G and an antibody having binding specificity for EGFR, PD-1 and / or PD-L1 and / or CD47 or SIRPα. Summary of the invention

[0031] Provided herein are methods and pharmaceutical compositions for treating a subject having cancer, the pharmaceutical compositions comprising an antibody that binds to HLA-G and an antibody that binds to EGFR, PD-1 or PD-L1 and / or CD47 or SIRPα.

[0032] The first aspect provides a pharmaceutical composition comprising an antibody that binds to HLA-G and an antibody that binds to epidermal growth factor receptor (EGFR). In some embodiments, the antibody that binds to HLA-G comprises or consists of a heavy chain variable region (VH) and a light chain variable region (VL), wherein VH and / or VL comprises:

[0033] a) a VHCDR1 having the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 11,

[0034] b) a VHCDR2 having the sequence shown in SEQ ID NO: 21 or SEQ ID NO: 31,

[0035] c) a VHCDR3 having the sequence shown in SEQ ID NO: 41,

[0036] d) VLCDR1 having the sequence shown in SEQ ID NO: 51,

[0037] e) VLCDR2 having the sequence shown in SEQ ID NO: 61, and

[0038] f) VLCDR3 having the sequence shown in SEQ ID NO:71.

[0039] In some embodiments, the antibody that binds to HLA-G comprises or consists of a heavy chain variable region (VH) and a light chain variable region (VL), the VH comprising, consisting of, or consisting essentially of a VH having a sequence as set forth in SEQ ID NO: 81, and the VL comprising, consisting of, or consisting essentially of a VL having a sequence as set forth in SEQ ID NO: 91. In some embodiments, the antibody that binds to HLA-G is an antibody comprising a heavy chain comprising or consisting of one or more molecules having a sequence comprising or consisting of SEQ ID NO: 101, and a light chain comprising or consisting of one or more molecules having a sequence comprising or consisting of SEQ ID NO: 111.

[0040] In some embodiments, the antibody that binds to epidermal growth factor receptor (EGFR) comprises or consists of a heavy chain variable region (VH) and a light chain variable region (VL), wherein VH and / or VL comprises:

[0041] a) a VHCDR1 having the sequence shown in SEQ ID NO: 9 or SEQ ID NO: 19,

[0042] b) a VHCDR2 having the sequence shown in SEQ ID NO: 29 or SEQ ID NO: 39,

[0043] c) a VHCDR3 having the sequence shown in SEQ ID NO: SEQ ID NO: 49,

[0044] d) VLCDR1 having the sequence shown in SEQ ID NO: SEQ ID NO: 59,

[0045] e) VLCDR2 having the sequence shown in SEQ ID NO: SEQ NO: 69, and

[0046] f) VLCDR3 having the sequence shown in SEQ ID NO:79.

[0047] In some embodiments, the antibody that binds to epidermal growth factor receptor (EGFR) comprises or consists of a heavy chain variable region (VH) and a light chain variable region (VL), VH and / or VL comprises, consists, or consists essentially of a VH having a sequence as shown in SEQ ID NO: 89, and VL comprises, consists, or consists essentially of a VL having a sequence as shown in SEQ ID NO: 99. In some embodiments, the antibody that binds to epidermal growth factor receptor (EGFR) is an antibody comprising a heavy chain and a light chain, the heavy chain comprising or consisting of one or more molecules having a sequence comprising or consisting of SEQ ID NO: 109, and the light chain comprising or consisting of one or more molecules having a sequence comprising or consisting of SEQ ID NO: 119.

[0048] The second aspect provides a pharmaceutical composition comprising an antibody that binds to HLA-G and an antibody that binds to PD-1 and / or PD-L1. In some embodiments, the antibody that binds to HLA-G comprises or consists of a heavy chain variable region (VH) and a light chain variable region (VL), wherein VH and / or VL comprises:

[0049] a) a VHCDR1 having the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 11,

[0050] b) a VHCDR2 having the sequence shown in SEQ ID NO: 21 or SEQ ID NO: 31,

[0051] c) a VHCDR3 having the sequence shown in SEQ ID NO: 41,

[0052] d) VLCDR1 having the sequence shown in SEQ ID NO: 51,

[0053] e) VLCDR2 having the sequence shown in SEQ ID NO: 61, and

[0054] f) VLCDR3 having the sequence shown in SEQ ID NO:71.

[0055] In some embodiments, the antibody that binds to HLA-G comprises or consists of a heavy chain variable region (VH) and a light chain variable region (VL), the VH comprising, consisting of, or consisting essentially of a VH having a sequence as set forth in SEQ ID NO: 81, and the VL comprising, consisting of, or consisting essentially of a VL having a sequence as set forth in SEQ ID NO: 91. In some embodiments, the antibody that binds to HLA-G is an antibody comprising a heavy chain comprising or consisting of one or more molecules having a sequence comprising or consisting of SEQ ID NO: 101, and a light chain comprising or consisting of one or more molecules having a sequence comprising or consisting of SEQ ID NO: 111.

[0056] In some embodiments, the antibody that binds to PD-1 and / or PD-L1 comprises or consists of a heavy chain variable region (VH) and a light chain variable region (VL), and VH and / or VL comprises:

[0057] a) a VHCDR1 having a sequence as shown in SEQ ID NO: 2-8 or SEQ ID NO: 12-18,

[0058] b) a VHCDR2 having a sequence as shown in SEQ ID NO: 22-28 or SEQ ID NO: 32-38,

[0059] c) a VHCDR3 having a sequence as shown in SEQ ID NO: 42-48,

[0060] d) a VLCDR1 having a sequence shown in SEQ ID NO: 52-58,

[0061] e) a VLCDR2 having a sequence as shown in SEQ ID NO: 62-68, and

[0062] f) VLCDR3 having the sequence shown in SEQ ID NO: 72-78.

[0063] In some embodiments, the antibody that binds to PD-1 and / or PD-L1 comprises or consists of a heavy chain variable region (VH) and a light chain variable region (VL), VH and / or VL comprises, consists of, or consists essentially of: a VH having a sequence as shown in any one of SEQ ID NOs: 82-88, and VL comprises, consists of, or consists essentially of: a VL having a sequence as shown in any one of SEQ ID NOs: 92-98. In some embodiments, the antibody that binds to PD-1 and / or PD-L1 is an antibody comprising a heavy chain and a light chain, the heavy chain comprising or consisting of: one or more molecules having a sequence as shown in any one of SEQ ID NOs: 102-108 or consisting thereof, and the light chain comprising or consisting of: one or more molecules having a sequence as shown in any one of SEQ ID NOs: 112-118 or consisting thereof.

[0064] The third aspect provides an antibody that binds to HLA-G and an antibody that binds to CD47 or SIRPα. In some embodiments, the antibody that binds to HLA-G comprises or consists of a heavy chain variable region (VH) and a light chain variable region (VL), wherein VH and / or VL comprises:

[0065] a) a VHCDR1 having the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 11,

[0066] b) a VHCDR2 having the sequence shown in SEQ ID NO: 21 or SEQ ID NO: 31,

[0067] c) a VHCDR3 having the sequence shown in SEQ ID NO: 41,

[0068] d) VLCDR1 having the sequence shown in SEQ ID NO: 51,

[0069] e) VLCDR2 having the sequence shown in SEQ ID NO: 61, and

[0070] f) VLCDR3 having the sequence shown in SEQ ID NO:71.

[0071] In some embodiments, the antibody that binds to HLA-G comprises or consists of a heavy chain variable region (VH) and a light chain variable region (VL), the VH comprising, consisting of, or consisting essentially of a VH having a sequence as set forth in SEQ ID NO: 81, and the VL comprising, consisting of, or consisting essentially of a VL having a sequence as set forth in SEQ ID NO: 91. In some embodiments, the antibody that binds to HLA-G is an antibody comprising a heavy chain comprising or consisting of one or more molecules having a sequence comprising or consisting of SEQ ID NO: 101, and a light chain comprising or consisting of one or more molecules having a sequence comprising or consisting of SEQ ID NO: 111.

[0072] In some embodiments, the antibody that binds to CD47 comprises or consists of a heavy chain variable region (VH) and a light chain variable region (VL), and VH and / or VL comprises:

[0073] a) a VHCDR1 having the sequence shown in SEQ ID NO: 10 or SEQ ID NO: 20,

[0074] b) a VHCDR2 having the sequence shown in SEQ ID NO: 30 or SEQ ID NO: 40,

[0075] c) a VHCDR3 having the sequence shown in SEQ ID NO: 50,

[0076] d) VLCDR1 having the sequence shown in SEQ ID NO: 60,

[0077] e) VLCDR2 having the sequence shown in SEQ ID NO: 70, and

[0078] f) VLCDR3 having the sequence shown in SEQ ID NO:80.

[0079] In some embodiments, the antibody that binds to CD47 comprises or consists of a heavy chain variable region (VH) and a light chain variable region (VL), the VH and / or VL comprising, consisting of, or consisting essentially of a VH having a sequence as set forth in SEQ ID NO: 90, and a VL comprising, consisting of, or consisting essentially of a VL having a sequence as set forth in SEQ ID NO: 100. In some embodiments, the antibody that binds to CD47 is an antibody comprising a heavy chain and a light chain, the heavy chain comprising or consisting of one or more molecules having a sequence comprising or consisting of SEQ ID NO: 110, and the light chain comprising or consisting of one or more molecules having a sequence comprising or consisting of SEQ ID NO: 120.

[0080] A fourth aspect provides a method for treating a subject with cancer, the method comprising administering to the subject any of the pharmaceutical compositions described herein. In some embodiments, the cancer is a solid cancer. In some embodiments, the cancer is a blood cancer. In some embodiments, the subject is a human subject. In some embodiments, the method further comprises one or more of the following:

[0081] a) administering chemotherapy;

[0082] b) administering radiation therapy; and / or

[0083] c) administering one or more additional therapeutic agents.

[0084] A fifth aspect provides a method of modulating immune system function in a subject in need thereof, the method comprising the step of contacting a population of immune cells of the subject with any one of the pharmaceutical compositions provided herein. In some embodiments, the subject is a human subject.

[0085] In some embodiments, the pharmaceutical composition is administered in an amount sufficient to achieve 1, 2, 3, 4, 5, 6, 7, or 8 of the following in a subject:

[0086] a) Suppression of immunosuppression;

[0087] b) reduce the level of regulatory T cells;

[0088] c) increasing the activity of myeloid cells, cytotoxic T lymphocytes, helper T cells, NK cells, T cells, B cells, neutrophils, monocytes, macrophages and / or dendritic cells;

[0089] d) increase phagocytic activity;

[0090] e) inhibit metastasis;

[0091] f) inhibiting tumor growth;

[0092] g) inducing tumor regression; and / or

[0093] h) Enhancement of ILT2 + CD8 + T cell degranulation.

