Combination therapy including anti-HLA-G antibody and anti-EGFR antibody, anti-PD1 or anti-PD-L1 antibody, and / or anti-CD47

Abstract

Provided herein is a combination therapy comprising 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. A 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). A 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.

Description

[Technical field]

[0001] Related Applications This application claims priority to U.S. Provisional Application No. 63 / 329,065, filed April 8, 2022, the entire contents of which are incorporated herein by reference.

[0002] Field Provided herein is a combination therapy comprising an antibody with binding specificity for HLA-G and an antibody with binding specificity for EGFR, PD-1 and / or PD-L1, and / or CD47 or SIRPα.

[0003] Sequence Listing This application has been submitted electronically in .XML format and contains a sequence listing which is incorporated herein 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 a part of the present specification and is incorporated herein by reference in its entirety. [Background technology]

[0004] background 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 non-classical class I heavy chain paralogs of HLA. HLA-G is a heterodimer consisting of a heavy chain and a light chain (beta-2 microglobulin). Membrane-bound and soluble forms of HLA-G exist.

[0005] HLA-G is normally expressed at the maternal-fetal interface and other immune privileged sites. HLA-G plays a role in immune tolerance during pregnancy and is expressed by villous trophoblast cells in the placenta, but not classical MHC class I genes (HLA-A and HLA-B). Since HLA-G was first identified in placental samples, many studies have evaluated its role in pregnancy disorders such as preeclampsia and recurrent pregnancy loss. 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.

[0006] HLA-G has been shown to be immunosuppressive. By binding to receptors expressed on various 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 suppresses phagocytosis and induces the generation or proliferation of regulatory T cells.

[0007] HLA-G mediates immune functions through at least three ITIM-containing inhibitory receptors, ILT2, ILT4, and KIR2DL4. For example, in lymphoid and myeloid cells, HLA-G mediates functions through ILT2. In myeloid cells, HLA-G mediates functions through ILT4. In decidual NK cells, HLA-G mediates immune functions through KIR2DL4 and ILT2.

[0008] HLA-G is an immune checkpoint target. HLA-G can directly inhibit immune cell function through receptor binding and / or nibbling, and impairing chemotaxis. HLA-G can confer higher invasive and metastatic potential to tumor cells. HLA-G promotes evasion of tumor immune surveillance and enhances metastasis and progression of malignant tumors. During tumor progression, HLA-G has other effects such as inhibiting immune cell cytolysis, inducing immune cell apoptosis, and / or generating regulatory cells through receptor binding and / or nibbling.

[0009] HLA-G expression is upregulated in a wide range 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 blood cancer. HLA-G has also been found to correlate with clinical parameters in advanced disease, such as tumor metastasis, poor prognosis, immune evasion, and tumor invasiveness.

[0010] PD-1 is a membrane protein of 268 amino acids. PD-1 contains an extracellular IgV domain, a transmembrane region, and an intracellular tail. The tail contains two phosphorylation sites located at the immunoreceptor tyrosine-based inhibitory motif and the immunoreceptor tyrosine-based switch motif. It has been suggested that PD-1 negatively regulates T cell receptor signaling.

[0011] 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 has a role in regulating immune system responses by downregulating immune responses and promoting self-tolerance by suppressing T cell inflammatory activity. This prevents autoimmune diseases, but may also prevent the immune system from killing cancer cells.

[0012] PD-1 is recognized as a key player in immune regulation and the maintenance of peripheral tolerance. PD-1 can be seen as an immune checkpoint and operates through several different mechanisms. For example, PD-1 promotes apoptosis of antigen-specific T cells in lymph nodes. In addition, PD-1 reduces apoptosis in regulatory T cells (anti-inflammatory, suppressive T cells).

[0013] 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 that play important roles throughout 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 that essentially turns off or inhibits the signal.

[0014] Cancer cells exploit this system by promoting high levels of PD-L1 expression, which allows them to gain control of the PD-1 pathway and turn off T cells expressing PD-1, thus suppressing anti-cancer immune responses. PD-L1 correlates with poor prognosis in ovarian, renal, colorectal, pancreatic, liver, and melanoma cancers. Similarly, PD-1 expression on tumor-infiltrating lymphocytes indicates dysfunctional T cells in breast cancer and melanoma, and correlates with poor prognosis in renal cancer.

[0015] PD-1 therapies that "unblock" a pre-existing immune response or unblock the initiation of an immune response are effective, but in some cases, only a subgroup of subjects respond. Additionally, even in the responding population, the response is not always complete or optimal.

[0016] The epidermal growth factor receptor (EGFR; ErbB-1; HER1 in humans) is a transmembrane protein that is the 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, a subfamily of four closely related receptor tyrosine kinases: EGFR (ErbB-1), HER2 / neu (ErbB-2), Her3 (ErbB-3), and Her4 (ErbB-4). In many cancer types, mutations affecting the expression or activity of EGFR can result in more aggressive cancers. Lack of signaling of EGFR and other receptor tyrosine kinases in humans has been associated with diseases such as Alzheimer's disease, while overexpression has been associated with the development of a wide variety of tumors. Interference with EGFR signaling, either 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 EGFR-expressing tumors and improve the patient's condition.

[0017] EGFR is a transmembrane protein that is activated by the binding of its specific ligands, including epidermal growth factor and transforming growth factor alpha (TGFα). ErbB2 has no known direct activating ligand and may be in a constitutively activated state, or it may become active upon heterodimerization with other family members, such as EGFR. Upon activation by its growth factor ligands, EGFR undergoes a transition from an inactive monomeric form to an active homodimer.

[0018] EGFR dimerization stimulates its intrinsic intracellular protein-tyrosine kinase activity. This results in the autophosphorylation of several tyrosine (Y) residues in the C-terminal domain of EGFR. These include Y992, Y1045, Y1068, Y1148, and Y1173. Autophosphorylation triggers downstream activation and signaling by several other proteins that associate with the phosphorylated tyrosines through their phosphotyrosine-binding SH2 domains. These downstream signaling proteins initiate several signaling cascades, mainly the MAPK, Akt, and JNK pathways, leading to 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 tyrosine residues of other receptors with which it is aggregated, and itself can be activated in that manner.

[0019] Mutations that result in overexpression (known as upregulation or amplification) of EGFR have been associated with many cancers, including adenocarcinoma of the lung (40% of cases), anal cancer, glioblastoma (50%), and epithelial tumors of the head and neck (80-100%). These somatic mutations involving EGFR lead to its constant activation, resulting in uncontrolled cell division. In glioblastoma, a specific mutation of EGFR, called EGFRvIII, is often observed. Mutation, amplification, or misregulation of EGFR or family members is involved in approximately 30% of all epithelial cancers.

[0020] CD47 (cluster of differentiation 47), also known as integrin-associated protein (IAP), is a transmembrane protein that in humans is encoded by the CD47 gene. CD47 belongs to the immunoglobulin superfamily and is a partner of membrane integrins, and also binds to the ligands thrombospondin-1 (TSP-1) and signal regulatory protein alpha (SIRPα). CD47 is involved in a range of cellular processes including apoptosis, proliferation, adhesion, and migration. Furthermore, CD47 plays an important 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.

[0021] CD47 is a 50 kDa membrane receptor with an extracellular N-terminal IgV domain, five transmembrane domains, and a short C-terminal intracellular tail. Four alternatively spliced ​​isoforms of CD47 exist that differ only in the length of their cytoplasmic tails.

[0022] Form 2 is the most widely expressed form found in all circulating and immune cells. The second most abundant isoform is Form 4, which is expressed primarily in the brain and peripheral nervous system. Only keratinocytes express significant amounts of Form 1.

[0023] CD47 is a high affinity receptor for thrombospondin-1 (TSP-1), a secreted glycoprotein that plays a role in vascular development and angiogenesis, in which TSP1 / CD47 interaction inhibits nitric oxide signaling in vascular cells at multiple levels. 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 regulating angiogenesis and inflammation.

[0024] CD47 interacts with signal-regulatory protein alpha (SIRPα), an inhibitory transmembrane receptor present on myeloid cells. CD47 / SIRPα interaction results in bidirectional signaling, resulting in distinct intercellular responses including inhibition of phagocytosis, stimulation of cell-cell fusion, and T cell activation. CD47 binds to several membrane integrins, most commonly integrin α. V β3. These interactions result in CD47 / integrin complexes that influence a range of cellular functions, including adhesion, spreading, and migration.

[0025] CD47 was first identified as a tumor antigen in human ovarian cancer in the 1980s. Since then, CD47 has been found to be expressed in multiple human tumor types, including acute myeloid leukemia (AML), chronic myeloid leukemia, acute lymphoblastic leukemia (ALL), non-Hodgkin's lymphoma (NHL), multiple myeloma (MM), bladder cancer, and other solid tumors. CD47 is also highly expressed in pediatric and adult brain tumors.

[0026] High levels of CD47 allow cancer cells to avoid phagocytosis. This is due to engagement of SIRP-α on macrophages by CD47. Engagement of SIRP-α leads to inhibition of phagocytosis.

[0027] Anti-CD47 antibody-mediated phagocytosis of cancer by macrophages can initiate anti-tumor T-cell immune responses. Anti-CD47 antibody treatment not only enables macrophage phagocytosis of cancer cells, but also promotes the activation of cancer-specific lymphocytes: cancer cells display mutated proteins to which the immune system can now respond. There is a need for combination therapies comprising antibodies with binding specificity for HLA-G and antibodies with binding specificity for EGFR, PD-1 and / or PD-L1, and / or CD47 or SIRPα. [Prior art documents] [Non-patent literature]

[0028] [Non-Patent Document 1] Michita,Rafael Tomoyaet al.,Human Immunology.2016,77(10):892-897 Summary of the Invention

[0029] overview Provided herein are methods and pharmaceutical compositions for the treatment of a subject suffering from cancer 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.

