CD47 and PD-L1 specific antibodies

Abstract

To provide antibodies that bind specifically to CD47 and PD-L1, a nucleic acid that codes for the antibody or an antigen-binding fragment thereof, an expression vector, a method of producing the antibody, and the use of the antibodies and compositions in cancer treatment.SOLUTION: The present invention provides a monoclonal antibody, which specifically binds to CD47 and PD-L1, comprising one binding site to CD47, and at least one binding site to PD-L1.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to the field of biotechnology, in particular to antibodies or antigen-binding fragments thereof and their uses. More specifically, the present invention relates to antibodies that specifically bind to CD47 and PD-L1. The present invention also relates to nucleic acids encoding said antibodies or antigen-binding fragments thereof, expression vectors, methods for obtaining said antibodies, and the use of said antibodies and compositions in cancer therapy. [Background technology]

[0002] The provision of two separate signals to T cells is a widespread model of lymphocytic activation of T lymphocytes remaining on antigen-presenting cells (APCs). This model is sufficient to provide self-from-nonself discrimination and immune tolerance. The primary or antigen-specific signal is delivered through the T cell receptor (TCR) after recognition of foreign antigenic peptides presented in the context of major histocompatibility complex (MHC). The secondary or costimulatory signal is delivered to T cells by costimulatory molecules expressed on antigen-presenting cells (APCs) and induces T cells to stimulate clonal expansion, cytokine secretion and effector functions. In the absence of costimulation, T cells may become immune to antigenic stimulation, which may trigger an effective immune response, and this may further lead to exhaustion or tolerance to foreign antigens.

[0003] In the two-signal model, T cells receive both signals: positive and negative secondary costimulatory signals. The control of these positive and negative signals is crucial to maximize the host's protective immune response while maintaining immune tolerance and preventing autoimmunity. The negative secondary signal appears to be necessary for T cell tolerance induction, while the positive signal stimulates T cell activation. Although the simple two-signal model still provides a valid explanation for naive lymphocytes, immune responses are dynamic processes, and costimulatory signals for antigen-exposed T cells can also be provided. The mechanism of costimulation is of interest from a therapeutic perspective, since it has been shown that manipulating costimulatory signals provides a means to either enhance or terminate immune responses. In recent years, it has been found that T cell dysfunction or anergy occurs concomitantly with the induction and persistence of the inhibitory receptor, programmed cell death polypeptide 1 (PD-1). As a result, therapeutic targeting of PD-1, and other molecules that signal through interactions with PD-1, such as programmed cell death ligand 1 (PD-L1) or programmed cell death ligand 2 (PD-L2), is an area of ​​considerable interest.

[0004] PD-L1 is overexpressed in multiple malignancies and is often associated with poor prognosis. Interestingly, the majority of tumor-infiltrating T lymphocytes express predominantly PD-1, in contrast to T lymphocytes in normal tissues and peripheral blood T lymphocytes, indicating that positive regulation of PD-1 on tumor-reactive T cells may contribute to impaired immune response. This may be due to the exploitation of the PD-L1 signaling pathway mediated by tumor cells expressing PD-L1 and interacting with PD-1-expressing T cells, with an overall attenuation of T cell activation and evasion of immune surveillance. Thus, inhibition of PD-L1 / PD-1 interaction may enhance CD8+ T cell-mediated killing of tumors.

[0005] Therapeutic targeting of PD-1, as well as other molecules that signal through interactions with PD-1, such as PD-L1 and PD-L2, is an area of ​​great interest. Blockade of PD-L1 signaling has been suggested as a means of increasing T cell immunity (e.g., antitumor immunity) for the treatment of cancer and infections, including acute and chronic infections. Inhibitors that block the PD-L1 / PD-1 interaction are known, inter alia, from WO2001014557, WO2002086083, WO2007005874, WO2010036959, WO2010077634 and WO2011066389. However, optimal therapeutic agents targeting this pathway have not yet been commercialized, and this represents a significant unmet medical need.