[0094] In some embodiments, increasing phagocytic activity comprises increasing the percentage of phagocytosis compared to a pharmaceutical composition without an antibody or a pharmaceutical composition with an antibody that binds to HLA-G. In some embodiments, the method further comprises one or more of the following:

[0095] a) administering chemotherapy;

[0096] b) administering radiation therapy; and / or

[0097] c) administering one or more additional therapeutic agents. BRIEF DESCRIPTION OF THE DRAWINGS

[0098] Figure 1 The results showed that the combination of an antibody (Fab fragment) binding to HLA-G and cetuximab significantly increased macrophages' affinity for HLA-G compared with cetuximab alone. + Phagocytosis of A549 target cells. No activity was observed in antibodies bound to HLA-G compared to untreated controls. Data are shown as mean ± standard deviation (n = 2 replicates). **p < 0.01 using Student's t-test.

[0099] Figure 2A and Figure 2B The combination of an antibody that binds to HLA-G with pembrolizumab or atezolizumab enhanced ILT2 compared to either agent alone. + CD8 + T cell degranulation (CD107a). Isotype-matched antibody was used as negative control. Graphs show mean ± SD from 1 representative donor (n = 2 replicate wells).

[0100] Figure 3A and Figure 3B It was demonstrated that the combination of an antibody binding to HLA-G and mololimab increased macrophage phagocytosis of HT-1376 and JEG-3 cells expressing endogenous HLA-G, respectively, compared to either agent alone. Figure 3C and Figure 3DIt was shown that the triple combination of an antibody that binds to HLA-G with mololimab and cetuximab was more active in inducing phagocytosis of A549 and SK-OV-3 cells engineered to express HLA-G than single or double-dose treatment. Isotype antibodies were used as negative controls. Data are shown as mean ± standard deviation (n = 2 replicates). DETAILED DESCRIPTION

[0101] Provided herein are combination therapies involving an antibody having binding specificity for HLA-G and an antibody having binding specificity for EGFR, PD-1 and / or PD-L1 and / or CD47 or SIRPα.

[0102] 1. Definition

[0103] Unless otherwise defined, all technical terms, symbols and other scientific terms used herein are intended to have the meanings commonly understood by those skilled in the art to which the present invention belongs. In some cases, for clarity and / or ease of reference, the terms with commonly understood meanings are defined herein, and such definitions are included herein and are not necessarily understood to represent differences compared with the meanings commonly understood in the art. The techniques and procedures described herein or cited are generally fully understood by those skilled in the art and are generally used to utilize using conventional methods, for example, the widely used molecular cloning methods described below: Sambrook et al., "Molecular Cloning: Laboratory Manual (Molecular Cloning: A Laboratory Manual)", 2nd edition, (1989), Cold Spring Harbor Laboratory Press (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY) of Cold Spring Harbor, New York. Where appropriate, unless otherwise noted, the procedures involving the use of commercially available kits and reagents are generally performed according to the schemes and / or parameters defined by the manufacturer.

[0104] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0105] The terms "HLA-G," "human leukocyte antigen G," and "HLA-G histocompatibility antigen class I G" are used interchangeably herein. Unless otherwise indicated, the terms encompass any variants, isoforms, and species homologs of human HLA-G that are naturally expressed by cells or expressed by cells transfected with the HLA-G gene.

[0106] The terms "PD-1", "programmed cell death protein 1" and "cluster of differentiation 279" are used interchangeably herein. Unless otherwise indicated, the terms include any variants, isoforms and species homologs of human PD-1 that are naturally expressed by cells or expressed by cells transfected with the PD-1 gene.

[0107] The terms "PD-L1", "programmed death-ligand 1", "cluster of differentiation 274", "B7 homolog 1" and "B7-H1" are used interchangeably herein. Unless otherwise indicated, the terms include any variants, isoforms and species homologs of human PD-L1 that are naturally expressed by cells or expressed by cells transfected with the PD-L1 gene.

[0108] The terms "EGFR", "epidermal growth factor receptor", "ErbB-1" and "HER1" are used interchangeably herein. Unless otherwise indicated, the terms include any variants, isoforms and species homologs of human EGFR that are naturally expressed by cells or expressed by cells transfected with the EGFR gene.

[0109] The terms "CD47", "cluster of differentiation 47" and "integrin-associated protein" are used interchangeably herein. Unless otherwise indicated, the terms include any variants, isoforms and species homologs of human CD47 that are naturally expressed by cells or expressed by cells transfected with the CD47 gene.

[0110] The terms "signal regulatory protein alpha" or "SIRPα" are used interchangeably herein.

[0111] The term "immunoglobulin" refers to a class of structurally related proteins that typically include two pairs of polypeptide chains: a pair of light (L) chains and a pair of heavy (H) chains. In a complete immunoglobulin, all four of these chains are interconnected by disulfide bonds. The structure of immunoglobulins has been well characterized. See, for example, Paul, Fundamental Immunology, 7th edition, Chapter 5 (2013) Lippincott Williams & Wilkins, Philadelphia, PA. In short, each heavy chain typically includes a heavy chain variable region (V H ) and the heavy chain constant region (C H ). The heavy chain constant region usually contains three domains CH1, CH2 and CH3. Each light chain typically contains a light chain variable region (VL) and a light chain constant region. The light chain constant region typically contains one domain, abbreviated as CL.

[0112] The term "antibody" describes a type of immunoglobulin molecule and is used herein in its broadest sense. Antibodies specifically include complete antibodies (e.g., complete immunoglobulins) and antibody fragments. Antibodies contain at least one antigen binding domain. An example of an antigen binding domain is an antigen binding domain formed by a VH-VL dimer.

[0113] V H Area and V L The V regions can be further subdivided into regions of high variability ("hypervariable regions (HVRs)"; also called "complementarity determining regions" (CDRs), interspersed with more conserved regions). The more conserved regions are called framework regions (FR). Each V H and V L Typically, it contains three CDRs and four FRs arranged in the following order (from N-terminus to C-terminus): FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The CDRs are involved in antigen binding and confer antigen specificity and binding affinity to the antibody. See Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition (1991), Public Health Service, National Institutes of Health, Bethesda, MD, which is incorporated by reference in its entirety.

[0114] The light chains from any vertebrate species can be assigned to one of two types, called kappa and lambda, based on the sequence of the constant domains.

[0115] Heavy chains from any vertebrate species can be assigned to one of five different classes (or isotypes): IgA, IgD, IgE, IgG, and IgM. These classes are also named α, δ, ε, γ, and μ, respectively. The IgG and IgA classes are further divided into subclasses based on differences in sequence and function. Humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

[0116] The amino acid sequence boundaries of a CDR can be determined by one of skill in the art using any of a number of known numbering schemes, including those described in Kabat et al., supra ("Kabat" numbering scheme); Al-Lazikani et al., 1997, J. Mol. Biol., 273:927-948 ("Chothia" numbering scheme); MacCallum et al., 1996, J. Mol. Biol., 262:732-745 ("Contact" numbering scheme); Lefranc et al., Dev. Comp. Immunol., 2003, 27:55-77 ("IMGT" numbering scheme); and Honegge and Pluckthun, J. Mol. Biol., 2001, 309:657-70 ("AHo" numbering scheme), each of which is incorporated by reference in its entirety.

[0117] Table 1 provides the positions of CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2 and CDR-H3 identified by the Kabat and Chothia schemes. For CDR-H1, the residue numbering is provided using both the Kabat and Chothia numbering schemes.

[0118] Unless otherwise indicated, the numbering scheme used herein to identify specific CDRs is the Kabat numbering scheme. Variants and equivalent antibodies having the Chothia numbering scheme are intended to be within the scope of the present invention.

[0119] Table 1. Residues in CDRs according to Kabat and Chothia numbering schemes.

[0120] CDR Kabat Chothia L1 L24-L34 L24-L34 L2 L50-L56 L50-L56 L3 L89-L97 L89-L97 H1(Kabat number) H31-H35B H26-H32 or H34* H1 (Chothia number) H31-H35 H26-H32 H2 H50-H65 H52-H56 H3 H95-H102 H95-H102

[0121] *When numbering using the Kabat numbering convention, the C-terminus of CDR-H1 varies between H32 and H34 depending on the length of the CDR.

[0122] "Antibody fragments" include a portion of an intact antibody, such as an antigen binding region or a variable region of an intact antibody. Antibody fragments include, for example, Fv fragments, Fab fragments, F(ab')2 fragments, Fab' fragments, scFv (sFv) fragments, and scFv-Fc fragments. In some embodiments, antibodies that bind to HLA-G, antibodies that bind to PD-1, and / or antibodies that bind to PD-L1, antibodies that bind to EGFR, and / or antibodies that bind to CD47 or SIRPα include antibody fragments of each of the specified antibodies.

[0123] An "Fv" fragment comprises a non-covalently linked dimer of one heavy-chain and one light-chain variable domain.

[0124] In addition to the heavy and light chain variable domains, the "Fab" fragment contains the constant domain of the light chain and the first constant domain of the heavy chain (C H1 ). Fab fragments can be produced, for example, by papain digestion of full-length antibodies.

[0125] "F(ab')2" fragments contain two Fab' fragments linked by a disulfide bond near the hinge region. F(ab')2 fragments can be produced, for example, by pepsin digestion of intact antibodies. F(ab') fragments can be dissociated, for example, by treatment with B-mercaptoethanol.

[0126] "Single-chain Fv" or "sFv" or "scFv" antibody fragments contain a VH domain and a VL domain in a single polypeptide chain. The VH and VL are typically connected by a peptide linker. See Pluckthun A. (1994). Antibodies from Escherichia coli. In Rosenberg M. and Moore GP (eds.), The Pharmacology of Monoclonal Antibodies, Vol. 113 (pp. 269-315). Springer-Verlag, New York, which is incorporated by reference in its entirety. "scFv-Fc" fragments contain a scFv connected to an Fc domain. For example, the Fc domain can be connected to the C-terminus of the scFv. Depending on the orientation of the variable domains in the scFv (i.e., V H -V L or V L -V H ), the Fc domain can follow the V H or V L Any suitable Fc domain known in the art or described herein may be used.

[0127] The term "monoclonal antibody" refers to an antibody from a substantially homogeneous antibody population. A substantially homogeneous antibody population includes antibodies that are substantially similar and that are bound to the same epitope, except for variants that may occur during the monoclonal antibody production process. Such variants are usually present in small quantities. Monoclonal antibodies are usually obtained by comprising a process for selecting a single antibody from a variety of antibodies. For example, the selection process can be to select a unique clone from a plurality of clones, such as a pool of hybridoma clones, phage clones, yeast clones, bacterial clones or other recombinant DNA clones. Selected antibodies can be further changed, for example, to improve affinity to the target ("affinity maturation"), to humanize antibodies, to improve their production in cell culture and / or to reduce their immunogenicity in a subject.

[0128] With respect to the binding of an antibody to a target molecule, the term "binding" or "binding to" a specific antigen (e.g., a polypeptide target) or an epitope on a specific antigen refers to binding that differs from non-selective interactions to a measurable degree. For example, binding can be measured by determining the binding of a molecule compared to the binding of a control molecule. Binding can also be determined by competition with a control molecule similar to the target, such as an excess of unlabeled target. In that case, if the binding of the labeled target to the probe is competitively inhibited by an excess of unlabeled target, binding is indicated.