[0030] A 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 the VH and / or the VL are a) a VHCDR1 having the sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 11, b) a VH CDR2 having the sequence set forth in SEQ ID NO: 21 or SEQ ID NO: 31; c) a VH CDR3 having the sequence set forth in SEQ ID NO: 41; d) VLCDR1 having the sequence set forth in SEQ ID NO: 51; e) VLCDR2 having the sequence set forth in SEQ ID NO: 61, and f) comprises a VLCDR3 having the sequence set forth in SEQ ID NO: 71.

[0031] In some embodiments, an antibody that binds to HLA-G comprises or consists of a heavy chain variable region (VH) and a light chain variable region (VL), where the VH comprises, consists of, or consists essentially of a VH having the sequence set forth in SEQ ID NO: 81, and the VL comprises, consists of, or consists essentially of a VL having the sequence set forth in SEQ ID NO: 91. In some embodiments, an antibody that binds to HLA-G is an antibody comprising a heavy chain and a light chain, where the heavy chain comprises or consists of one or more molecules having a sequence comprising or consisting of SEQ ID NO: 101, and the light chain comprises or consists of one or more molecules having a sequence comprising or consisting of SEQ ID NO: 111.

[0032] In some embodiments, an 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 the VH and / or the VL is a) a VHCDR1 having the sequence set forth in SEQ ID NO: 9 or SEQ ID NO: 19, b) a VH CDR2 having the sequence set forth in SEQ ID NO: 29 or SEQ ID NO: 39; c) a VH CDR3 having the sequence set forth in SEQ ID NO: 49; d) VLCDR1 having the sequence set forth in SEQ ID NO: 59; e) VLCDR2 having the sequence set forth in SEQ ID NO: 69, and f) comprises a VLCDR3 having the sequence set forth in SEQ ID NO: 79.

[0033] In some embodiments, an antibody that binds to epidermal growth factor receptor (EGFR) comprises, consists of, or consists essentially of a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and / or VL comprises, consists of, or consists essentially of a VH having the sequence set forth in SEQ ID NO: 89, and a VL having the sequence set forth in SEQ ID NO: 99. In some embodiments, an antibody that binds to epidermal growth factor receptor (EGFR) is an antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises or consists of one or more molecules having a sequence comprising or consisting of SEQ ID NO: 109, and the light chain comprises or consists of one or more molecules having a sequence comprising or consisting of SEQ ID NO: 119.

[0034] A 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 the VH and / or the VL are a) a VHCDR1 having the sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 11, b) a VH CDR2 having the sequence set forth in SEQ ID NO: 21 or SEQ ID NO: 31; c) a VH CDR3 having the sequence set forth in SEQ ID NO: 41; d) VLCDR1 having the sequence set forth in SEQ ID NO: 51; e) VLCDR2 having the sequence set forth in SEQ ID NO: 61, and f) comprises a VLCDR3 having the sequence set forth in SEQ ID NO: 71.

[0035] In some embodiments, an antibody that binds to HLA-G comprises or consists of a heavy chain variable region (VH) and a light chain variable region (VL), where the VH comprises, consists of, or consists essentially of a VH having the sequence set forth in SEQ ID NO: 81, and the VL comprises, consists of, or consists essentially of a VL having the sequence set forth in SEQ ID NO: 91. In some embodiments, an antibody that binds to HLA-G is an antibody comprising a heavy chain and a light chain, where the heavy chain comprises or consists of one or more molecules having a sequence comprising or consisting of SEQ ID NO: 101, and the light chain comprises or consists of one or more molecules having a sequence comprising or consisting of SEQ ID NO: 111.

[0036] In some embodiments, an 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), wherein the VH and / or VL are a) VHCDR1 having a sequence set forth in SEQ ID NO: 2 to 8 or SEQ ID NO: 12 to 18, b) VH CDR2 having a sequence set forth in SEQ ID NOs: 22 to 28 or 32 to 38; c) VHCDR3 having a sequence set forth in SEQ ID NOs: 42 to 48; d) VLCDR1 having a sequence set forth in SEQ ID NOs: 52 to 58; e) VLCDR2 having the sequence set forth in SEQ ID NOs: 62 to 68, and f) Contains a VLCDR3 having the sequence set forth in SEQ ID NOs: 72 to 78.

[0037] In some embodiments, an antibody that binds PD-1 and / or PD-L1 comprises or consists of a heavy chain variable region (VH) and a light chain variable region (VL), where the VH and / or VL comprises, consists of, or consists essentially of a VH having a sequence set forth in one of SEQ ID NOs: 82-88, and the VL comprises, consists of, or consists essentially of a VL having a sequence set forth in any one of SEQ ID NOs: 92-98. In some embodiments, an antibody that binds PD-1 and / or PD-L1 is an antibody comprising a heavy chain and a light chain, where the heavy chain comprises or consists of one or more molecules having a sequence comprising or consisting of a sequence set forth in any one of SEQ ID NOs: 102-108, and the light chain comprises or consists of one or more molecules having a sequence comprising or consisting of a sequence set forth in any one of SEQ ID NOs: 112-118.

[0038] A 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 the VH and / or the VL are: a) a VHCDR1 having the sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 11, b) a VH CDR2 having the sequence set forth in SEQ ID NO: 21 or SEQ ID NO: 31; c) a VH CDR3 having the sequence set forth in SEQ ID NO: 41; d) VLCDR1 having the sequence set forth in SEQ ID NO: 51; e) VLCDR2 having the sequence set forth in SEQ ID NO: 61, and f) comprises a VLCDR3 having the sequence set forth in SEQ ID NO: 71.

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

[0040] In some embodiments, an antibody that binds CD47 comprises or consists of a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH and / or the VL is a) a VHCDR1 having the sequence set forth in SEQ ID NO: 10 or SEQ ID NO: 20, b) a VH CDR2 having the sequence set forth in SEQ ID NO: 30 or SEQ ID NO: 40; c) a VH CDR3 having the sequence set forth in SEQ ID NO: 50; d) VLCDR1 having the sequence set forth in SEQ ID NO: 60; e) VLCDR2 having the sequence set forth in SEQ ID NO: 70, and f) comprises a VLCDR3 having the sequence set forth in SEQ ID NO: 80.

[0041] In some embodiments, an antibody that binds CD47 comprises or consists of a heavy chain variable region (VH) and a light chain variable region (VL), where the VH and / or VL comprises, consists of, or consists essentially of a VH having the sequence set forth in SEQ ID NO: 90 and a VL comprises, consists of, or consists essentially of a VL having the sequence set forth in SEQ ID NO: 100. In some embodiments, an antibody that binds CD47 is an antibody comprising a heavy chain and a light chain, where the heavy chain comprises or consists of one or more molecules having a sequence comprising or consisting of SEQ ID NO: 110 and the light chain comprises or consists of one or more molecules having a sequence comprising or consisting of SEQ ID NO: 120.

[0042] A fourth aspect provides a method for treating a subject suffering from cancer, 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 hematological cancer. In some embodiments, the subject is a human subject. In some embodiments, the method comprises the steps of: a) administering chemotherapy; b) administering radiation therapy; and / or c) administering one or more additional therapeutic agents.

[0043] A fifth aspect provides a method of modulating immune system function in a subject in need thereof, comprising contacting a population of immune cells of the subject with any of the pharmaceutical compositions provided herein. In some embodiments, the subject is a human subject.

[0044] In some embodiments, the pharmaceutical composition is administered to a subject: a) inhibition of immunosuppression; b) a reduction in the levels 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) increased phagocytic activity; e) inhibition of metastasis; f) inhibition of tumor growth; g) induction of tumor regression, and / or h) ILT2 + CD8 + The antibody is administered in an amount sufficient to achieve one, two, three, four, five, six, seven, or eight of the following: enhancement of T cell degranulation.

[0045] In some embodiments, the increase in phagocytic activity comprises an increase in percent phagocytosis compared to a pharmaceutical composition that does not include an antibody or a pharmaceutical composition that includes an antibody that binds to HLA-G. a) administering chemotherapy; b) administering radiation therapy; and / or c) administering one or more additional therapeutic agents. [Brief description of the drawings]

[0046] [Figure 1] Figure 1 demonstrates that an antibody (Fab fragment) that binds HLA-G in combination with cetuximab significantly increased macrophage phagocytosis of HLA-G+A549 target cells compared to cetuximab alone. No activity was observed from the antibody that binds HLA-G compared to untreated controls. Data are presented as mean ± standard deviation (n=2 replicates). **p<0.01 using Student's t-test. [Figure 2A] Figures 2A and 2B show that an antibody that binds HLA-G in combination with pembrolizumab or atezolizumab, respectively, enhanced ILT2+CD8+ T cell degranulation (CD107a) over either single agent alone. Isotype-matched antibodies were used as negative controls. Graphs show the mean ± standard deviation (n=2 replicate wells) from one representative donor. [Figure 2B]Figures 2A and 2B show that an antibody that binds HLA-G in combination with pembrolizumab or atezolizumab, respectively, enhanced ILT2+CD8+ T cell degranulation (CD107a) over either single agent alone. Isotype-matched antibodies were used as negative controls. Graphs show the mean ± standard deviation (n=2 replicate wells) from one representative donor. [Figure 3A] Figures 3A and 3B demonstrate that an antibody that binds HLA-G in combination with magrolimab increased macrophage phagocytosis of endogenous HLA-G-expressing HT-1376 and JEG-3 cells, respectively, compared to either single agent alone. [Figure 3B] Figures 3A and 3B demonstrate that an antibody that binds HLA-G in combination with magrolimab increased macrophage phagocytosis of endogenous HLA-G-expressing HT-1376 and JEG-3 cells, respectively, compared to either single agent alone. [Figure 3C] Figures 3C and 3D show that the triple combination of an antibody that binds HLA-G together with magrolimab and cetuximab is more active than single or double therapy in inducing phagocytosis of A549 and SK-OV-3 cells engineered to express HLA-G. An isotype antibody was used as a negative control. Data are presented as mean ± standard deviation (n = 2 replicates). [Figure 3D] Figures 3C and 3D show that the triple combination of an antibody that binds HLA-G together with magrolimab and cetuximab is more active than single or double therapy in inducing phagocytosis of A549 and SK-OV-3 cells engineered to express HLA-G. An isotype antibody was used as a negative control. Data are presented as mean ± standard deviation (n = 2 replicates). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0047] Provided herein is a combination therapy comprising an antibody with binding specificity for HLA-G and an antibody with binding specificity for EGFR, PD-1 and / or PD-L1, and / or CD47 or SIRPα.