[0006] CD47 is a cell surface glycoprotein that binds to SIRPα (also known as SHPS-1) and SIRPγ on corresponding cells. This interaction may lead to negative regulation of immune cell function or mediate cell adhesion and migration. The use of CD47 as a biological agent in the treatment of autoimmune disorders (WO1999 / 040940) has been proposed. In contrast, there is very little data on the possible use of CD47 ligands, such as SIRPα, for similar therapeutic purposes. One explanation is that CD47 is ubiquitously expressed, which may hinder the use of CD47-binding polypeptides as potential drugs. Data published by Yu et al. (J Invest Dermatol, 126:797-807 (2006)) suggest that a fusion protein consisting of the extracellular domain of SIRPα fused to an immunoglobulin Fc domain can prevent migration of skin-derived dendritic cells (DCs) to draining lymph nodes in mice, and thereby attenuate (at least partially) contact hypersensitivity responses in mice. DC migration and function are important for immune or inflammatory responses. In painful conditions, these exacerbated DC responses can lead to the maintenance of disease. Interfering with migration of pathogenic DCs from tissues to lymphoid organs would be an attractive opportunity to stop the vicious cycle that drives autoimmune or inflammatory diseases.

[0007] CD47, also known as integrin-associated protein (IAP), ovarian cancer antigen OA3, Rh-associated antigen and MER6, is a transmembrane receptor that spans the membrane several times and belongs to the immunoglobulin superfamily. CD47 expression and / or activity have been observed in several diseases and disorders. Therefore, there is a need for CD47-targeting therapies. Furthermore, due to the expression of CD47 on platelets, there is also a need for CD47-targeting therapies (e.g., antibodies) that do not cause significant levels of platelet depletion, hemagglutination, red blood cell depletion, and / or anemia when administered to subjects.

[0008] Known antibodies that inhibit the interaction between CD47 and SIRPα ligand have been described in the following sources: applications, WO2014123580, WO2013119714, WO2015191861, WO2011143624, WO / 2014 / 093678, WO2017053423.

[0009] Various sources describing multispecific antibodies are also known, for example WO / 2014 / 087248 describes bispecific antibodies specific for CD47 and CD19, and WO2016023001 describes bispecific antibodies specific for CD47 and PD1. However, the production and possible effective use of multispecific antibodies that specifically bind to CD47 and PD-L1 has not been described. [Prior art documents] [Patent documents]

[0010] [Patent Document 1] WO2001014557 [Patent Document 2] WO2002086083 [Patent Document 3] WO2007005874 [Patent Document 4] WO2010036959 [Patent Document 5] WO2010077634 [Patent Document 6] WO2011066389 [Patent Document 7] WO1999 / 040940 [Patent Document 8] WO2014123580 [Patent Document 9] WO2013119714 [Patent Document 10] WO2015191861 [Patent Document 11] WO2011143624 [Patent Document 12] WO / 2014 / 093678 [Patent Document 13] WO2017053423 [Patent Document 14] WO / 2014 / 087248 [Patent Document 15] WO2016023001 [Non-patent literature]

[0011] [Non-Patent Document 1] Yu et al. (J Invest Dermatol, 126:797-807(2006) Summary of the Invention [Problem to be solved by the invention]

[0012] In this regard, the generation of novel antibodies that effectively bind to CD47 and PD-L1 is pertinent. [Means for solving the problem]

[0013] The present invention relates to binding molecules, such as antibodies, directed to bind to CD47 and PD-L1. Such antibodies may be used to treat diseases or disorders mediated by CD47 and PD-L1.

[0014] In one aspect, the invention relates to a monoclonal antibody that specifically binds to CD47 and PD-L1 and contains one binding site for CD47 and at least one binding site for PD-L1.

[0015] In some embodiments, the antibodies of the present invention are full-length antibodies or antigen-binding fragments thereof. In some embodiments, the antibodies of the invention comprise one or two binding sites for PD-L1.

[0016] In some embodiments, the binding site of the antibody of the invention for CD47 inhibits the interaction between the CD47 receptor and a SIRPα ligand, and / or the binding site for PD-L1 inhibits the interaction between PD-L1 and the PD-1 receptor.