[0129] In certain embodiments, "treating" or "treatment" of any cancer refers to ameliorating a cancer present in a subject. In another embodiment, "treating" or "treatment" comprises ameliorating at least one physical parameter, which may not be discernible by the subject. In yet another embodiment, "treating" or "treatment" comprises modulating the cancer physically (e.g., stabilization of discernible symptoms) or physiologically (e.g., stabilization of a physical parameter), or both.

[0130] As used herein, the term "subject" means a mammal or a human. In some embodiments, the subject includes but is not limited to monkeys, dogs, cats, mice, rats, cows, horses, camels, birds, goats and sheep.

[0131] 2. Antibody Combination

[0132] Provided herein are methods and antibody combinations for treating cancer or other diseases. The antibody combination combines an antibody that binds to HLA-G with an antibody that has binding specificity to EGFR, PD-1 and / or PD-L1 and / or CD47 or SIRPα.

[0133] CDR-H1+CDR-H2+CDR-H3 region of antibody

[0134] In some embodiments, an antibody that binds to HLA-G comprises a VH sequence comprising: a CDR-H1 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NO: 1 or SEQ ID NO: 11; a CDR-H2 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NO: 21 or SEQ ID NO: 31; and a CDR-H3 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NO: 41. In some embodiments, the CDR-H1 sequence, the CDR-H2 sequence, and the CDR-H3 sequence are all from a single illustrative VH sequence provided in the present disclosure. H For example, in some embodiments, CDR-H1, CDR-H2, and CDR-H3 are all derived from a single illustrative V sequence having SEQ ID NO: 81. H sequence.

[0135] In some embodiments, the antibody that binds to EGFR comprises V H sequence, the V H The sequence comprises: a CDR-H1 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NO: 9 or SEQ ID NO: 19; a CDR-H2 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NO: 29 or SEQ ID NO: 39; and a CDR-H3 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NO: 49. In some embodiments, the CDR-H1 sequence, the CDR-H2 sequence, and the CDR-H3 sequence are all from a single illustrative V sequence provided in the present disclosure. H For example, in some embodiments, CDR-H1, CDR-H2, and CDR-H3 are all derived from a single illustrative V sequence selected from SEQ ID NO: 89. H sequence.

[0136] In some embodiments, the antibody that binds to PD-1 comprises V H sequence, the V HThe sequence comprises: a CDR-H1 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 2-5 or SEQ ID NOs: 12-15; a CDR-H2 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 22-25 or SEQ ID NOs: 32-35; and a CDR-H3 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 42-45. In some embodiments, the CDR-H1 sequence, the CDR-H2 sequence, and the CDR-H3 sequence are all from a single illustrative V sequence provided in the present disclosure. H For example, in some embodiments, CDR-H1, CDR-H2, and CDR-H3 are all derived from a single illustrative V sequence selected from SEQ ID NOs: 82-85. H sequence.

[0137] In some embodiments, the antibody that binds to PD-1 comprises V H sequence, the V H The sequence comprises: a CDR-H1 sequence comprising, consisting of, or consisting essentially of a sequence comprising SEQ ID NO: 2 or SEQ ID NO: 12; a CDR-H2 sequence comprising, consisting of, or consisting essentially of a sequence comprising SEQ ID NO: 22 or SEQ ID NO: 32; and a CDR-H3 sequence comprising, consisting of, or consisting essentially of a sequence comprising SEQ ID NO: 42. In some embodiments, the antibody that binds to PD-1 comprises V H sequence, the V H The sequence comprises: a CDR-H1 sequence comprising, consisting of, or consisting essentially of a sequence comprising SEQ ID NO: 3 or SEQ ID NO: 13; a CDR-H2 sequence comprising, consisting of, or consisting essentially of a sequence comprising SEQ ID NO: 23 or SEQ ID NO: 33; and a CDR-H3 sequence comprising, consisting of, or consisting essentially of a sequence comprising SEQ ID NO: 43. In some embodiments, the antibody that binds to PD-1 comprises V H sequence, the V HThe sequence comprises: a CDR-H1 sequence comprising, consisting of, or consisting essentially of a sequence comprising SEQ ID NO: 4 or SEQ ID NO: 14; a CDR-H2 sequence comprising, consisting of, or consisting essentially of a sequence comprising SEQ ID NO: 24 or SEQ ID NO: 34; and a CDR-H3 sequence comprising, consisting of, or consisting essentially of a sequence comprising SEQ ID NO: 44. In some embodiments, the antibody that binds to PD-1 comprises V H sequence, the V H The sequence comprises: a CDR-H1 sequence, wherein the CDR-H1 sequence comprises, consists of, or consists essentially of a sequence comprising SEQ ID NO: 5 or SEQ ID NO: 15; a CDR-H2 sequence, wherein the CDR-H2 sequence comprises, consists of, or consists essentially of a sequence comprising SEQ ID NO: 25 or SEQ ID NO: 35; and a CDR-H3 sequence, wherein the CDR-H3 sequence comprises, consists of, or consists essentially of a sequence comprising SEQ ID NO: 45.

[0138] In some embodiments, the CDR-H1 sequence, CDR-H2 sequence, and CDR-H3 sequence are all derived from a single illustrative V sequence provided in the present disclosure. H For example, in some embodiments, CDR-H1, CDR-H2, and CDR-H3 are all derived from a single V sequence having SEQ ID NO: 82. H In some embodiments, CDR-H1, CDR-H2, and CDR-H3 are all derived from a single V having SEQ ID NO: 83. H In some embodiments, CDR-H1, CDR-H2, and CDR-H3 are all derived from a single V having SEQ ID NO: 84. H In some embodiments, CDR-H1, CDR-H2, and CDR-H3 are all derived from a single V having SEQ ID NO: 85. H sequence.

[0139] In some embodiments, the antibody that binds to PD-L1 comprises V H sequence, the V HThe sequence comprises: a CDR-H1 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 6-8 or SEQ ID NOs: 16-18; a CDR-H2 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 26-28 or SEQ ID NOs: 36-38; and a CDR-H3 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 46-48. In some embodiments, the CDR-H1 sequence, the CDR-H2 sequence, and the CDR-H3 sequence are all from a single illustrative V sequence provided in the present disclosure. H For example, in some embodiments, CDR-H1, CDR-H2, and CDR-H3 are all derived from a single illustrative V sequence selected from SEQ ID NOs: 86-88. H sequence.

[0140] In some embodiments, the antibody that binds to PD-L1 comprises V H sequence, the V H The sequence comprises: a CDR-H1 sequence comprising, consisting of, or consisting essentially of a sequence comprising SEQ ID NO: 6 or SEQ ID NO: 16; a CDR-H2 sequence comprising, consisting of, or consisting essentially of a sequence comprising SEQ ID NO: 26 or SEQ ID NO: 36; and a CDR-H3 sequence comprising, consisting of, or consisting essentially of a sequence comprising SEQ ID NO: 46. In some embodiments, the antibody that binds to PD-L1 comprises V H sequence, the V H The sequence comprises: a CDR-H1 sequence comprising, consisting of, or consisting essentially of a sequence comprising SEQ ID NO: 7 or SEQ ID NO: 17; a CDR-H2 sequence comprising, consisting of, or consisting essentially of a sequence comprising SEQ ID NO: 27 or SEQ ID NO: 37; and a CDR-H3 sequence comprising, consisting of, or consisting essentially of a sequence comprising SEQ ID NO: 47. In some embodiments, the antibody that binds to PD-L1 comprises V H sequence, the V HThe sequence comprises: a CDR-H1 sequence, wherein the CDR-H1 sequence comprises, consists of, or consists essentially of a sequence comprising SEQ ID NO: 8 or SEQ ID NO: 18; a CDR-H2 sequence, wherein the CDR-H2 sequence comprises, consists of, or consists essentially of a sequence comprising SEQ ID NO: 28 or SEQ ID NO: 38; and a CDR-H3 sequence, wherein the CDR-H3 sequence comprises, consists of, or consists essentially of a sequence comprising SEQ ID NO: 48.

[0141] In some embodiments, the CDR-H1 sequence, CDR-H2 sequence, and CDR-H3 sequence are all derived from a single illustrative V sequence provided in the present disclosure. H In some embodiments, CDR-H1, CDR-H2, and CDR-H3 are all derived from a single V having SEQ ID NO: 86. H In some embodiments, CDR-H1, CDR-H2, and CDR-H3 are all derived from a single V having SEQ ID NO: 87. H In some embodiments, CDR-H1, CDR-H2, and CDR-H3 are all derived from a single V sequence having SEQ ID NO: 88. H sequence.

[0142] In some embodiments, the antibody that binds to CD47 comprises V H sequence, the V H The sequence comprises: a CDR-H1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 10 or SEQ ID NO: 20; a CDR-H2 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 30 or SEQ ID NO: 40; and a CDR-H3 sequence comprising, consisting of, or consisting essentially of sequence SEQ ID NO: 50. In some embodiments, the CDR-H1 sequence, the CDR-H2 sequence, and the CDR-H3 sequence are all from a single illustrative V sequence provided in the present disclosure. H For example, in some embodiments, CDR-H1, CDR-H2, and CDR-H3 are all derived from a single illustrative V sequence having SEQ ID NO: 90. H sequence.

[0143] In some embodiments, the antibody that binds to HLA-G comprises a V H sequence, the V HThe sequence comprises: a CDR-H1 sequence comprising SEQ ID NO: 1 or SEQ ID NO: 11; a CDR-H2 sequence comprising SEQ ID NO: 21 or SEQ ID NO: 31; and a CDR-H3 sequence comprising SEQ ID NO: 41, and the antibody that binds to EGFR comprises V H Sequence contains V H sequence, the V H The sequences include: a CDR-H1 sequence comprising SEQ ID NO:9 or SEQ ID NO:19; a CDR-H2 sequence comprising SEQ ID NO:29 or SEQ ID NO:39; and a CDR-H3 sequence comprising SEQ ID NO:49.