[0048] 1.Definition Unless otherwise defined, all technical terms, notations, and other scientific terms used herein are intended to have the meaning commonly understood by those skilled in the art to which this invention belongs. In some cases, terms with commonly understood meanings are defined herein for clarity and / or ease of reference, and the inclusion of such definitions herein should not necessarily be interpreted as representing a difference from what is commonly understood in the art. The techniques and procedures described or referenced herein are generally understood and commonly used by those skilled in the art using conventional methodologies in use, such as the widely used molecular cloning methodologies described in, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual 2nd ed. (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Where appropriate, unless otherwise noted, procedures involving the use of commercially available kits and reagents are generally carried out according to manufacturer-defined protocols and / or parameters.

[0049] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0050] The terms "HLA-G", "human leukocyte antigen G" and "HLA-G histocompatibility antigen, class I, G" are used interchangeably herein. Unless otherwise specified, the terms include any variants, isoforms, and species homologs of human HLA-G naturally expressed by a cell or expressed by a cell transfected with the HLA-G gene.

[0051] The terms "PD-1," "programmed cell death protein 1," and "cluster of differentiation 279" are used interchangeably herein. Unless otherwise specified, the terms include any variants, isoforms, and species homologs of human PD-1 naturally expressed by a cell or expressed by a cell transfected with the PD-1 gene.

[0052] The terms "PD-L1", "programmed death-ligand 1", "cluster of differentiation 274", "B7 homolog 1", and "B7-H1" are used interchangeably herein. Unless otherwise specified, the terms include any variants, isoforms, and species homologs of human PD-L1 naturally expressed by cells or expressed by cells transfected with the PD-L1 gene.

[0053] The terms "EGFR," "epidermal growth factor receptor," "ErbB-1," and "HER1" are used interchangeably herein. Unless otherwise specified, the terms include any variants, isoforms, and species homologs of human EGFR expressed naturally by a cell or expressed by a cell transfected with the EGFR gene.

[0054] The terms "CD47," "cluster of differentiation 47," and "integrin-associated protein" are used interchangeably herein. Unless otherwise specified, the terms include any variants, isoforms, and species homologs of human CD47 naturally expressed by cells or expressed by cells transfected with the CD47 gene.

[0055] The terms "signal regulatory protein alpha" or "SIRPα" are used interchangeably herein.

[0056] The term "immunoglobulin" generally refers to a class of structurally related proteins that contain two pairs of polypeptide chains: one pair of light (L) chains and one pair of heavy (H) chains. In "intact immunoglobulins", all four of these chains are interconnected by disulfide bonds. The structure of immunoglobulins has been well characterized. See, e.g., Paul, Fundamental Immunology 7th ed., Ch. 5 (2013) Lippincott Williams & Wilkins, Philadelphia, PA. Briefly, each heavy chain typically comprises a heavy chain variable region (V H ) and heavy chain constant region (C H ). The heavy chain constant region typically 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 CL.

[0057] The term "antibody" describes a type of immunoglobulin molecule and is used herein in the broadest sense. Specifically, antibodies include intact antibodies (e.g., intact immunoglobulins) and antibody fragments. An antibody comprises at least one antigen-binding domain. One example of an antigen-binding domain is the antigen-binding domain formed by a VH-VL dimer.

[0058] V H and V L The regions can be further subdivided into regions of hypervariability ("hypervariable regions (HVRs)"; also called "complementarity determining regions" (CDRs)), interspersed with more conserved regions. The more conserved regions are called framework regions (FRs). Each V H and V Lgenerally 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 ed. (1991) Public Health Service, National Institutes of Health, Bethesda, MD, which is incorporated by reference in its entirety.

[0059] 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 domain.

[0060] 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 designated α, δ, ε, γ, and μ, respectively. The IgG and IgA classes are further divided into subclasses based on sequence and functional differences. Humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

[0061] The amino acid sequence boundaries of the CDRs can be determined by one of skill in the art using any of the many known numbering schemes, including those described by Kabat et al., supra (the "Kabat" numbering scheme), Al-Lazikani et al., 1997, J. Mol. Biol., 273:927-948 (the "Chothia" numbering scheme), MacCallum et al., 1996, J. Mol. Biol. 262:732-745 (the "Contact" numbering scheme), Lefranc et al., Dev. Comp. Immunol., 2003, 27:55-77 (the "IMGT" numbering scheme), and Honegge and Pluckthun, J. Mol. Biol., 2001, 309:657-70 (the "AHo" numbering scheme), each of which is incorporated by reference in its entirety.

[0062] Table 1 provides the positions of CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 as identified by the Kabat and Chothia schemes. For CDR-H1, residue numbering is provided using both the Kabat and Chothia numbering schemes.

[0063] Unless otherwise specified, the numbering scheme used herein to identify specific CDRs is the Kabat numbering scheme. Variants and equivalent antibodies according to the Chothia numbering scheme are intended to be within the scope of the present invention. [Table 1]

[0064] An "antibody fragment" includes a portion of an intact antibody, such as the antigen-binding or variable region of the 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, the antibody that binds to HLA-G, the antibody that binds to PD-1, and / or the antibody that binds to PD-L1, the antibody that binds to EGFR, and / or the antibody that binds to CD47 or SIRPα includes antibody fragments of each of the particular antibodies.

[0065] An "Fv" fragment comprises a non-covalently associated dimer of one heavy- and one light-chain variable domain.

[0066] A "Fab" fragment contains, in addition to the heavy and light chain variable domains, the constant domain of the light chain and the first constant domain of the heavy chain (C H1 ). Fab fragments can be generated, for example, by papain digestion of a full-length antibody.

[0067] "F(ab')2" fragments contain two Fab' fragments joined by disulfide bonds near the hinge region. F(ab')2 fragments can be generated, for example, by pepsin digestion of an intact antibody. F(ab') fragments can be dissociated, for example, by treatment with B-mercaptoethanol.

[0068] A "single-chain Fv" or "sFv" or "scFv" antibody fragment comprises a VH domain and a VL domain in a single polypeptide chain. The VH and VL are generally linked by a peptide linker. See Pluckthun A. (1994). Antibodies from Escherichia coli. In Rosenberg M. & Moore GP (Eds.), The Pharmacology of Monoclonal Antibodies vol. 113 (pp. 269-315). Springer-Verlag, New York, which is incorporated herein by reference in its entirety. An "scFv-Fc" fragment comprises an scFv attached to an Fc domain. For example, the Fc domain may be attached to the C-terminus of the scFv. The Fc domain may vary depending on the orientation of the variable domains in the scFv (i.e., V, VL, VH, VL ... H -V L Or V L -V H ) depending on V H Or V L Any suitable Fc domain known in the art or described herein may be used.

[0069] The term "monoclonal antibody" refers to an antibody from a population of substantially homogeneous antibodies. A population of substantially homogeneous antibodies includes antibodies that are substantially similar and bind to the same epitope, except for variants that may normally arise during the production of monoclonal antibodies. Such variants are generally present only in minor amounts. Monoclonal antibodies are typically obtained by a process that includes the selection of a single antibody from a plurality of antibodies. For example, the selection process can be the selection of 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. The selected antibody can be further modified, for example, to improve affinity for the target ("affinity maturation"), to humanize the antibody, to improve its production in cell culture, and / or to reduce its immunogenicity in a subject.

[0070] With respect to the binding of an antibody to a target molecule, the term "binding" or "binding" to a particular antigen (e.g., a polypeptide target) or epitope on a particular antigen refers to binding that is measurably different from non-selective interactions. Binding can be measured, for example, 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 that is similar to the target, such as an excess of unlabeled target. In that case, binding is indicated if the binding of the labeled target to the probe is competitively inhibited by an excess of unlabeled target.

[0071] "Treating" or "treatment" of any cancer, in certain embodiments, refers to ameliorating the cancer present in a subject. In another embodiment, "treating" or "treatment" includes improving at least one physical parameter, which may not be discernible by the subject. In yet another embodiment, "treating" or "treatment" includes modulating the cancer, either physically (e.g., stabilization of a discernible symptom) or physiologically (e.g., stabilization of a physical parameter), or both.

[0072] As used herein, the term "subject" means a mammal or a human. In some embodiments, subjects include, but are not limited to, monkeys, dogs, cats, mice, rats, cows, horses, camels, birds, goats, and sheep.

[0073] 2. Antibody Combinations Provided herein are methods and antibody combinations for the treatment of cancer or other diseases, the antibody combinations combining antibodies that bind to HLA-G and antibodies with binding specificity for EGFR, PD-1 and / or PD-L1, and / or CD47 or SIRPα.

[0074] CDR-H1+CDR-H2+CDR-H3 region of an antibody In some embodiments, an antibody that binds to HLA-G comprises a VH sequence that comprises a CDR-H1 sequence that comprises, consists of, or consists essentially of a sequence selected from SEQ ID NO:1 or SEQ ID NO:11, a CDR-H2 sequence that comprises, consists of, or consists essentially of a sequence selected from SEQ ID NO:21 or SEQ ID NO:31, and a CDR-H3 sequence that comprises, consists of, or consists essentially of a sequence selected from SEQ ID NO:41. In some embodiments, the CDR-H1, CDR-H2, and CDR-H3 sequences are all selected from a single exemplary VH sequence provided in the present disclosure. H For example, in some embodiments, CDR-H1, CDR-H2, and CDR-H3 are all from a single exemplary VHV sequence having SEQ ID NO:81. H It is from an array.