[0144] In some embodiments, the antibody that binds to HLA-G comprises a V H sequence, the V H The sequence comprises: a CDR-H1 sequence comprising SEQ ID NO: 1 or SEQ ID NO: 11; a CDR-H2 sequence comprising SEQ ID NO: 21 or SEQ ID NO: 31; and a CDR-H3 sequence comprising SEQ ID NO: 41, and the antibody that binds to PD-1 comprises V H Sequence contains V H sequence, the V H The sequence comprises: a CDR-H1 sequence comprising SEQ ID NO: 2 or SEQ ID NO: 12; a CDR-H2 sequence comprising SEQ ID NO: 22 or SEQ ID NO: 32; and a CDR-H3 sequence comprising SEQ ID NO: 42. In some embodiments, the antibody that binds to HLA-G comprises V H sequence, the V H The sequence comprises: a CDR-H1 sequence comprising SEQ ID NO: 1 or SEQ ID NO: 11; a CDR-H2 sequence comprising SEQ ID NO: 21 or SEQ ID NO: 31; and a CDR-H3 sequence comprising SEQ ID NO: 41, and the antibody that binds to PD-1 comprises V H sequence, the V H The sequence comprises: a CDR-H1 sequence comprising SEQ ID NO: 3 or SEQ ID NO: 13; a CDR-H2 sequence comprising SEQ ID NO: 23 or SEQ ID NO: 33; and a CDR-H3 sequence comprising SEQ ID NO: 43. In some embodiments, the antibody that binds to HLA-G comprises V H sequence, the V HThe sequence comprises: a CDR-H1 sequence comprising SEQ ID NO: 1 or SEQ ID NO: 11; a CDR-H2 sequence comprising SEQ ID NO: 21 or SEQ ID NO: 31; and a CDR-H3 sequence comprising SEQ ID NO: 41, and the antibody that binds to PD-1 comprises V H sequence, the V H The sequence comprises: a CDR-H1 sequence comprising SEQ ID NO: 4 or SEQ ID NO: 14; a CDR-H2 sequence comprising SEQ ID NO: 24 or SEQ ID NO: 34; and a CDR-H3 sequence comprising SEQ ID NO: 44. In some embodiments, the antibody that binds to HLA-G comprises V H sequence, the V H The sequence comprises: a CDR-H1 sequence comprising SEQ ID NO: 1 or SEQ ID NO: 11; a CDR-H2 sequence comprising SEQ ID NO: 21 or SEQ ID NO: 31; and a CDR-H3 sequence comprising SEQ ID NO: 41, and the antibody that binds to PD-1 comprises V H sequence, the V H The sequences include: a CDR-H1 sequence comprising SEQ ID NO:5 or SEQ ID NO:15; a CDR-H2 sequence comprising SEQ ID NO:25 or SEQ ID NO:35; and a CDR-H3 sequence comprising SEQ ID NO:45.

[0145] In some embodiments, the antibody that binds to HLA-G comprises a V H sequence, the V H The sequence comprises: a CDR-H1 sequence comprising SEQ ID NO: 1 or SEQ ID NO: 11; a CDR-H2 sequence comprising SEQ ID NO: 21 or SEQ ID NO: 31; and a CDR-H3 sequence comprising SEQ ID NO: 41, and the antibody that binds to PD-L1 comprises V H sequence, the V H The sequence comprises: a CDR-H1 sequence comprising SEQ ID NO: 6 or SEQ ID NO: 16; a CDR-H2 sequence comprising SEQ ID NO: 26 or SEQ ID NO: 36; and a CDR-H3 sequence comprising SEQ ID NO: 46. In some embodiments, the antibody that binds to HLA-G comprises V H sequence, the V HThe sequence comprises: a CDR-H1 sequence comprising SEQ ID NO: 1 or SEQ ID NO: 11; a CDR-H2 sequence comprising SEQ ID NO: 21 or SEQ ID NO: 31; and a CDR-H3 sequence comprising SEQ ID NO: 41, and the antibody that binds to PD-L1 comprises V H sequence, the V H The sequence comprises: a CDR-H1 sequence comprising SEQ ID NO: 7 or SEQ ID NO: 17; a CDR-H2 sequence comprising SEQ ID NO: 27 or SEQ ID NO: 37; and a CDR-H3 sequence comprising SEQ ID NO: 47. In some embodiments, the antibody that binds to HLA-G comprises a V H sequence, the V H The sequence comprises: a CDR-H1 sequence comprising SEQ ID NO: 1 or SEQ ID NO: 11; a CDR-H2 sequence comprising SEQ ID NO: 21 or SEQ ID NO: 31; and a CDR-H3 sequence comprising SEQ ID NO: 41, and the antibody that binds to PD-L1 comprises V H sequence, the V H The sequences include: a CDR-H1 sequence comprising SEQ ID NO: 8 or SEQ ID NO: 18; a CDR-H2 sequence comprising SEQ ID NO: 28 or SEQ ID NO: 38; and a CDR-H3 sequence comprising SEQ ID NO: 48.

[0146] In some embodiments, the antibody that binds to HLA-G comprises a V H sequence, the V H The sequence comprises: a CDR-H1 sequence comprising SEQ ID NO: 1 or SEQ ID NO: 11; a CDR-H2 sequence comprising SEQ ID NO: 21 or SEQ ID NO: 31; and a CDR-H3 sequence comprising SEQ ID NO: 41, and the antibody that binds to CD47 comprises V H sequence, the V H The sequences include: a CDR-H1 sequence comprising SEQ ID NO: 10 or SEQ ID NO: 20; a CDR-H2 sequence comprising SEQ ID NO: 30 or SEQ ID NO: 40; and a CDR-H3 sequence comprising SEQ ID NO: 50.

[0147] V H sequence

[0148] In some embodiments, the antibody that binds to HLA-G comprises a V Hsequence, the V H The sequence comprises, consists of, or consists essentially of: SEQ ID NO: 81. In some embodiments, the antibody that binds to EGFR comprises V H sequence, the V H The sequence comprises, consists of, or consists essentially of: SEQ ID NO: 89. In some embodiments, the antibody that binds to PD-1 comprises V H sequence, the V H The sequence comprises, consists of, or consists essentially of: SEQ ID NO: 82. In some embodiments, the antibody that binds to PD-1 comprises V H sequence, the V H The sequence comprises, consists of, or consists essentially of: SEQ ID NO: 83. In some embodiments, the antibody that binds to PD-1 comprises V H sequence, the V H The sequence comprises, consists of, or consists essentially of: SEQ ID NO: 84. In some embodiments, the antibody that binds to PD-1 comprises V H sequence, the V H The sequence comprises, consists of, or consists essentially of: SEQ ID NO:85.

[0149] In some embodiments, the antibody that binds to PD-L1 comprises V H sequence, the V H The sequence comprises, consists of, or consists essentially of: SEQ ID NO: 86. In some embodiments, the antibody that binds to PD-L1 comprises V H sequence, the V H The sequence comprises, consists of, or consists essentially of: SEQ ID NO: 87. In some embodiments, the antibody that binds to PD-L1 comprises V H sequence, the V H The sequence comprises, consists of, or consists essentially of: SEQ ID NO: 88. In some embodiments, the antibody that binds to CD47 comprises V H sequence, the V H The sequence comprises, consists of, or consists essentially of: SEQ ID NO:90.

[0150] CDR-L1+CDR-L2+CDR-L3 region of an antibody

[0151] In some embodiments, the antibody that binds to HLA-G comprises a V L sequence, the V LThe sequences include: a CDR-L1 sequence comprising, consisting of, or consisting essentially of: SEQ ID NO: 51; a CDR-L2 sequence comprising, consisting of, or consisting essentially of: a sequence selected from SEQ ID NO: 61; and a CDR-L3 sequence comprising, consisting of, or consisting essentially of: a sequence selected from SEQ ID NO: 71. In some embodiments, the CDR-L1 sequence, the CDR-L2 sequence, and the CDR-L3 sequence are all from a single illustrative V sequence provided in the present disclosure. L For example, in some embodiments, CDR-L1, CDR-L2, and CDR-L3 are all derived from a single illustrative V sequence having SEQ ID NO: 91. L sequence.

[0152] In some embodiments, the antibody that binds to EGFR comprises V L sequence, the V L The sequences include: a CDR-L1 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 59; a CDR-L2 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 69; and a CDR-L3 sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 79. In some embodiments, the CDR-L1 sequence, the CDR-L2 sequence, and the CDR-L3 sequence are all from a single illustrative V sequence provided in the present disclosure. L For example, in some embodiments, CDR-L1, CDR-L2, and CDR-L3 are all derived from a single illustrative V sequence having SEQ ID NO: 99. L sequence.

[0153] In some embodiments, the antibody that binds to PD-1 comprises V L sequence, the V LThe sequence comprises: a CDR-L1 sequence comprising, consisting of, or consisting essentially of a sequence selected from any one of SEQ ID NOs: 52-55; a CDR-L2 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 62-65; and a CDR-L3 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: SEQ ID NOs: 72-75. In some embodiments, the CDR-L1 sequence, the CDR-L2 sequence, and the CDR-L3 sequence are all from a single illustrative V sequence provided in the present disclosure. L For example, in some embodiments, CDR-L1, CDR-L2, and CDR-L3 are all derived from a single illustrative V sequence selected from SEQ ID NOs: 92-95. H sequence.

[0154] In some embodiments, the antibody that binds to PD-1 comprises V L sequence, the V L The sequence comprises: a CDR-L1 sequence comprising, consisting of, or consisting essentially of a sequence comprising SEQ ID NO: 52; a CDR-L2 sequence comprising, consisting of, or consisting essentially of a sequence comprising SEQ ID NO: 62; and a CDR-L3 sequence comprising, consisting of, or consisting essentially of a sequence comprising SEQ ID NO: 72. In some embodiments, the antibody that binds to PD-1 comprises V L sequence, the V L The sequence comprises: a CDR-L1 sequence comprising, consisting of, or consisting essentially of a sequence comprising SEQ ID NO: 53; a CDR-L2 sequence comprising, consisting of, or consisting essentially of a sequence comprising SEQ ID NO: 63; and a CDR-L3 sequence comprising, consisting of, or consisting essentially of a sequence comprising SEQ ID NO: 73. In some embodiments, the antibody that binds to PD-1 comprises V L sequence, the V LThe sequence comprises: a CDR-L1 sequence comprising, consisting of, or consisting essentially of a sequence comprising SEQ ID NO: 54; a CDR-L2 sequence comprising, consisting of, or consisting essentially of a sequence comprising SEQ ID NO: 64; and a CDR-L3 sequence comprising, consisting of, or consisting essentially of a sequence comprising SEQ ID NO: 74. In some embodiments, the antibody that binds to PD-1 comprises V L sequence, the V L The sequence comprises: a CDR-L1 sequence, which comprises, consists of, or is mainly composed of: a sequence comprising SEQ ID NO: 55; a CDR-L2 sequence, which comprises, consists of, or is mainly composed of: a sequence comprising SEQ ID NO: 65; and a CDR-L3 sequence, which comprises, consists of, or is mainly composed of: a sequence comprising SEQ ID NO: 75.

[0155] In some embodiments, the CDR-L1 sequence, CDR-L2 sequence, and CDR-L3 sequence are all derived from a single illustrative V sequence provided in the present disclosure. L For example, in some embodiments, CDR-L1, CDR-L2, and CDR-L3 are all derived from a single V having SEQ ID NO: 92. L In some embodiments, CDR-H1, CDR-H2, and CDR-H3 are all derived from a single V having SEQ ID NO: 93. L In some embodiments, CDR-H1, CDR-H2, and CDR-H3 are all derived from a single V sequence having SEQ ID NO: 94. L In some embodiments, CDR-H1, CDR-H2, and CDR-H3 are all derived from a single V sequence having SEQ ID NO: 95. L sequence.