[0075] In some embodiments, an antibody that binds EGFR 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. H In some embodiments, the CDR-H1 sequence, the CDR-H2 sequence, and the CDR-H3 sequence all comprise a single exemplary VH sequence provided in the present disclosure. H For example, in some embodiments, CDR-H1, CDR-H2, and CDR-H3 are all selected from a single exemplary V sequence selected from SEQ ID NO:89. H It is from an array.

[0076] In some embodiments, an antibody that binds to PD-1 comprises a CDR-H1 sequence that comprises, consists of, or consists essentially of a sequence selected from SEQ ID NOs: 2-5 or 12-15; a CDR-H2 sequence that comprises, consists of, or consists essentially of a sequence selected from SEQ ID NOs: 22-25 or 32-35; and a CDR-H3 sequence that comprises, consists of, or consists essentially of a sequence selected from SEQ ID NOs: 42-45. H In some embodiments, the CDR-H1 sequence, the CDR-H2 sequence, and the CDR-H3 sequence all comprise a single exemplary VH sequence provided in the present disclosure. H For example, in some embodiments, CDR-H1, CDR-H2, and CDR-H3 are all selected from a single exemplary V sequence selected from SEQ ID NOs: 82-85. H It is from an array.

[0077] In some embodiments, an antibody that binds to PD-1 comprises a CDR-H1 sequence that comprises, consists of, or consists essentially of a sequence that comprises SEQ ID NO:2 or SEQ ID NO:12, a CDR-H2 sequence that comprises, consists of, or consists essentially of a sequence that comprises SEQ ID NO:22 or SEQ ID NO:32, and a CDR-H3 sequence that comprises, consists of, or consists essentially of a sequence that comprises SEQ ID NO:42. H In some embodiments, an antibody that binds to PD-1 comprises a CDR-H1 sequence that comprises, consists of, or consists essentially of a sequence that includes SEQ ID NO:3 or SEQ ID NO:13, a CDR-H2 sequence that comprises, consists of, or consists essentially of a sequence that includes SEQ ID NO:23 or SEQ ID NO:33, and a CDR-H3 sequence that comprises, consists of, or consists essentially of a sequence that includes SEQ ID NO:43. HIn some embodiments, an antibody that binds to PD-1 comprises a CDR-H1 sequence that comprises, consists of, or consists essentially of a sequence that includes SEQ ID NO:4 or SEQ ID NO:14, a CDR-H2 sequence that comprises, consists of, or consists essentially of a sequence that includes SEQ ID NO:24 or SEQ ID NO:34, and a CDR-H3 sequence that comprises, consists of, or consists essentially of a sequence that includes SEQ ID NO:44. H In some embodiments, an antibody that binds to PD-1 comprises a CDR-H1 sequence that comprises, consists of, or consists essentially of a sequence that includes SEQ ID NO:5 or SEQ ID NO:15, a CDR-H2 sequence that comprises, consists of, or consists essentially of a sequence that includes SEQ ID NO:25 or SEQ ID NO:35, and a CDR-H3 sequence that comprises, consists of, or consists essentially of a sequence that includes SEQ ID NO:45. H Contains arrays.

[0078] In some embodiments, the CDR-H1 sequence, the CDR-H2 sequence, and the CDR-H3 sequence are all selected from a single exemplary VH sequence provided in the present disclosure. H For example, in some embodiments, CDR-H1, CDR-H2, and CDR-H3 are all from a single V sequence having SEQ ID NO: 82. H In some embodiments, CDR-H1, CDR-H2, and CDR-H3 are all from a single V sequence having SEQ ID NO: 83. H In some embodiments, CDR-H1, CDR-H2, and CDR-H3 are all from a single V sequence having SEQ ID NO: 84. H In some embodiments, CDR-H1, CDR-H2, and CDR-H3 are all from a single VHV having SEQ ID NO: 85. H It is from an array.

[0079] In some embodiments, an antibody that binds PD-L1 comprises a CDR-H1 sequence that comprises, consists of, or consists essentially of a sequence selected from SEQ ID NO:6-8 or SEQ ID NO:16-18; a CDR-H2 sequence that comprises, consists of, or consists essentially of a sequence selected from SEQ ID NO:26-28 or SEQ ID NO:36-38; and a CDR-H3 sequence that comprises, consists of, or consists essentially of a sequence selected from SEQ ID NO:46-48. H In some embodiments, the CDR-H1 sequence, the CDR-H2 sequence, and the CDR-H3 sequence all comprise a single exemplary VH sequence provided in the present disclosure. H For example, in some embodiments, CDR-H1, CDR-H2, and CDR-H3 are all selected from a single exemplary V sequence selected from SEQ ID NOs: 86-88. H It is from an array.

[0080] In some embodiments, an antibody that binds PD-L1 comprises a CDR-H1 sequence that comprises, consists of, or consists essentially of a sequence comprising SEQ ID NO:6 or SEQ ID NO:16, a CDR-H2 sequence that comprises, consists of, or consists essentially of a sequence comprising SEQ ID NO:26 or SEQ ID NO:36, and a CDR-H3 sequence that comprises, consists of, or consists essentially of a sequence comprising SEQ ID NO:46. H In some embodiments, an antibody that binds PD-L1 comprises a CDR-H1 sequence that comprises, consists of, or consists essentially of a sequence comprising SEQ ID NO:7 or SEQ ID NO:17, a CDR-H2 sequence that comprises, consists of, or consists essentially of a sequence comprising SEQ ID NO:27 or SEQ ID NO:37, and a CDR-H3 sequence that comprises, consists of, or consists essentially of a sequence comprising SEQ ID NO:47. HIn some embodiments, an antibody that binds PD-L1 comprises a CDR-H1 sequence that comprises, consists of, or consists essentially of a sequence comprising SEQ ID NO:8 or SEQ ID NO:18, a CDR-H2 sequence that comprises, consists of, or consists essentially of a sequence comprising SEQ ID NO:28 or SEQ ID NO:38, and a CDR-H3 sequence that comprises, consists of, or consists essentially of a sequence comprising SEQ ID NO:48. H Contains arrays.

[0081] In some embodiments, the CDR-H1 sequence, the CDR-H2 sequence, and the CDR-H3 sequence are all selected from a single exemplary VH sequence provided in the present disclosure. H In some embodiments, CDR-H1, CDR-H2, and CDR-H3 are all from a single V sequence having SEQ ID NO:86. H In some embodiments, CDR-H1, CDR-H2, and CDR-H3 are all from a single VHVHVHV having SEQ ID NO:87. H In some embodiments, CDR-H1, CDR-H2, and CDR-H3 are all from a single V sequence having SEQ ID NO:88. H It is from an array.

[0082] In some embodiments, an antibody that binds CD47 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 the sequence SEQ ID NO:50. H In some embodiments, the CDR-H1 sequence, the CDR-H2 sequence, and the CDR-H3 sequence all comprise a single exemplary VH sequence provided in the present disclosure. H For example, in some embodiments, CDR-H1, CDR-H2, and CDR-H3 are all from a single exemplary VHV sequence having SEQ ID NO: 90. H It is from an array.

[0083] In some embodiments, an antibody that binds to HLA-G 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. H and an antibody that binds to EGFR, comprising 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. H V containing arrays H Contains arrays.

[0084] In some embodiments, an antibody that binds to HLA-G 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. H and an antibody that binds to PD-1 comprising 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. H V containing arrays H In some embodiments, an antibody that binds to HLA-G 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. H and an antibody that binds to PD-1 comprising 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. H In some embodiments, an antibody that binds to HLA-G 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. H and an antibody that binds to PD-1 comprising 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. HIn some embodiments, an antibody that binds to HLA-G 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. H and an antibody that binds to PD-1 comprising 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. H Contains arrays.

[0085] In some embodiments, an antibody that binds to HLA-G 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. H and an antibody that binds to PD-L1 comprising 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. H In some embodiments, an antibody that binds to HLA-G 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. H and an antibody that binds to PD-L1 comprising 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. H In some embodiments, an antibody that binds to HLA-G 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. H and an antibody that binds to PD-L1 comprising 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. H Contains arrays.

[0086] In some embodiments, an antibody that binds to HLA-G 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. H and an antibody that binds to CD47, comprising 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. H Contains arrays.

[0087] V H array In some embodiments, an antibody that binds to HLA-G comprises, consists of, or consists essentially of SEQ ID NO:81. H In some embodiments, an antibody that binds to EGFR comprises, consists of, or consists essentially of SEQ ID NO:89. H In some embodiments, an antibody that binds to PD-1 comprises, consists of, or consists essentially of SEQ ID NO:82. H In some embodiments, an antibody that binds to PD-1 comprises the V sequence comprising, consisting of, or consisting essentially of SEQ ID NO:83. H In some embodiments, an antibody that binds to PD-1 comprises the V sequence comprising, consisting of, or consisting essentially of SEQ ID NO:84. H In some embodiments, an antibody that binds to PD-1 comprises a V sequence comprising, consisting of, or consisting essentially of SEQ ID NO:85. H Contains arrays.

[0088] In some embodiments, an antibody that binds PD-L1 comprises, consists of, or consists essentially of SEQ ID NO:86. H In some embodiments, an antibody that binds PD-L1 comprises, consists of, or consists essentially of SEQ ID NO:87. HIn some embodiments, an antibody that binds PD-L1 comprises, consists of, or consists essentially of SEQ ID NO:88. H In some embodiments, an antibody that binds to CD47 comprises the V sequence comprising, consisting of, or consisting essentially of SEQ ID NO:90. H Contains arrays.