[0156] In some embodiments, the antibody that binds to PD-L1 comprises V L sequence, the V LThe sequences include: a CDR-L1 sequence comprising, consisting of, or consisting essentially of a sequence selected from any one of SEQ ID NOs: 56-58; a CDR-L2 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: 66-68; and a CDR-L3 sequence comprising, consisting of, or consisting essentially of a sequence selected from SEQ ID NOs: SEQ ID NOs: 76-78. In some embodiments, the CDR-L1 sequence, the CDR-L2 sequence, and the CDR-L3 sequence are all from a single illustrative V sequence provided in the present disclosure. L For example, in some embodiments, CDR-L1, CDR-L2, and CDR-L3 are all derived from a single illustrative V sequence selected from SEQ ID NOs: 96-98. H sequence.

[0157] In some embodiments, the antibody that binds to PD-L1 comprises V L sequence, the V L The sequence comprises: a CDR-L1 sequence comprising, consisting of, or consisting essentially of a sequence comprising SEQ ID NO: 56; a CDR-L2 sequence comprising, consisting of, or consisting essentially of a sequence comprising SEQ ID NO: 66; and a CDR-L3 sequence comprising, consisting of, or consisting essentially of a sequence comprising SEQ ID NO: 76. In some embodiments, the antibody that binds to PD-L1 comprises V L sequence, the V L The sequence comprises: a CDR-L1 sequence comprising, consisting of, or consisting essentially of a sequence comprising SEQ ID NO: 57; a CDR-L2 sequence comprising, consisting of, or consisting essentially of a sequence comprising SEQ ID NO: 67; and a CDR-L3 sequence comprising, consisting of, or consisting essentially of a sequence comprising SEQ ID NO: 77. In some embodiments, the antibody that binds to PD-L1 comprises V L sequence, the V LThe sequence comprises: a CDR-L1 sequence, which comprises, consists of, or is mainly composed of: a sequence comprising SEQ ID NO: 58; a CDR-L2 sequence, which comprises, consists of, or is mainly composed of: a sequence comprising SEQ ID NO: 68; and a CDR-L3 sequence, which comprises, consists of, or is mainly composed of: a sequence comprising SEQ ID NO: 78.

[0158] In some embodiments, the CDR-L1 sequence, CDR-L2 sequence, and CDR-L3 sequence are all derived from a single illustrative V sequence provided in the present disclosure. L For example, in some embodiments, CDR-L1, CDR-L2, and CDR-L3 are all derived from a single V having SEQ ID NO: 96. L In some embodiments, CDR-H1, CDR-H2, and CDR-H3 are all derived from a single V sequence having SEQ ID NO: 97. L In some embodiments, CDR-H1, CDR-H2, and CDR-H3 are all derived from a single V sequence having SEQ ID NO: 98. L sequence.

[0159] In some embodiments, the antibody that binds to CD47 comprises V L sequence, the V L The sequence comprises: a CDR-L1 sequence comprising, consisting of, or consisting essentially of: SEQ ID NO: 60; a CDR-L2 sequence comprising, consisting of, or consisting essentially of: a sequence selected from SEQ ID NO: 70; and a CDR-L3 sequence comprising, consisting of, or consisting essentially of: a sequence selected from SEQ ID NO: 80. In some embodiments, the CDR-L1 sequence, the CDR-L2 sequence, and the CDR-L3 sequence are all from a single illustrative V sequence provided in the present disclosure. L For example, in some embodiments, CDR-L1, CDR-L2, and CDR-L3 are all derived from a single illustrative V sequence having SEQ ID NO: 100. L sequence.

[0160] V L sequence

[0161] In some embodiments, the antibody that binds to HLA-G comprises a V L sequence, the V LThe sequence comprises, consists of, or consists essentially of: sequence SEQ ID NO: 91. In some embodiments, the antibody that binds to EGFR comprises V L sequence, the V L The sequence comprises, consists of, or consists essentially of: sequence SEQ ID NO: 99. In some embodiments, the antibody that binds to PD-1 comprises V L sequence, the V L The sequence comprises, consists of, or consists essentially of: sequence SEQ ID NO: 92. In some embodiments, the antibody that binds to PD-1 comprises V L sequence, the V L The sequence comprises, consists of, or consists essentially of: sequence SEQ ID NO: 93. In some embodiments, the antibody that binds to PD-1 comprises V L sequence, the V L The sequence comprises, consists of, or consists essentially of: sequence SEQ ID NO: 94. In some embodiments, the antibody that binds to PD-1 comprises V L sequence, the V L The sequence comprises, consists of or consists essentially of: sequence SEQ ID NO:95.

[0162] In some embodiments, the antibody that binds to PD-L1 comprises V L sequence, the V L The sequence comprises, consists of, or consists essentially of: sequence SEQ ID NO: 96. In some embodiments, the antibody that binds to PD-L1 comprises V L sequence, the V L The sequence comprises, consists of, or consists essentially of: sequence SEQ ID NO: 97. In some embodiments, the antibody that binds to PD-1 comprises V L sequence, the V L The sequence comprises, consists of, or consists essentially of: sequence SEQ ID NO: 98. In some embodiments, the antibody that binds to CD47 comprises V L sequence, the V L The sequence comprises, consists of, or consists essentially of: SEQ ID NO:100.

[0163] V H -V L right

[0164] In some embodiments, the antibody that binds to HLA-G comprises a V H Sequence and V Lsequence, the V H The sequence comprises, consists of, or consists essentially of: SEQ ID NO: 81, and the V L The sequence comprises, consists of, or consists essentially of: SEQ ID NO: 91. In some embodiments, the antibody that binds to EGFR comprises V H Sequence and V L sequence, the V H The sequence comprises, consists of, or consists essentially of: SEQ ID NO: 89, and the V L The sequence comprises, consists of, or consists essentially of: SEQ ID NO: 99. In some embodiments, the antibody that binds to CD47 comprises V H Sequence and V L sequence, the V H The sequence comprises, consists of, or consists essentially of: SEQ ID NO: 90, and the V L The sequence comprises, consists of, or consists essentially of: SEQ ID NO:100.

[0165] In some embodiments, the antibody that binds to PD-1 comprises V H Sequence and V L sequence, the V H The sequence comprises, consists of, or consists essentially of a sequence selected from any one of SEQ ID NOs: 82-85, and the V L The sequence comprises, consists of, or consists essentially of a sequence selected from any one of SEQ ID NOs: 92-95. In some embodiments, the antibody that binds to PD-1 comprises V H Sequence and V L sequence, the V H The sequence comprises, consists of, or consists essentially of: SEQ ID NO: 82, and the V L The sequence comprises, consists of, or consists essentially of: SEQ ID NO: 92. In some embodiments, the antibody that binds to PD-1 comprises V H Sequence and V L sequence, the V H The sequence comprises, consists of, or consists essentially of: SEQ ID NO: 83, and the V L The sequence comprises, consists of, or consists essentially of: SEQ ID NO: 93. In some embodiments, the antibody that binds to PD-1 comprises V H Sequence and V L sequence, the VH The sequence comprises, consists of, or consists essentially of: SEQ ID NO: 84, and the V L The sequence comprises, consists of, or consists essentially of: SEQ ID NO: 94. In some embodiments, the antibody that binds to PD-1 comprises V H Sequence and V L sequence, the V H The sequence comprises, consists of, or consists essentially of: SEQ ID NO: 85, and the V L The sequence comprises, consists of, or consists essentially of: SEQ ID NO:95.

[0166] In some embodiments, the antibody that binds to PD-L1 comprises V H Sequence and V L sequence, the V H The sequence comprises, consists of, or consists essentially of a sequence selected from any one of SEQ ID NOs: 86-88, and the V L The sequence comprises, consists of, or consists essentially of a sequence selected from any one of SEQ ID NOs: 96-98. In some embodiments, the antibody that binds to PD-L1 comprises V H Sequence and V L sequence, the V H The sequence comprises, consists of, or consists essentially of: SEQ ID NO: 86, and the V L The sequence comprises, consists of, or consists essentially of: SEQ ID NO: 96. In some embodiments, the antibody that binds to PD-L1 comprises V H Sequence and V L sequence, the V H The sequence comprises, consists of, or consists essentially of: SEQ ID NO: 87, and the V L The sequence comprises, consists of, or consists essentially of: SEQ ID NO: 97. In some embodiments, the antibody that binds to PD-L1 comprises V H Sequence and V L sequence, the V H The sequence comprises, consists of, or consists essentially of: SEQ ID NO: 88, and the V L The sequence comprises, consists of, or consists essentially of: SEQ ID NO:99.

[0167] CDR-H1+CDR-H2+CDR-H3+CDR-L1+CDR-L2+CDR-L3

[0168] In some embodiments, the antibody that binds to HLA-G comprises or consists of: a heavy chain variable region (V H ) and light chain variable region (V L ), where V H and / or V L Include:

[0169] a) a VHCDR1 having the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 11,

[0170] b) a VHCDR2 having the sequence shown in SEQ ID NO: 21 or SEQ ID NO: 31,

[0171] c) a VHCDR3 having the sequence shown in SEQ ID NO: 41,

[0172] d) VLCDR1 having the sequence shown in SEQ ID NO: 51,

[0173] e) VLCDR2 having the sequence shown in SEQ ID NO: 61, and

[0174] f) VLCDR3 having the sequence shown in SEQ ID NO:71.

[0175] In some embodiments, the antibody that binds to EGFR comprises or consists of: a heavy chain variable region (V H ) and light chain variable region (V L ), where V H and / or V L Include:

[0176] a) a VHCDR1 having the sequence shown in SEQ ID NO: 9 or SEQ ID NO: 19,

[0177] b) a VHCDR2 having the sequence shown in SEQ ID NO: 29 or SEQ ID NO: 39,

[0178] c) a VHCDR3 having the sequence shown in SEQ ID NO: 49,

[0179] d) VLCDR1 having the sequence shown in SEQ ID NO: 59,

[0180] e) VLCDR2 having the sequence shown in SEQ ID NO: 69, and

[0181] f) VLCDR3 having the sequence shown in SEQ ID NO:79.

[0182] In some embodiments, the antibody that binds to PD-1 comprises or consists of: a heavy chain variable region (V H ) and light chain variable region (V L ), where V H and / or V L Include:

[0183] a) a VHCDR1 having a sequence shown in any one of SEQ ID NOs: 2-5 or SEQ ID NOs: 12-15,

[0184] b) a VHCDR2 having a sequence as shown in any one of SEQ ID NOs: 22-25 or SEQ ID NOs: 32-35,

[0185] c) a VHCDR3 having a sequence as shown in any one of SEQ ID NOs: 42-45,

[0186] d) a VLCDR1 having a sequence as shown in any one of SEQ ID NOs: 52-55,

[0187] e) a VLCDR2 having a sequence as shown in any one of SEQ ID NOs: 62-65, and

[0188] f) VLCDR3 having the sequence shown in any one of SEQ ID NOs: 72-75.

[0189] In some embodiments, the antibody that binds to PD-1 comprises V H sequence and VL sequence, the V H The sequence comprises: a CDR-H1 sequence comprising SEQ ID NO: 2 or SEQ ID NO: 12; a CDR-H2 sequence comprising SEQ ID NO: 22 or SEQ ID NO: 32; and a CDR-H3 sequence comprising SEQ ID NO: 42, and the VL sequence comprises: a CDR-L1 sequence comprising SEQ ID NO: 52; a CDR-L2 sequence comprising SEQ ID NO: 62; and a CDR-L3 sequence SEQ ID NO: 72. In some embodiments, the antibody that binds to PD-1 comprises a V H sequence and VL sequence, the V HThe sequence comprises: a CDR-H1 sequence comprising SEQ ID NO: 3 or SEQ ID NO: 13; a CDR-H2 sequence comprising SEQ ID NO: 23 or SEQ ID NO: 33; and a CDR-H3 sequence comprising SEQ ID NO: 43, and the VL sequence comprises: a CDR-L1 sequence comprising SEQ ID NO: 53; a CDR-L2 sequence comprising SEQ ID NO: 63; and a CDR-L3 sequence SEQ ID NO: 73. In some embodiments, the antibody that binds to PD-1 comprises a V H sequence and VL sequence, the V H The sequence comprises: a CDR-H1 sequence comprising SEQ ID NO: 4 or SEQ ID NO: 14; a CDR-H2 sequence comprising SEQ ID NO: 24 or SEQ ID NO: 34; and a CDR-H3 sequence comprising SEQ ID NO: 44, and the VL sequence comprises: a CDR-L1 sequence comprising SEQ ID NO: 54; a CDR-L2 sequence comprising SEQ ID NO: 64; and a CDR-L3 sequence SEQ ID NO: 74. In some embodiments, the antibody that binds to PD-1 comprises a V H sequence and VL sequence, the V H The sequence comprises: a CDR-H1 sequence comprising SEQ ID NO:5 or SEQ ID NO:15; a CDR-H2 sequence comprising SEQ ID NO:25 or SEQ ID NO:35; and a CDR-H3 sequence comprising SEQ ID NO:45, and the VL sequence comprises: a CDR-L1 sequence comprising SEQ ID NO:55; a CDR-L2 sequence comprising SEQ ID NO:65; and a CDR-L3 sequence SEQ ID NO:75.

[0190] In some embodiments, the antibody that binds to PD-L1 comprises or consists of: a heavy chain variable region (V H ) and light chain variable region (V L ), where V H and / or V L Include:

[0191] a) a VHCDR1 having a sequence shown in any one of SEQ ID NOs: 6-8 or SEQ ID NOs: 16-18,

[0192] b) a VHCDR2 having a sequence as shown in any one of SEQ ID NOs: 26-28 or SEQ ID NOs: 38-38,

[0193] c) a VHCDR3 having a sequence as shown in any one of SEQ ID NOs: 46-48,

[0194] d) a VLCDR1 having a sequence as shown in any one of SEQ ID NOs: 56-58,

[0195] e) a VLCDR2 having a sequence as shown in any one of SEQ ID NOs: 66-68, and

[0196] f) VLCDR3 having the sequence shown in any one of SEQ ID NOs: 76-78.

[0197] In some embodiments, the antibody that binds to PD-L1 comprises V H sequence and VL sequence, the V H The sequence comprises: a CDR-H1 sequence comprising SEQ ID NO: 6 or SEQ ID NO: 16; a CDR-H2 sequence comprising SEQ ID NO: 26 or SEQ ID NO: 36; and a CDR-H3 sequence comprising SEQ ID NO: 46, and the VL sequence comprises: a CDR-L1 sequence comprising SEQ ID NO: 56; a CDR-L2 sequence comprising SEQ ID NO: 66; and a CDR-L3 sequence SEQ ID NO: 76. In some embodiments, the antibody that binds to PD-L1 comprises a V H sequence and VL sequence, the V H The sequence comprises: a CDR-H1 sequence comprising SEQ ID NO: 7 or SEQ ID NO: 17; a CDR-H2 sequence comprising SEQ ID NO: 27 or SEQ ID NO: 37; and a CDR-H3 sequence comprising SEQ ID NO: 47, and the VL sequence comprises: a CDR-L1 sequence comprising SEQ ID NO: 57; a CDR-L2 sequence comprising SEQ ID NO: 67; and a CDR-L3 sequence SEQ ID NO: 77. In some embodiments, the antibody that binds to PD-L1 comprises a V H sequence and VL sequence, the V H The sequence comprises: a CDR-H1 sequence comprising SEQ ID NO:8 or SEQ ID NO:18; a CDR-H2 sequence comprising SEQ ID NO:28 or SEQ ID NO:38; and a CDR-H3 sequence comprising SEQ ID NO:48, and the VL sequence comprises: a CDR-L1 sequence comprising SEQ ID NO:58; a CDR-L2 sequence comprising SEQ ID NO:68; and a CDR-L3 sequence SEQ ID NO:78.

[0198] In some embodiments, the antibody that binds to CD47 comprises or consists of: a heavy chain variable region (V H ) and light chain variable region (V L ), where V H and / or V L Include:

[0199] a) a VHCDR1 having the sequence shown in SEQ ID NO: 10 or SEQ ID NO: 20,

[0200] b) a VHCDR2 having the sequence shown in SEQ ID NO: 30 or SEQ ID NO: 40,

[0201] c) a VHCDR3 having the sequence shown in SEQ ID NO: 50,

[0202] d) VLCDR1 having the sequence shown in SEQ ID NO: 60,

[0203] e) VLCDR2 having the sequence shown in SEQ ID NO: 70, and

[0204] f) VLCDR3 having the sequence shown in SEQ ID NO:80.

[0205] HC+LC

[0206] In some embodiments, the antibody that binds to HLA-G, EGFR, PD-1, PD-L1 and / or CD47 comprises or consists of one or more heavy chains consisting of an HC sequence and one or more light chains consisting of an LC sequence. In some embodiments, the antibody that binds to HLA-G, EGFR, PD-1, PD-L1 and / or CD47 or SIRPα comprises or consists of two identical heavy chains and two identical light chains, the two identical heavy chains comprising, consisting of, or consisting essentially of an HC sequence, the two identical light chains comprising, consisting of, or consisting essentially of an LC sequence.

[0207] In some embodiments, the HC sequence of the antibody that binds to HLA-G is a HC sequence comprising, consisting of, or consisting essentially of: SEQ ID NO: 101, and the LC sequence of the antibody that binds to HLA-G is a LC sequence comprising, consisting of, or consisting essentially of: any one of SEQ ID NO: 111. In some embodiments, the HC sequence of the antibody that binds to EGFR is a HC sequence comprising, consisting of, or consisting essentially of: SEQ ID NO: 109, and the LC sequence of the antibody that binds to EGFR is a LC sequence comprising, consisting of, or consisting essentially of: SEQ ID NO: 119. In some embodiments, the HC sequence of the antibody that binds to CD47 is a HC sequence comprising, consisting of, or consisting essentially of: SEQ ID NO: 110, and the LC sequence of the antibody that binds to PD-1 is a LC sequence comprising, consisting of, or consisting essentially of: SEQ ID NO: 120.

[0208] In some embodiments, the HC sequence of the antibody that binds to PD-1 is a HC sequence comprising, consisting of, or consisting essentially of: any one of SEQ ID NOs: 102-105, and the LC sequence of the antibody that binds to PD-1 is a LC sequence comprising, consisting of, or consisting essentially of: any one of SEQ ID NOs: 112-115. In some embodiments, the HC sequence of the antibody that binds to PD-1 is a HC sequence comprising, consisting of, or consisting essentially of: SEQ ID NO: 102, and the LC sequence of the antibody that binds to PD-1 is a LC sequence comprising, consisting of, or consisting essentially of: SEQ ID NO: 112. In some embodiments, the HC sequence of the antibody that binds to PD-1 is a HC sequence comprising, consisting of, or consisting essentially of: SEQ ID NO: 103, and the LC sequence of the antibody that binds to PD-1 is a LC sequence comprising, consisting of, or consisting essentially of: SEQ ID NO: 113. In some embodiments, the HC sequence of the antibody that binds to PD-1 is a HC sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 104, and the LC sequence of the antibody that binds to PD-1 is a LC sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 114. In some embodiments, the HC sequence of the antibody that binds to PD-1 is a HC sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 105, and the LC sequence of the antibody that binds to PD-1 is a LC sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 115.

[0209] In some embodiments, the HC sequence of the antibody that binds to PD-L1 is a HC sequence comprising, consisting of, or consisting essentially of any one of SEQ ID NOs: 106-108, and the LC sequence of the antibody that binds to PD-1 is a LC sequence comprising, consisting of, or consisting essentially of any one of SEQ ID NOs: 118-118. In some embodiments, the HC sequence of the antibody that binds to PD-L1 is a HC sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 106, and the LC sequence of the antibody that binds to PD-L1 is a LC sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 116. In some embodiments, the HC sequence of the antibody that binds to PD-L1 is a HC sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 107, and the LC sequence of the antibody that binds to PD-L1 is a LC sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 117. In some embodiments, the HC sequence of the antibody that binds to PD-L1 is a HC sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 118, and the LC sequence of the antibody that binds to PD-L1 is a LC sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 118.

[0210] In some embodiments, the HC sequence of the antibody that binds to HLA-G is a HC sequence comprising or consisting of SEQ ID NO: 101, and the LC sequence is a LC sequence comprising or consisting of SEQ ID NO: 111, and the HC sequence of the antibody that binds to PD-1 is a HC sequence comprising or consisting of SEQ ID NO: 102, and the LC sequence is a LC sequence comprising or consisting of SEQ ID NO: 112. In some embodiments, the HC sequence of the antibody that binds to HLA-G is a HC sequence comprising or consisting of SEQ ID NO: 101, and the LC sequence is a LC sequence comprising or consisting of SEQ ID NO: 111, and the HC sequence of the antibody that binds to PD-1 is a HC sequence comprising or consisting of SEQ ID NO: 103, and the LC sequence is a LC sequence comprising or consisting of SEQ ID NO: 113. In some embodiments, the HC sequence of the antibody that binds to HLA-G is a HC sequence comprising or consisting of SEQ ID NO: 101, and the LC sequence is a LC sequence comprising or consisting of SEQ ID NO: 111, and the HC sequence of the antibody that binds to PD-1 is a HC sequence comprising or consisting of SEQ ID NO: 104, and the LC sequence is a LC sequence comprising or consisting of SEQ ID NO: 114. In some embodiments, the HC sequence of the antibody that binds to HLA-G is a HC sequence comprising or consisting of SEQ ID NO: 101, and the LC sequence is a LC sequence comprising or consisting of SEQ ID NO: 111, and the HC sequence of the antibody that binds to PD-1 is a HC sequence comprising or consisting of SEQ ID NO: 105, and the LC sequence is a LC sequence comprising or consisting of SEQ ID NO: 115.