[0089] CDR-L1+CDR-L2+CDR-L3 region of the antibody In some embodiments, an antibody that binds to HLA-G comprises 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. L In some embodiments, the CDR-L1 sequence, the CDR-L2 sequence, and the CDR-L3 sequence all comprise a single exemplary V sequence provided in the present disclosure. L For example, in some embodiments, CDR-L1, CDR-L2, and CDR-L3 are all from a single exemplary V sequence having SEQ ID NO:91. L It is from an array.

[0090] In some embodiments, an antibody that binds EGFR comprises a CDR-L1 sequence that comprises, consists of, or consists essentially of SEQ ID NO:59, a CDR-L2 sequence that comprises, consists of, or consists essentially of SEQ ID NO:69, and a CDR-L3 sequence that comprises, consists of, or consists essentially of SEQ ID NO:79. L In some embodiments, the CDR-L1 sequence, the CDR-L2 sequence, and the CDR-L3 sequence all comprise a single exemplary V sequence provided in the present disclosure. L For example, in some embodiments, CDR-L1, CDR-L2, and CDR-L3 are all from a single exemplary V sequence having SEQ ID NO:99. L It is from an array.

[0091] In some embodiments, an antibody that binds to PD-1 comprises a CDR-L1 sequence that comprises, consists of, or consists essentially of a sequence selected from any one of SEQ ID NOs: 52-55, a CDR-L2 sequence that comprises, consists of, or consists essentially of a sequence selected from SEQ ID NOs: 62-65, and a CDR-L3 sequence that comprises, consists of, or consists essentially of a sequence selected from SEQ ID NOs: 72-75. L In some embodiments, the CDR-L1 sequence, the CDR-L2 sequence, and the CDR-L3 sequence all comprise a single exemplary V sequence provided in the present disclosure. L For example, in some embodiments, CDR-L1, CDR-L2, and CDR-L3 are all selected from a single exemplary V sequence selected from SEQ ID NOs: 92-95. H It is from an array.

[0092] In some embodiments, an antibody that binds to PD-1 comprises a CDR-L1 sequence that comprises, consists of, or consists essentially of a sequence that comprises SEQ ID NO:52, a CDR-L2 sequence that comprises, consists of, or consists essentially of a sequence that comprises SEQ ID NO:62, and a CDR-L3 sequence that comprises, consists of, or consists essentially of a sequence that comprises SEQ ID NO:72. L In some embodiments, an antibody that binds to PD-1 comprises a CDR-L1 sequence that comprises, consists of, or consists essentially of a sequence that comprises SEQ ID NO:53, a CDR-L2 sequence that comprises, consists of, or consists essentially of a sequence that comprises SEQ ID NO:63, and a CDR-L3 sequence that comprises, consists of, or consists essentially of a sequence that comprises SEQ ID NO:73. L In some embodiments, an antibody that binds to PD-1 comprises a CDR-L1 sequence that comprises, consists of, or consists essentially of a sequence that comprises SEQ ID NO:54, a CDR-L2 sequence that comprises, consists of, or consists essentially of a sequence that comprises SEQ ID NO:64, and a CDR-L3 sequence that comprises, consists of, or consists essentially of a sequence that comprises SEQ ID NO:74.L In some embodiments, an antibody that binds to PD-1 comprises a CDR-L1 sequence that comprises, consists of, or consists essentially of a sequence that comprises SEQ ID NO:55, a CDR-L2 sequence that comprises, consists of, or consists essentially of a sequence that comprises SEQ ID NO:65, and a CDR-L3 sequence that comprises, consists of, or consists essentially of a sequence that comprises SEQ ID NO:75. L Contains arrays.

[0093] In some embodiments, the CDR-L1 sequence, the CDR-L2 sequence, and the CDR-L3 sequence are all selected from a single exemplary V L For example, in some embodiments, CDR-L1, CDR-L2, and CDR-L3 are all from a single V sequence having SEQ ID NO:92. L In some embodiments, CDR-H1, CDR-H2, and CDR-H3 are all from a single V sequence having SEQ ID NO:93. L In some embodiments, CDR-H1, CDR-H2, and CDR-H3 are all from a single V sequence having SEQ ID NO:94. L In some embodiments, CDR-H1, CDR-H2, and CDR-H3 are all from a single VHVHVHV having SEQ ID NO:95. L It is from an array.

[0094] In some embodiments, an antibody that binds PD-L1 comprises a CDR-L1 sequence that comprises, consists of, or consists essentially of a sequence selected from any one of SEQ ID NOs: 56-58, a CDR-L2 sequence that comprises, consists of, or consists essentially of a sequence selected from SEQ ID NOs: 66-68, and a CDR-L3 sequence that comprises, consists of, or consists essentially of a sequence selected from SEQ ID NOs: 76-78. L In some embodiments, the CDR-L1 sequence, the CDR-L2 sequence, and the CDR-L3 sequence all comprise a single exemplary V sequence provided in the present disclosure. LFor example, in some embodiments, CDR-L1, CDR-L2, and CDR-L3 are all selected from a single exemplary V sequence selected from SEQ ID NOs: 96-98. H It is from an array.

[0095] In some embodiments, an antibody that binds PD-L1 comprises a CDR-L1 sequence that comprises, consists of, or consists essentially of a sequence comprising SEQ ID NO:56, a CDR-L2 sequence that comprises, consists of, or consists essentially of a sequence comprising SEQ ID NO:66, and a CDR-L3 sequence that comprises, consists of, or consists essentially of a sequence comprising SEQ ID NO:76. L In some embodiments, an antibody that binds PD-L1 comprises a CDR-L1 sequence that comprises, consists of, or consists essentially of a sequence comprising SEQ ID NO:57, a CDR-L2 sequence that comprises, consists of, or consists essentially of a sequence comprising SEQ ID NO:67, and a CDR-L3 sequence that comprises, consists of, or consists essentially of a sequence comprising SEQ ID NO:77. L In some embodiments, an antibody that binds PD-L1 comprises a CDR-L1 sequence that comprises, consists of, or consists essentially of a sequence comprising SEQ ID NO:58, a CDR-L2 sequence that comprises, consists of, or consists essentially of a sequence comprising SEQ ID NO:68, and a CDR-L3 sequence that comprises, consists of, or consists essentially of a sequence comprising SEQ ID NO:78. L Contains arrays.

[0096] In some embodiments, the CDR-L1 sequence, the CDR-L2 sequence, and the CDR-L3 sequence are all selected from a single exemplary V L For example, in some embodiments, CDR-L1, CDR-L2, and CDR-L3 are all from a single V sequence having SEQ ID NO:96. L In some embodiments, CDR-H1, CDR-H2, and CDR-H3 are all from a single V sequence having SEQ ID NO:97. LIn some embodiments, CDR-H1, CDR-H2, and CDR-H3 are all from a single V sequence having SEQ ID NO:98. L It is from an array.

[0097] In some embodiments, an antibody that binds CD47 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. L In some embodiments, the CDR-L1 sequence, the CDR-L2 sequence, and the CDR-L3 sequence all comprise a single exemplary V sequence provided in the present disclosure. L For example, in some embodiments, CDR-L1, CDR-L2, and CDR-L3 are all from a single exemplary V having the sequence MO100. L It is from an array.

[0098] V L array In some embodiments, an antibody that binds to HLA-G comprises, consists of, or consists essentially of the sequence SEQ ID NO:91. L In some embodiments, an antibody that binds to EGFR comprises, consists of, or consists essentially of the sequence SEQ ID NO:99. L In some embodiments, an antibody that binds to PD-1 comprises, consists of, or consists essentially of the sequence SEQ ID NO:92. L In some embodiments, an antibody that binds to PD-1 comprises, consists of, or consists essentially of the sequence SEQ ID NO:93. L In some embodiments, an antibody that binds to PD-1 comprises, consists of, or consists essentially of the sequence SEQ ID NO:94. LIn some embodiments, an antibody that binds to PD-1 comprises, consists of, or consists essentially of the sequence SEQ ID NO:95. L Contains arrays.

[0099] In some embodiments, an antibody that binds PD-L1 comprises, consists of, or consists essentially of the sequence SEQ ID NO:96. L In some embodiments, an antibody that binds PD-L1 comprises, consists of, or consists essentially of the sequence SEQ ID NO:97. L In some embodiments, an antibody that binds to PD-1 comprises, consists of, or consists essentially of the sequence SEQ ID NO:98. L In some embodiments, an antibody that binds to CD47 comprises the V sequence comprising, consisting of, or consisting essentially of SEQ ID NO: 100. L Contains arrays.

[0100] V H -V L versus In some embodiments, the antibody that binds to HLA-G is H Sequence and V L Contains the sequence, V H The sequence comprises, consists of, or essentially consists of SEQ ID NO: 81, L The sequence comprises, consists of, or consists essentially of SEQ ID NO: 91. In some embodiments, the antibody that binds to EGFR comprises the sequence H Sequence and V L Contains the sequence, V H The sequence comprises, consists of, or consists essentially of SEQ ID NO:89, L The sequence comprises, consists of, or consists essentially of SEQ ID NO: 99. In some embodiments, the antibody that binds to CD47 comprises the sequence H Sequence and V L Contains the sequence, V H The sequence comprises, consists of, or essentially consists of SEQ ID NO: 90, LThe sequence comprises, consists of, or consists essentially of SEQ ID NO:100.

[0101] In some embodiments, the antibody that binds PD-1 is H Sequence and V L Contains the sequence, V H The sequence comprises, consists of, or essentially consists of a sequence selected from any one of SEQ ID NOs: 82 to 85, L In some embodiments, the antibody that binds to PD-1 comprises, consists of, or consists essentially of a sequence selected from any one of SEQ ID NOs: 92-95. H Sequence and V L Contains the sequence, V H The sequence comprises, consists of, or essentially consists of SEQ ID NO: 82, L The sequence comprises, consists of, or consists essentially of SEQ ID NO: 92. In some embodiments, the antibody that binds to PD-1 has the sequence H Sequence and V L Contains the sequence, V H The sequence comprises, consists of, or essentially consists of SEQ ID NO: 83, L The sequence comprises, consists of, or consists essentially of SEQ ID NO: 93. In some embodiments, the antibody that binds to PD-1 has the sequence H Sequence and V L Contains the sequence, V H The sequence comprises, consists of, or essentially consists of SEQ ID NO: 84, L The sequence comprises, consists of, or consists essentially of SEQ ID NO: 94. In some embodiments, the antibody that binds to PD-1 has the sequence H Sequence and V L Contains the sequence, V H The sequence comprises, consists of, or essentially consists of SEQ ID NO: 85, L The sequence comprises, consists of, or consists essentially of SEQ ID NO:95.