[0211] In some embodiments, the HC sequence of the antibody that binds to HLA-G is a HC sequence comprising or consisting of SEQ ID NO: 101, and the LC sequence is a LC sequence comprising or consisting of SEQ ID NO: 111, and the HC sequence of the antibody that binds to PD-L1 is a HC sequence comprising or consisting of SEQ ID NO: 106, and the LC sequence is a LC sequence comprising or consisting of SEQ ID NO: 116. In some embodiments, the HC sequence of the antibody that binds to HLA-G is a HC sequence comprising or consisting of SEQ ID NO: 101, and the LC sequence is a LC sequence comprising or consisting of SEQ ID NO: 111, and the HC sequence of the antibody that binds to PD-L1 is a HC sequence comprising or consisting of SEQ ID NO: 107, and the LC sequence is a LC sequence comprising or consisting of SEQ ID NO: 117. In some embodiments, the HC sequence of the antibody that binds to HLA-G is a HC sequence comprising or consisting of SEQ ID NO: 101, and the LC sequence is a LC sequence comprising or consisting of SEQ ID NO: 111, and the HC sequence of the antibody that binds to PD-L1 is a HC sequence comprising or consisting of SEQ ID NO: 108, and the LC sequence is a LC sequence comprising or consisting of SEQ ID NO: 118.

[0212] In some embodiments, the HC sequence of the antibody that binds to HLA-G is a HC sequence comprising or consisting of SEQ ID NO: 101, and the LC sequence is a LC sequence comprising or consisting of SEQ ID NO: 111, and the HC sequence of the antibody that binds to EGFR is a HC sequence comprising or consisting of SEQ ID NO: 109, and the LC sequence is a LC sequence comprising or consisting of SEQ ID NO: 119. In some embodiments, the HC sequence of the antibody that binds to HLA-G is a HC sequence comprising or consisting of SEQ ID NO: 101, and the LC sequence is a LC sequence comprising or consisting of SEQ ID NO: 111, and the HC sequence of the antibody that binds to CD47 is a HC sequence comprising or consisting of SEQ ID NO: 110, and the LC sequence is a LC sequence comprising or consisting of SEQ ID NO: 120.

[0213] Glycosylation variants

[0214] In certain embodiments, the antibodies of the invention may be altered to increase, decrease or eliminate the extent of their glycosylation. Glycosylation of polypeptides is typically "N-linked" or "O-linked".

[0215] "N-linked" glycosylation refers to the attachment of a carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, are the recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Thus, the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site.

[0216] "O-linked" glycosylation refers to the attachment of one of the sugars N-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used.

[0217] Addition or deletion of N-linked glycosylation sites for antibodies can be achieved by changing the amino acid sequence so that one or more of the above-mentioned tripeptide sequences are generated or deleted. Addition or deletion of O-linked glycosylation sites can be achieved by addition, deletion or substitution of one or more serine or threonine residues in the sequence of the antibody or by addition, deletion or substitution of one or more serine or threonine residues in the sequence of the antibody (as the case may be).

[0218] In certain embodiments, the antibodies are glycosylated. In certain embodiments, the antibodies are deglycosylated. Carbohydrates can be removed by standard techniques. In certain embodiments, the antibodies are aglycosylated, for example, by expressing the antibodies in a non-glycosylated system.

[0219] Antibody preparation

[0220] HLA-G, PD-1, PD-L1 and / or CD47 or SIRPα antigens can be used to generate antibodies. They can be complete proteins or fragments of antigens. They can be in the form of isolated proteins or can be expressed by cells. Other forms of antigens that can be used to generate antibodies will be obvious to those skilled in the art.

[0221] The DNA encoding the antibody can be easily separated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that can be specifically bound to the heavy chain and light chain of the encoding monoclonal antibody). Therefore, hybridoma cells can be used as a useful source of DNA encoding antibodies with desired properties. After separation, the DNA can be placed in an expression vector and then transfected into a host cell such as a myeloma cell that does not produce antibodies in addition, such as bacteria (e.g., Escherichia coli), yeast (e.g., Saccharomyces or Pichia sp.), COS cells, Chinese hamster ovary (CHO) cells, or cells, to produce monoclonal antibodies. Other ways that can be used to produce antibodies will be apparent to those skilled in the art.

[0222] cancer

[0223] For cancer, the pharmaceutical compositions of the present invention are generally administered to humans or other organisms or animals in pharmaceutically acceptable dosage forms. Any suitable cancer can be treated with the antibodies provided herein. In some embodiments, the cancer is a blood cancer. In some embodiments, the cancer is a solid cancer.

[0224] In some embodiments, the compositions and methods increase macrophage phagocytosis. In some embodiments, the compositions and methods enhance ILT2 + CD8 + T cell degranulation (CD107a). In some embodiments, macrophages increase phagocytosis of lung cancer cells. In some embodiments, lung cancer cells are HLA-G + EGFR + Lung cancer cells. In some embodiments, macrophages are resistant to HLA-G + A549、HLA-G + Increased phagocytosis of SK-OV-3, HT-1376 and / or JEG-3 cells.

[0225] Examples

[0226] Example 1: The combination of an antibody (Fab fragment) that binds to HLA-G and cetuximab increases macrophages' affinity for HLA-G compared to either agent alone + EGFR + Phagocytosis of lung cancer cells.

[0227] To determine the potential clinical benefit of combining antibodies that bind to HLA-G with cetuximab, Fab fragments of antibodies that bind to HLA-G were evaluated in a macrophage assay in which cetuximab was used to induce expression of EGFR in macrophages engineered to express HLA-G. + Phagocytosis of A549 lung cancer cells.

[0228] CD14-enriched cells were cultured in complete RPMI (cRPMI) with recombinant human M-CSF at 37°C, 5% CO2 for 7 days. + The cells were differentiated into adherent macrophages. After 7 days, the cells were harvested, washed, and resuspended in cRPMI. The cells were plated at 50,000 cells / well in 50 μl cRPMI in 96-well round-bottom plates and incubated at 37°C, 5% CO2 before being combined with target cells.

[0229] At 37°C, use CellTrace TM Target cells (A549 lung cancer cells engineered to express HLA-G) were stained with BD Fc Block 500 (CTV, 1:1000) in PBS, washed, and plated at 25,000 cells per well in cRPMI. The cells were then incubated with antibodies (antibodies that bind to HLA-G Fab and / or cetuximab) for 1 hour at 37°C, 5% CO2. The mixture of A549 cells and antibodies was then combined with macrophages and incubated for 2 hours at 37°C, 5% CO2. After incubation for 2 hours, the cells were washed with wash buffer / BD Fc Block TM Cells were blocked with 6% mouse serum for 20 minutes and then stained with anti-CD11b antibody and live / dead discrimination dye for 20 minutes. Cells were then washed, resuspended in wash buffer, and analyzed on a BD Fortessa flow cytometer.

[0230] Sample data were exported as FCS files and analyzed using FlowJo software v10. The presence of CTV in macrophages was assessed as a marker for CTV. + Readout of phagocytic uptake of target cells. Live cells directed against CD11b + / CTV + Gates were set and reported as a percentage of total viable macrophages.

[0231] Figure 1 The results showed that the combination of HLA-G-binding antibody Fab and cetuximab significantly increased the phagocytosis of target cells compared to cetuximab alone. No activity was observed for the HLA-G-binding antibody Fab compared to the untreated control.

[0232] Example 2: Combination of an antibody binding to HLA-G with pembrolizumab or atezolizumab enhances ILT2 compared to either drug alone + CD8 + T cell degranulation (CD107a)

[0233] To demonstrate that anti-PD-1 antibody (pembrolizumab) or anti-PD-L1 antibody (atezolizumab) can be used in combination with anti-HLA-G antibody to enhance the functional activity of human T cells, 721.221PD-L1 + HLA-G + Cells and human CD8 + T cell co-culture.

[0234] The isolated CD8 + T cells were plated at 50,000 cells / well in cRPMI in 96-well round-bottom plates. + T cells were incubated with ImmunoCult human CD3 / CD28 T cell activator and pembrolizumab for 1 hour at 37°C, 5% CO2. Target cells (721.221 engineered to express PD-L1 and HLA-G) were plated at 50,000 cells / well in cRPMI and then incubated with antibodies that bind to HLA-G or a combination of antibodies that bind to HLA-G and atezolizumab for 1 hour at 37°C, 5% CO2. CD8 + The mixture of T cells was combined with target cells and incubated at 37°C, 5% CO2 for 24 hours. + The final ratio of T cells to target cells was 1:1.

[0235] After 24 h of incubation, a mixture containing eFluor660αCD107a antibody at a final concentration of 1:100 and monensin at a final concentration of 1:1000 was added to the CD8 + T cell-target cell co-cultures were added and the cells were incubated with the mixture at 37°C, 5% CO2 for 4 hours. After incubation, the cells were washed once in 4°C wash buffer (phosphate buffered saline, 2% FBS, 2mM EDTA) and then washed with wash buffer / BD Fc Block TM / 6% mouse serum blocking cells for 20 minutes. The staining solution was then added to cells consisting of 1:2000 fixable viability dye eFluorTM 780 (Invitrogen) and fluorescently labeled αILT2, αCD8 and αPD-1 antibodies for 25 minutes at 4°C. The cells were washed again in 4°C wash buffer and then resuspended in a fixation / permeabilization buffer consisting of 1 part of fixation / permeabilization concentrate and 3 parts of fixation / permeabilization diluent (Invitrogen) for 30 minutes at 4°C. The cells were then washed with permeabilization buffer (Invitrogen), followed by a final wash in 4°C wash buffer and resuspended in wash buffer.

[0236] Cells were analyzed in ILT2 by flow cytometry analysis on a BD LSR Fortessa+ CD8 + The percentage of CD107a expression on T cells. Figure 2A and 2B As shown, the combination of HLA-G-binding antibodies with pembrolizumab or atezolizumab enhanced ILT2 compared with either drug alone. + CD8 + Degranulation of T cells.

[0237] Example 3: The combination of an antibody that binds to HLA-G and mololimab increases macrophage cytotoxicity to HLA-G compared to either agent alone + A549, HLA-G + Phagocytosis of SK-OV-3, HT-1376, and JEG-3 target cells. The three-dose combination showed increased activity compared to the single-dose or double-dose treatment groups.

[0238] To determine the potential clinical benefit of antibodies that bind to HLA-G in combination with mololimab and / or cetuximab, the agents were evaluated in a macrophage assay that measured phagocytosis of A549 lung cancer cells and SK-OV-3 ovarian cancer cells engineered to express HLA-G, as well as HT-1376 bladder cancer and JEG-3 choriocarcinoma cells that endogenously express HLA-G.

[0239] CD14 cells were enriched by incubation in complete RPMI (cRPMI) with recombinant human M-CSF for 7 days at 37°C, 5% CO2. + The cells were differentiated into adherent macrophages. After 7 days, the cells were harvested, washed, and resuspended in cRPMI. The cells were plated in 96-well round-bottom plates at 100,000 macrophages per well in 50 μl cRPMI and incubated at 37°C, 5% CO2 before merging with target cells (A549, SK-OV-3, HT-1376, JEG-3 cell lines).