[0102] In some embodiments, the antibody that binds PD-L1 isH Sequence and V L Contains the sequence, V H The sequence comprises, consists of, or essentially consists of a sequence selected from any one of SEQ ID NOs: 86 to 88, L In some embodiments, the antibody that binds to PD-L1 comprises, consists of, or consists essentially of a sequence selected from any one of SEQ ID NOs: 96-98. H Sequence and V L Contains the sequence, V H The sequence comprises, consists of, or essentially consists of SEQ ID NO: 86, L The sequence comprises, consists of, or consists essentially of SEQ ID NO: 96. In some embodiments, the antibody that binds PD-L1 has the sequence H Sequence and V L Contains the sequence, V H The sequence comprises, consists of, or essentially consists of SEQ ID NO: 87, L The sequence comprises, consists of, or consists essentially of SEQ ID NO: 97. In some embodiments, the antibody that binds PD-L1 has the sequence H Sequence and V L Contains the sequence, V H The sequence comprises, consists of, or essentially consists of SEQ ID NO: 88, L The sequence comprises, consists of, or consists essentially of SEQ ID NO:99.

[0103] CDR-H1+CDR-H2+CDR-H3+CDR-L1+CDR-L2+CDR-L3 In some embodiments, an antibody that binds to HLA-G comprises a heavy chain variable region (V H ) and the light chain variable region (V L ), or consisting of V H and / or V L but, a) a VHCDR1 having the sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 11, b) a VH CDR2 having the sequence set forth in SEQ ID NO: 21 or SEQ ID NO: 31; c) a VH CDR3 having the sequence set forth in SEQ ID NO: 41; d) VLCDR1 having the sequence set forth in SEQ ID NO: 51; e) VLCDR2 having the sequence set forth in SEQ ID NO: 61, and f) comprises a VLCDR3 having the sequence set forth in SEQ ID NO: 71.

[0104] In some embodiments, the antibody that binds EGFR comprises a heavy chain variable region (V H ) and the light chain variable region (V L ), V H and / or V L but, a) a VHCDR1 having the sequence set forth in SEQ ID NO: 9 or SEQ ID NO: 19, b) a VH CDR2 having the sequence set forth in SEQ ID NO: 29 or SEQ ID NO: 39; c) a VH CDR3 having the sequence set forth in SEQ ID NO: 49; d) VLCDR1 having the sequence set forth in SEQ ID NO: 59; e) VLCDR2 having the sequence set forth in SEQ ID NO: 69, and f) comprises a VLCDR3 having the sequence set forth in SEQ ID NO: 79.

[0105] In some embodiments, an antibody that binds to PD-1 comprises a heavy chain variable region (V H ) and the light chain variable region (V L ), or consisting of V H and / or V L but, a) VHCDR1 having a sequence set forth in any one of SEQ ID NOs: 2 to 5 or 12 to 15; b) VH CDR2 having a sequence set forth in any one of SEQ ID NOs: 22 to 25 or 32 to 35; c) VHCDR3 having a sequence set forth in any one of SEQ ID NOs: 42 to 45; d) VLCDR1 having a sequence set forth in any one of SEQ ID NOs: 52 to 55; e) VLCDR2 having a sequence set forth in any one of SEQ ID NOs: 62 to 65, and f) Contains a VLCDR3 having a sequence set forth in any one of SEQ ID NOs: 72 to 75.

[0106] In some embodiments, an antibody that binds to PD-1 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. H and a VL sequence comprising the sequence SEQ ID NO: 52, a CDR-L2 sequence comprising SEQ ID NO: 62, and a CDR-L3 sequence comprising the sequence SEQ ID NO: 72. In some embodiments, an antibody that binds PD-1 comprises a VL sequence comprising 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. H and a VL sequence comprising 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, an antibody that binds PD-1 comprises a VL sequence comprising 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. H and a VL sequence comprising SEQ ID NO:5, a CDR-L1 sequence comprising SEQ ID NO:54, a CDR-L2 sequence comprising SEQ ID NO:64, and a CDR-L3 sequence comprising SEQ ID NO:74. In some embodiments, an antibody that binds PD-1 comprises a VL sequence comprising 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. H and a VL sequence comprising the sequence, a CDR-L1 sequence comprising SEQ ID NO:55, a CDR-L2 sequence comprising SEQ ID NO:65, and a CDR-L3 sequence comprising SEQ ID NO:75.

[0107] In some embodiments, an antibody that binds PD-L1 comprises a heavy chain variable region (V H ) and the light chain variable region (V L ), or consisting of V H and / or V Lbut, a) VHCDR1 having a sequence set forth in any one of SEQ ID NOs: 6 to 8 or 16 to 18; b) VHCDR2 having a sequence set forth in any one of SEQ ID NOs: 26 to 28 or 38 to 38; c) VHCDR3 having a sequence set forth in any one of SEQ ID NOs: 46 to 48; d) VLCDR1 having a sequence set forth in any one of SEQ ID NOs: 56 to 58; e) VLCDR2 having a sequence set forth in any one of SEQ ID NOs: 66 to 68, and f) Contains a VLCDR3 having a sequence set forth in any one of SEQ ID NOs: 76 to 78.

[0108] In some embodiments, an antibody that binds PD-L1 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. H and a VL sequence comprising the sequence SEQ ID NO: 56, a CDR-L2 sequence comprising SEQ ID NO: 66, and a CDR-L3 sequence comprising the sequence SEQ ID NO: 76. In some embodiments, an antibody that binds PD-L1 comprises a VL sequence comprising 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. H and a VL sequence comprising SEQ ID NO: 57, a CDR-L1 sequence comprising SEQ ID NO: 57, a CDR-L2 sequence comprising SEQ ID NO: 67, and a CDR-L3 sequence comprising SEQ ID NO: 77. In some embodiments, an antibody that binds PD-L1 comprises a VL sequence comprising 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. H and a VL sequence comprising the sequence, a CDR-L1 sequence comprising SEQ ID NO:58, a CDR-L2 sequence comprising SEQ ID NO:68, and a CDR-L3 sequence comprising SEQ ID NO:78.

[0109] In some embodiments, an antibody that binds to CD47 comprises a heavy chain variable region (V H ) and the light chain variable region (V L ), or consisting of V H and / or V L but, a) a VHCDR1 having the sequence set forth in SEQ ID NO: 10 or SEQ ID NO: 20, b) a VH CDR2 having the sequence set forth in SEQ ID NO: 30 or SEQ ID NO: 40; c) a VH CDR3 having the sequence set forth in SEQ ID NO: 50; d) VLCDR1 having the sequence set forth in SEQ ID NO: 60; e) VLCDR2 having the sequence set forth in SEQ ID NO: 70, and f) comprises a VLCDR3 having the sequence set forth in SEQ ID NO: 80.

[0110] HC+LC In some embodiments, antibodies that bind HLA-G, EGFR, PD-1, PD-L1, and / or CD47 comprise or consist 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, antibodies that bind HLA-G, EGFR, PD-1, PD-L1, and / or CD47 or SIRPα comprise or consist of two identical heavy chains that comprise, consist, or consist essentially of an HC sequence and two identical light chains that comprise, consist, or consist essentially of an LC sequence.

[0111] In some embodiments, the HC sequence of the antibody that binds HLA-G is a HC sequence that comprises, consists of, or consists essentially of SEQ ID NO: 101 and the LC sequence of the antibody that binds HLA-G is a LC sequence that comprises, consists of, or consists essentially of any one of SEQ ID NO: 111. In some embodiments, the HC sequence of the antibody that binds EGFR is a HC sequence that comprises, consists of, or consists essentially of SEQ ID NO: 109 and the LC sequence of the antibody that binds EGFR is a LC sequence that comprises, consists of, or consists essentially of SEQ ID NO: 119. In some embodiments, the HC sequence of the antibody that binds CD47 is a HC sequence that comprises, consists of, or consists essentially of SEQ ID NO: 110 and the LC sequence of the antibody that binds PD-1 is a LC sequence that comprises, consists of, or consists essentially of SEQ ID NO: 120.

[0112] In some embodiments, the HC sequence of the antibody that binds PD-1 is a HC sequence that comprises, consists of, or consists essentially of any one of SEQ ID NOs: 102-105, and the LC sequence of the antibody that binds PD-1 is a LC sequence that comprises, consists of, or consists essentially of any one of SEQ ID NOs: 112-115. In some embodiments, the HC sequence of the antibody that binds PD-1 is a HC sequence that comprises, consists of, or consists essentially of SEQ ID NO: 102, and the LC sequence of the antibody that binds PD-1 is a LC sequence that comprises, consists of, or consists essentially of SEQ ID NO: 112. In some embodiments, the HC sequence of the antibody that binds PD-1 is a HC sequence that comprises, consists of, or consists essentially of SEQ ID NO: 103, and the LC sequence of the antibody that binds PD-1 is a LC sequence that comprises, consists of, or consists essentially of SEQ ID NO: 113. In some embodiments, the HC sequence of the antibody that binds PD-1 is a HC sequence that comprises, consists of, or consists essentially of SEQ ID NO: 104 and the LC sequence of the antibody that binds PD-1 is a LC sequence that comprises, consists of, or consists essentially of SEQ ID NO: 114. In some embodiments, the HC sequence of the antibody that binds PD-1 is a HC sequence that comprises, consists of, or consists essentially of SEQ ID NO: 105 and the LC sequence of the antibody that binds PD-1 is a LC sequence that comprises, consists of, or consists essentially of SEQ ID NO: 115.