[0240] At 37°C, use CellTrace TM Target cells were stained with BD Fc Block 5000 (CTV, 1:1000) in PBS, washed, and plated at 50,000 target cells per well in cRPMI. The cells were then incubated with antibodies (antibodies that bind to HLA-G and / or mololimab and / or cetuximab) for 1 hour at 37°C, 5% CO2. The mixture of target cells and antibodies was then combined with macrophages and incubated for 2 hours at 37°C, 5% CO2. After incubation for 2 hours, the cells were washed with wash buffer / BD Fc Block TMCells were blocked with 6% mouse serum for 20 minutes and then stained with anti-CD11b antibody and live / dead discrimination dye for 20 minutes. Cells were then washed, resuspended in wash buffer, and analyzed on a BD Fortessa flow cytometer.

[0241] Sample data were exported as FCS files and analyzed using FlowJo software v10. The presence of CTV in macrophages was assessed as a marker for CTV. + Readout of phagocytic uptake by target cells. Targeted to CD11b on live cells + / CTV + Gates were set and reported as a percentage of total viable macrophages.

[0242] Figure 3A and Figure 3B It was demonstrated that the combination of an antibody binding to HLA G and mololimab increased macrophage phagocytosis of HT1376 and JEG-3 cells expressing endogenous HLA-G, respectively, compared to either agent alone. Figure 3C and Figure 3D The results showed that a three-dose combination of an antibody that binds to HLA-G with mololimab and cetuximab was more active than single-dose or double-dose treatment in inducing phagocytosis of A549 and SK-OV-3 cells engineered to express HLA-G. Isotype antibodies were used as negative controls.

[0243] Example S: Sequence Listing S provides the sequences mentioned herein.

[0244]

[0245]

[0246]

[0247]

[0248]

[0249]

[0250]

[0251]

[0252]

[0253]

[0254]

[0255]

[0256]

[0257] Equivalent

[0258] The disclosure set forth above may encompass a variety of different inventions with independent uses. Although each of these inventions has been disclosed in its preferred form, the specific embodiments disclosed and shown herein should not be considered to have a restrictive meaning, because a large number of variants are possible. The subject matter of the present invention includes all novel and non-obvious combinations and sub-combinations of various elements, features, functions and / or properties disclosed herein. The following claims specifically point out certain combinations and sub-combinations that are considered novel and non-obvious. Inventions embodied in other combinations and sub-combinations of features, functions, elements and / or properties may be claimed in this application, an application claiming priority to this application, or a related application. Whether for different inventions or for the same invention, and whether broader, narrower, equal or different than the original claims, such claims are also considered to be included in the subject matter of the invention disclosed herein.

Claims

1. A method for treating a subject having cancer, the method comprising administering to the subject: an antibody that binds to HLA-G and an antibody that binds to epidermal growth factor receptor (EGFR).

2. The method of claim 1, wherein the antibody that binds to HLA-G comprises or consists of a heavy chain variable region (VH) and a light chain variable region (VL), wherein VH and / or VL comprises: a) a VHCDR1 having a sequence as shown in either SEQ ID NO: 1 or SEQ ID NO: 11, b) a VHCDR2 having a sequence as shown in either SEQ ID NO: 21 or SEQ ID NO: 31, c) a VHCDR3 having the sequence shown in any one of SEQ ID NO: 41, d) a VLCDR1 having a sequence as shown in any one of SEQ ID NO: 51, e) a VLCDR2 having a sequence as shown in any one of SEQ ID NO: 61, and f) VLCDR3 having the sequence shown in any one of SEQ ID NO:

71.

3. The method of claim 1 or claim 2, wherein the antibody that binds to HLA-G comprises or consists of a heavy chain variable region (VH) and a light chain variable region (VL), wherein VH comprises, consists of, or consists essentially of SEQ ID NO: 81, and wherein VL comprises, consists of, or consists essentially of a VL having a sequence as shown in SEQ ID NO:

91.

4. The method according to any one of claims 1 to 3, wherein the antibody that binds to epidermal growth factor receptor (EGFR) comprises or consists of a heavy chain variable region (VH) and a light chain variable region (VL), VH and / or VL comprising: a) a VHCDR1 having the sequence shown in SEQ ID NO: 9 or SEQ ID NO: 19, b) a VHCDR2 having the sequence shown in SEQ ID NO: 29 or SEQ ID NO: 39, c) a VHCDR3 having the sequence shown in SEQ ID NO: SEQ ID NO: 49, d) VLCDR1 having the sequence shown in SEQ ID NO: SEQ ID NO: 59, e) VLCDR2 having the sequence shown in SEQ ID NO: SEQ NO: 69, and f) VLCDR3 having the sequence shown in SEQ ID NO:

79.

5. The method according to any one of claims 1 to 4, wherein the antibody that binds to epidermal growth factor receptor (EGFR) comprises or consists of a heavy chain variable region (VH) and a light chain variable region (VL), and VH and / or VL comprises, consists of, or consists essentially of: A VH having the sequence shown in SEQ ID NO:89, and a VL comprising, consisting of, or mainly consisting of a VL having the sequence shown in SEQ ID NO:

99.

6. A method for treating a subject having cancer, the method comprising administering to the subject: an antibody that binds to HLA-G and an antibody that binds to PD-1 or PD-L1.

7. The method of claim 6, wherein the antibody that binds to HLA-G comprises or consists of a heavy chain variable region (VH) and a light chain variable region (VL), wherein VH and / or VL comprises: a) a VHCDR1 having a sequence as shown in either SEQ ID NO: 1 or SEQ ID NO: 11, b) a VHCDR2 having a sequence as shown in either SEQ ID NO: 21 or SEQ ID NO: 31, c) a VHCDR3 having the sequence shown in any one of SEQ ID NO: 41, d) a VLCDR1 having a sequence as shown in any one of SEQ ID NO: 51, e) a VLCDR2 having a sequence as shown in any one of SEQ ID NO: 61, and f) VLCDR3 having the sequence shown in any one of SEQ ID NO:

71.

8. The method of claim 6 or claim 7, wherein the antibody that binds to HLA-G comprises or consists of a heavy chain variable region (VH) and a light chain variable region (VL), wherein VH comprises, consists of, or consists essentially of SEQ ID NO: 81, and wherein VL comprises, consists of, or consists essentially of a VL having a sequence as shown in SEQ ID NO:

91.

9. The method according to any one of claims 6 to 8, wherein the antibody that binds to PD-1 and / or PD-L1 comprises or consists of a heavy chain variable region (VH) and a light chain variable region (VL), and VH and / or VL comprises: a) a VHCDR1 having a sequence as shown in SEQ ID NO: 2-8 or SEQ ID NO: 12-18, b) a VHCDR2 having a sequence as shown in SEQ ID NO: 22-28 or SEQ ID NO: 32-38, c) a VHCDR3 having a sequence as shown in SEQ ID NO: 42-48, d) a VLCDR1 having a sequence shown in SEQ ID NO: 52-58, e) a VLCDR2 having a sequence as shown in SEQ ID NO: 62-68, and f) VLCDR3 having the sequence shown in SEQ ID NO: 72-78.

10. The method according to any one of claims 6 to 9, wherein the antibody that binds to PD-1 and / or PD-L1 comprises or consists of a heavy chain variable region (VH) and a light chain variable region (VL), and VH and / or VL comprises, consists of, or consists mainly of: A VH having a sequence shown in SEQ ID NOs: 82-88, and a VL comprising, consisting of, or mainly consisting of a VL having a sequence shown in SEQ ID NOs: 92-98.

11. A method for treating a subject having cancer, the method comprising administering to the subject: an antibody that binds to HLA-G and an antibody that binds to CD47.

12. The method of claim 11, wherein the antibody that binds to HLA-G comprises or consists of a heavy chain variable region (VH) and a light chain variable region (VL), wherein VH and / or VL comprises: a) a VHCDR1 having a sequence as shown in either SEQ ID NO: 1 or SEQ ID NO: 11, b) a VHCDR2 having a sequence as shown in either SEQ ID NO: 21 or SEQ ID NO: 31, c) a VHCDR3 having the sequence shown in any one of SEQ ID NO: 41, d) a VLCDR1 having a sequence as shown in any one of SEQ ID NO: 51, e) a VLCDR2 having a sequence as shown in any one of SEQ ID NO: 61, and f) VLCDR3 having the sequence shown in any one of SEQ ID NO:

71.

13. The method of claim 11 or claim 12, wherein the antibody that binds to HLA-G comprises or consists of a heavy chain variable region (VH) and a light chain variable region (VL), wherein VH comprises, consists of, or consists essentially of SEQ ID NO: 81, and wherein VL comprises, consists of, or consists essentially of a VL having a sequence as shown in SEQ ID NO:

91.

14. The method according to any one of claims 11 to 13, wherein the antibody that binds to CD47 comprises or consists of a heavy chain variable region (VH) and a light chain variable region (VL), VH and / or VL comprising: a) a VHCDR1 having the sequence shown in SEQ ID NO: 10 or SEQ ID NO: 20, b) a VHCDR2 having the sequence shown in SEQ ID NO: 30 or SEQ ID NO: 40, c) a VHCDR3 having the sequence shown in SEQ ID NO: 50, d) VLCDR1 having the sequence shown in SEQ ID NO: 60, e) VLCDR2 having the sequence shown in SEQ ID NO: 70, and f) VLCDR3 having the sequence shown in SEQ ID NO:

80.

15. The method according to any one of claims 11 to 14, wherein the antibody that binds to CD47 comprises or consists of a heavy chain variable region (VH) and a light chain variable region (VL), VH and / or VL comprising, consisting of, or consisting essentially of: A VH having the sequence shown in SEQ ID NO:90, and a VL comprising, consisting of, or mainly consisting of a VL having the sequence shown in SEQ ID NO:

100.

16. A pharmaceutical composition for use in the method of any one of the preceding claims.

17. The method of any one of claims 1 to 15, wherein the cancer is a solid cancer.

18. The method of any one of claims 1 to 15, wherein the cancer is a hematological cancer.

19. The method according to any one of claims 1 to 15, wherein the subject is a human subject.

20. The method of any one of claims 1 to 15, wherein the pharmaceutical composition is administered in an amount sufficient to achieve 1, 2, 3, 4, 5, 6, 7, or 8 of the following in the subject: a) suppress immunosuppression; b) reduce the level of regulatory T cells; c) increasing the activity of myeloid cells, cytotoxic T lymphocytes, helper T cells, NK cells, T cells, B cells, neutrophils, monocytes, macrophages and / or dendritic cells; d) increase phagocytic activity; e) inhibit metastasis; f) inhibiting tumor growth; g) inducing tumor regression; and / or h) Enhancement of ILT2 + CD8 + T cell degranulation.

21. The method of claim 20, wherein increasing phagocytic activity comprises increasing the percentage of phagocytosis compared to a pharmaceutical composition without an antibody or a pharmaceutical composition with an antibody that binds to HLA-G.

22. The method according to any one of claims 1 to 15, wherein the method further comprises one or more of the following: a) administering chemotherapy; b) administering radiation therapy; and / or c) administering one or more additional therapeutic agents.

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