[0113] In some embodiments, the HC sequence of the antibody that binds PD-L1 is a HC sequence that comprises, consists of, or consists essentially of any one of SEQ ID NOs: 106-108 and the LC sequence of the antibody that binds PD-1 is a LC sequence that comprises, consists of, or consists essentially of any one of SEQ ID NOs: 118-118. In some embodiments, the HC sequence of the antibody that binds PD-L1 is a HC sequence that comprises, consists of, or consists essentially of SEQ ID NO: 106 and the LC sequence of the antibody that binds PD-L1 is a LC sequence that comprises, consists of, or consists essentially of SEQ ID NO: 116. In some embodiments, the HC sequence of the antibody that binds PD-L1 is a HC sequence that comprises, consists of, or consists essentially of SEQ ID NO: 107 and the LC sequence of the antibody that binds PD-L1 is a LC sequence that comprises, consists of, or consists essentially of SEQ ID NO: 117. In some embodiments, the HC sequence of the antibody that binds PD-L1 is a HC sequence that comprises, consists of, or consists essentially of SEQ ID NO:118 and the LC sequence of the antibody that binds PD-L1 is a LC sequence that comprises, consists of, or consists essentially of SEQ ID NO:118.

[0114] In some embodiments, the HC sequence of the antibody that binds to HLA-G is an HC sequence that comprises or consists of SEQ ID NO: 101, and the LC sequence is an LC sequence that comprises or consists of SEQ ID NO: 111, and the HC sequence of the antibody that binds to PD-1 is an HC sequence that comprises or consists of SEQ ID NO: 102, and the LC sequence is an LC sequence that comprises or consists of SEQ ID NO: 112. In some embodiments, the HC sequence of the antibody that binds to HLA-G is an HC sequence that comprises or consists of SEQ ID NO: 101, and the LC sequence is an LC sequence that comprises or consists of SEQ ID NO: 111, and the HC sequence of the antibody that binds to PD-1 is an HC sequence that comprises or consists of SEQ ID NO: 103, and the LC sequence is an LC sequence that comprises or consists of SEQ ID NO: 113. In some embodiments, the HC sequence of the antibody that binds to HLA-G is the HC sequence that comprises or consists of SEQ ID NO: 101, and the LC sequence is the LC sequence that comprises or consists of SEQ ID NO: 111, and the HC sequence of the antibody that binds to PD-1 is the HC sequence that comprises or consists of SEQ ID NO: 104, and the LC sequence is the LC sequence that comprises or consists of SEQ ID NO: 114. In some embodiments, the HC sequence of the antibody that binds to HLA-G is the HC sequence that comprises or consists of SEQ ID NO: 101, and the LC sequence is the LC sequence that comprises or consists of SEQ ID NO: 111, and the HC sequence of the antibody that binds to PD-1 is the HC sequence that comprises or consists of SEQ ID NO: 105, and the LC sequence is the LC sequence that comprises or consists of SEQ ID NO: 115.

[0115] In some embodiments, the HC sequence of the antibody that binds to HLA-G is the HC sequence that comprises or consists of SEQ ID NO: 101, and the LC sequence is the LC sequence that comprises or consists of SEQ ID NO: 111, and the HC sequence of the antibody that binds to PD-L1 is the HC sequence that comprises or consists of SEQ ID NO: 106, and the LC sequence is the LC sequence that comprises or consists of SEQ ID NO: 116. In some embodiments, the HC sequence of the antibody that binds to HLA-G is the HC sequence that comprises or consists of SEQ ID NO: 101, and the LC sequence is the LC sequence that comprises or consists of SEQ ID NO: 111, and the HC sequence of the antibody that binds to PD-L1 is the HC sequence that comprises or consists of SEQ ID NO: 107, and the LC sequence is the LC sequence that comprises or consists of SEQ ID NO: 117. In some embodiments, the HC sequence of the antibody that binds to HLA-G is a HC sequence that comprises or consists of SEQ ID NO:101 and the LC sequence is a LC sequence that comprises or consists of SEQ ID NO:111, and the HC sequence of the antibody that binds to PD-L1 is a HC sequence that comprises or consists of SEQ ID NO:108 and the LC sequence is a LC sequence that comprises or consists of SEQ ID NO:118.

[0116] In some embodiments, the HC sequence of an antibody that binds to HLA-G is an HC sequence that comprises or consists of SEQ ID NO: 101, and the LC sequence is an LC sequence that comprises or consists of SEQ ID NO: 111, and the HC sequence of an antibody that binds to EGFR is an HC sequence that comprises or consists of SEQ ID NO: 109, and the LC sequence is an LC sequence that comprises or consists of SEQ ID NO: 119. In some embodiments, the HC sequence of an antibody that binds to HLA-G is an HC sequence that comprises or consists of SEQ ID NO: 101, and the LC sequence is an LC sequence that comprises or consists of SEQ ID NO: 111, and the HC sequence of an antibody that binds to CD47 is an HC sequence that comprises or consists of SEQ ID NO: 110, and the LC sequence is an LC sequence that comprises or consists of SEQ ID NO: 120.

[0117] Glycosylation variants In certain embodiments, antibodies of the invention may be modified to increase, decrease, or eliminate the extent to which they are glycosylated. Glycosylation of polypeptides is typically either "N-linked" or "O-linked."

[0118] "N-linked" glycosylation refers to the attachment of the 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.

[0119] "O-linked" glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine can also be used.

[0120] Addition or deletion of N-linked glycosylation sites to the antibody can be accomplished by altering the amino acid sequence such that one or more of the above-described tripeptide sequences are created or removed. Addition or deletion of O-linked glycosylation sites can be accomplished by adding, deleting, or substituting one or more serine or threonine residues within or (as the case may be) to the sequence of the antibody.

[0121] In certain embodiments, the antibody is glycosylated. In certain embodiments, the antibody is deglycosylated. Carbohydrates can be removed by standard techniques. In certain embodiments, the antibody is aglycosylated, for example, by expression in a non-glycosylated system.

[0122] Preparation of antibodies HLA-G, PD-1, PD-L1, and / or CD47 or SIRPα antigens can be used to generate antibodies. They may be intact proteins or fragments of the antigens. They may be in the form of isolated proteins or expressed by cells. Other forms of antigens useful for generating antibodies will be apparent to those skilled in the art.

[0123] DNA encoding the antibody can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of the monoclonal antibody). Thus, hybridoma cells can serve as a useful source of DNA encoding antibodies with the desired properties. Once isolated, the DNA can be placed into an expression vector which can then be transfected into host cells such as bacteria (e.g., E. coli), yeast (e.g., Saccharomyces or Pichia sp.), COS cells, Chinese hamster ovary (CHO) cells, or non-antibody producing myeloma cells to produce the monoclonal antibody. Other methods for generating antibodies may be useful and will be apparent to those skilled in the art.

[0124] cancer In the case of cancer, the pharmaceutical compositions of the present invention are generally administered to humans or other organisms or animals in a pharma- ceutically acceptable form. Any suitable cancer can be treated with the antibodies provided herein. In some embodiments, the cancer is a hematological cancer. In some embodiments, the cancer is a solid cancer.

[0125] In some embodiments, the compositions and methods increase macrophage phagocytosis. In some embodiments, the compositions and methods increase ILT2 + CD8 + In some embodiments, the increased macrophage phagocytosis is macrophage phagocytosis of lung cancer cells. In some embodiments, the lung cancer cells are HLA-G + EGFR+ In some embodiments, the increased macrophage phagocytosis is associated with HLA-G + A549, HLA-G + Macrophage phagocytosis of SK-OV-3, HT-1376, and / or JEG-3 cells. EXAMPLES

[0126] Example 1: The combination of an antibody (Fab fragment) that binds to HLA-G with cetuximab inhibits HLA-G more effectively than either agent alone + EGFR + Increased macrophage phagocytosis of lung cancer cells. To determine the potential clinical benefit of an antibody that binds HLA-G in combination with cetuximab, a Fab fragment of an antibody that binds HLA-G was administered to EGFR1-targeted mice engineered to express HLA-G. + This was assessed in a macrophage assay using cetuximab to induce phagocytosis of A549 lung cancer cells.

[0127] CD14 + Enriched cells were differentiated into adherent macrophages by incubation in complete RPMI (cRPMI) containing recombinant human M-CSF for 7 days at 37° C., 5% CO2. Cells were harvested after 7 days, washed, and resuspended in cRPMI. Cells were seeded at 50,000 cells / well in 50 μl of cRPMI in 96-well round-bottom plates and incubated at 37° C., 5% CO2 before combining with target cells.

[0128] Target cells (A549 lung cancer cells engineered to express HLA-G) were stained with CellTrace™ Violet (CTV, 1:1000) in PBS at 37°C, washed, and seeded at 25,000 cells per well in cRPMI. Cells were then incubated with antibodies (antibodies that bind to HLA-G Fab and / or cetuximab) for 1 hour at 37°C and 5% CO2. The A549 cell and antibody mixture was then combined with macrophages and incubated for 2 hours at 37°C and 5% CO2. After the 2 hour incubation, cells were blocked with wash buffer / BD Fc Block™ / 6% mouse serum for 20 minutes and then stained with anti-CD11b antibody and live / dead dye for 20 minutes. Cells were then washed, resuspended in wash buffer, and analyzed on a BD Fortessa flow cytometer.

[0129] Sample data were exported as FCS files and analyzed using FlowJo software v10. + Macrophages were assessed as a readout for phagocytic uptake of target cells. Live cells were stained with CD11b + / CTV + Results were gated on and reported as a percentage of total viable macrophages.

[0130] Figure 1 shows that the combination of an antibody that binds to HLA-G Fab with cetuximab significantly increased target cell phagocytosis compared to cetuximab alone. No activity was observed with the antibody that binds to HLA-G Fab compared to untreated controls.

[0131] Example 2: Antibodies that bind HLA-G in combination with pembrolizumab or atezolizumab inhibit ILT2 more than either agent alone + CD8 + Enhanced T cell degranulation (CD107a) 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 human T cell functional activity,+ HLA-G + Cells were cultured using human CD8 + were co-cultured with T cells.

[0132] Isolated CD8 + T cells were seeded in 96-well round-bottom plates at 50,000 cells / well in cRPMI. + T cells were incubated with pembrolizumab plus ImmunoCult Human CD3 / CD28 T cell activator for 1 hour at 37°C, 5% CO2. Target cells (721.221 engineered to express PD-L1 and HLA-G) were seeded at 50,000 cells / well in cRPMI and then incubated with an antibody that binds HLA-G or a combination of an antibody that binds HLA-G and atezolizumab for 1 hour at 37°C, 5% CO2. CD8 + The mixture of T cells was combined with the target cells and incubated for 24 hours at 37°C and 5% CO2. + The final ratio of T cells to target cells was 1:1.

[0133] After 24 hours of incubation, a mixture containing eFluor660 αCD107a antibody at a final concentration of 1:100 and monensin at a final concentration of 1:1000 was added to CD8 +The mixture was added to the T cell-target cell co-culture and the cells were incubated with the mixture for 4 hours at 37°C, 5% CO2. Following incubation, the cells were washed once with 4°C wash buffer (phosphate buffered saline, 2% FBS, 2mM EDTA) and then the cells were blocked with wash buffer / BD Fc Block™ / 6% mouse serum for 20 minutes. A staining solution consisting of 1:2000 Fixable Viability Dye eFluor™ 780 (Invitrogen) and fluorescently labeled αILT2, αCD8, and αPD-1 antibodies was then added to the cells for 25 minutes at 4°C. The cells were washed again with 4°C wash buffer and then resuspended in fixation / permeabilization buffer consisting of 1 part Fixation / Permeabilization Concentrate and 3 parts 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 washing buffer at 4° C. and resuspended in washing buffer.

[0134] Flow cytometry analysis on the BD LSR Fortessa revealed that ILT2 + CD8 + The cells were analyzed for the percent expression of CD107a on T cells. As shown in Figures 2A and 2B, the combination of an antibody that binds to HLA-G with pembrolizumab or atezolizumab, respectively, significantly increased ILT2 expression over either agent alone. + CD8 + These results indicate that T cell degranulation was enhanced.

[0135] Example 3: The combination of an antibody that binds to HLA-G with magrolimab inhibits HLA-G over either agent alone + A549, HLA-G + Increased macrophage phagocytosis of SK-OV-3, HT-1376, and JEG-3 target cells. The triple combination showed increased activity compared to the single-agent or dual-agent treatment groups. To determine the potential clinical benefit of antibodies that bind HLA-G in combination with magrolimab and / or cetuximab, the agents were evaluated in macrophage assays measuring 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.

[0136] CD14 + Enriched cells were differentiated into adherent macrophages by incubation in complete RPMI (cRPMI) containing recombinant human M-CSF for 7 days at 37° C., 5% CO2. Cells were harvested after 7 days, washed, and resuspended in cRPMI. Cells were seeded at 100,000 macrophages per well in 50 μl of cRPMI in 96-well round-bottom plates and incubated at 37° C., 5% CO2 before combining with target cells (A549, SK-OV-3, HT-1376, JEG-3 cell lines).

[0137] Target cells were stained with CellTrace™ Violet (CTV, 1:1000) in PBS at 37°C, washed and seeded at 50,000 target cells in cRPMI per well. Cells were then incubated with antibodies (antibodies binding to HLA-G and / or magrolimab and / or cetuximab) at 37°C, 5% CO2 for 1 hour. The target cell and antibody mixture was then combined with macrophages and incubated at 37°C, 5% CO2 for 2 hours. After the 2 hour incubation, cells were blocked with wash buffer / BD Fc Block™ / 6% mouse serum for 20 minutes and then stained with anti-CD11b antibody and live / dead dye for 20 minutes. Cells were then washed, resuspended in wash buffer and analyzed on a BD Fortessa flow cytometer.

[0138] Sample data were exported as FCS files and analyzed using FlowJo software v10. + Macrophages were assessed as a readout for phagocytic uptake of target cells. Live cells were stained with CD11b+ / CTV + Results were gated on and reported as a percentage of total viable macrophages.

[0139] Figures 3A and 3B demonstrate that an antibody that binds HLA-G in combination with magrolimab increased macrophage phagocytosis of endogenous HLA-G-expressing HT1376 and JEG-3 cells, respectively, compared to either single agent alone. Figures 3C and 3D show that a triple combination of an antibody that binds HLA-G together with magrolimab and cetuximab has higher activity than single-agent or double-agent treatments to induce phagocytosis of A549 and SK-OV-3 cells engineered to express HLA-G. An isotype antibody was used as a negative control.

[0140] Example S: Sequence Table S provides the sequences referenced herein. [Table S-1] [Table S-2] [Table S-3] [Table S-4] [Table S-5] [Table S-6] [Table S-7] [Table S-8] [Table S-9] [Table S-10] [Table S-11] [Table S-12] [Table S-13] [Table S-14] [Table S-15]

[0141] Equivalent The disclosure described above may encompass multiple separate inventions with independent utility. Each of these inventions has been disclosed in its preferred form, but the specific embodiments thereof disclosed and illustrated herein are not to be considered in a limiting sense, as numerous variations are possible. The subject matter of the invention includes all novel and unobvious combinations and subcombinations of the various elements, features, functions, and / or properties disclosed herein. The following claims specifically set forth certain combinations and subcombinations that are considered novel and unobvious. The invention embodied in other combinations and subcombinations of features, functions, elements, and / or properties may be claimed in this application, in an application claiming priority from this application, or in a related application. Such claims, whether directed to different or the same invention, and whether broader, narrower, equal, or different in scope compared to the original claims, are also considered to be included within the subject matter of the invention of the disclosure.

Claims

1. A method for treating a subject suffering from cancer, 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 HLA-G-binding antibody comprises or consists of a heavy chain variable region (VH) and a light chain variable region (VL), wherein VH and / or VL are a) a VHCDR1 having a sequence as set forth in any one of SEQ ID NO: 1 or SEQ ID NO: 11; b) a VHCDR2 having the sequence set forth in any one of SEQ ID NO: 21 or SEQ ID NO: 31; c) a VHCDR3 having a sequence as set forth in any one of SEQ ID NOs: 41; d) a VLCDR1 having a sequence as set forth in any one of SEQ ID NOs: 51; e) a VLCDR2 having a sequence as set forth in any one of SEQ ID NOs: 61; and f) The method of claim 1, comprising a VLCDR3 having a sequence as set forth in any one of SEQ ID NOs:

71.

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

91.

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

79.

5. 5. The method of 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), wherein the VH and / or VL comprises, consists of, or consists essentially of a VH having the sequence set forth in SEQ ID NO:89 and a VL comprising, consisting of, or consisting essentially of a VL having the sequence set forth in SEQ ID NO:

99.

6. A method for treating a subject suffering from cancer, 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 HLA-G-binding antibody comprises or consists of a heavy chain variable region (VH) and a light chain variable region (VL), wherein VH and / or VL are a) a VHCDR1 having a sequence as set forth in any one of SEQ ID NO: 1 or SEQ ID NO: 11; b) a VHCDR2 having the sequence set forth in any one of SEQ ID NO: 21 or SEQ ID NO: 31; c) a VHCDR3 having a sequence as set forth in any one of SEQ ID NOs: 41; d) a VLCDR1 having a sequence as set forth in any one of SEQ ID NOs: 51; e) a VLCDR2 having a sequence as set forth in any one of SEQ ID NOs: 61; and f) The method of claim 6, comprising a VLCDR3 having a sequence as set forth in any one of SEQ ID NOs:

71.

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

91.

9. 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), wherein the VH and / or VL are: a) a VHCDR1 having the sequence set forth in SEQ ID NO: 2 to 8 or SEQ ID NO: 12 to 18; b) a VHCDR2 having a sequence as set forth in SEQ ID NO: 22-28 or SEQ ID NO: 32-38; c) a VHCDR3 having a sequence as set forth in SEQ ID NOs: 42-48; d) VLCDR1 having the sequence set forth in SEQ ID NO: 52-58; e) a VLCDR2 having the sequence set forth in SEQ ID NOs: 62-68; and The method according to any one of claims 6 to 8, comprising: f) a VLCDR3 having a sequence as set forth in SEQ ID NO: 72 to 78.

10. 10. The method of any one of claims 6-9, 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), wherein the VH and / or VL comprises, consists of, or consists essentially of a VH having a sequence set forth in SEQ ID NO:82-88, and the VL comprises, consists of, or consists essentially of a VL having a sequence set forth in SEQ ID NO:92-98.

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

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

71.

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

91.

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

80.

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

100.

16. 10. A pharmaceutical composition for use in a method according to 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 blood cancer.

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

20. The pharmaceutical composition is for administering to the subject: a) inhibition of immunosuppression; b) reducing the levels 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) increased phagocytic activity; e) inhibition of metastasis; f) inhibition of tumor growth; g) induction of tumor regression, and / or h) ILT2 ＋ CD8 ＋ 16. The method of any one of claims 1-15, wherein the antibody is administered in an amount sufficient to achieve one, two, three, four, five, six, seven, or eight of the following: enhanced T cell degranulation.

21. 21. The method of claim 20, wherein the increased phagocytic activity comprises an increased percent phagocytosis compared to a pharmaceutical composition that does not contain an antibody or a pharmaceutical composition that contains an antibody that binds to HLA-G.

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

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