[0017] In some embodiments, the binding site of the antibody of the present invention to CD47 comprises a heavy chain variable domain comprising CDR1, CDR2 and CDR3 sequences, wherein CDR1 is a sequence that is at least 80% homologous to a sequence selected from the group consisting of SEQ ID NOs: 1 to 4 below, i.e., CDR1 is a sequence selected from the group consisting of SEQ ID NOs: 1 to 4, or a sequence selected from the group consisting of SEQ ID NOs: 1 to 4 containing one or two substitutions, CDR2 is a sequence that is at least 80% homologous to a sequence selected from the group consisting of SEQ ID NOs: 6 to 15 below, i.e., CDR2 is a sequence selected from the group consisting of SEQ ID NOs: 6 to 15, or a sequence selected from the group consisting of SEQ ID NOs: 6 to 15 containing one, two, three, four or five substitutions, and CDR3 is a sequence that is at least 80% homologous to a sequence selected from the group consisting of SEQ ID NOs: 17 to 20 below, i.e., CDR3 is a sequence selected from the group consisting of SEQ ID NOs: 17 to 20, or a sequence selected from the group consisting of SEQ ID NOs: 17 to 20 containing one, two or three substitutions.

[0018] In some embodiments, the CD47-binding site of the antibody of the present invention comprises a heavy chain variable domain comprising CDR1, CDR2, and CDR3 sequences, wherein CDR1 is a sequence selected from the group consisting of SEQ ID NOs: 1 to 4 below, CDR2 is a sequence selected from the group consisting of SEQ ID NOs: 6 to 15 below, and CDR3 is a sequence selected from the group consisting of SEQ ID NOs: 17 to 20 below.

[0019] In some embodiments, the CD47-binding site of the antibody of the present invention comprises a heavy chain variable domain of claim 4, and a light chain variable domain comprising CDR1, CDR2 and CDR3 sequences, wherein CDR1 is a sequence that is at least 80% homologous to a sequence selected from the group of SEQ ID NOs: 22 to 34 below, i.e., CDR1 is a sequence selected from the group of SEQ ID NOs: 22 to 34, or a sequence selected from the group of SEQ ID NOs: 22 to 34 containing one or two substitutions, CDR2 is a sequence that is at least 80% homologous to a sequence selected from the group of SEQ ID NOs: 36 to 48 below, i.e., CDR2 is a sequence selected from the group including SEQ ID NOs: 36 to 48, or a sequence selected from the group of SEQ ID NOs: 36 to 48 containing one, two or three substitutions, and CDR3 is a sequence that is at least 80% homologous to a sequence selected from the group of SEQ ID NOs: 50 to 64 below, i.e., CDR3 is a sequence selected from the group of SEQ ID NOs: 50 to 64, or a sequence selected from the group of SEQ ID NOs: 50 to 64 containing one or two substitutions.

[0020] In some embodiments, the binding site of the antibody of the present invention to CD47 comprises a heavy chain variable domain of claim 4 and a light chain variable domain comprising CDR1, CDR2 and CDR3 sequences, wherein CDR1 is a sequence selected from the group consisting of SEQ ID NOs: 22 to 34 below, CDR2 is a sequence selected from the group consisting of SEQ ID NOs: 36 to 48 below, and CDR3 is a sequence selected from the group consisting of SEQ ID NOs: 50 to 64 below.

[0021] In some embodiments, the binding site for CD47 of the antibody of the present invention comprises a heavy chain variable domain comprising a sequence that is at least 90% homologous to a sequence selected from the group consisting of SEQ ID NOs: 66 to 88 below, and a light chain variable domain comprising a sequence that is at least 90% homologous to a sequence selected from the group consisting of SEQ ID NOs: 89 to 106 below.

[0022] In some embodiments, the binding site of the antibody of the present invention to CD47 comprises a heavy chain variable domain comprising a sequence selected from the group consisting of SEQ ID NOs: 66 to 88, and a light chain variable domain comprising a sequence selected from the group consisting of SEQ ID NOs: 89 to 106.

[0023] In some embodiments, the binding site of the antibodies of the invention to PD-L1 comprises a heavy chain variable domain comprising a sequence that is at least 80% homologous to the following sequences: SEQ ID NO:5, SEQ ID NO:16, and SEQ ID NO:21, i.e. comprising the amino acid sequences of SEQ ID NO:5, 16 and 21, or comprising the amino acid sequence of SEQ ID NO:5 with one substitution, SEQ ID NO:16 with one, two or three substitutions, SEQ ID NO:21 with one, two or three substitutions, and a light chain variable domain comprising a sequence that is at least 80% homologous to the following sequences: SEQ ID NO:35, SEQ ID NO:49, and SEQ ID NO:65, i.e. comprising the amino acid sequence of SEQ ID NO:35, 49 and 65, or comprising the amino acid sequence of SEQ ID NO:35 with one, two or three substitutions, SEQ ID NO:49 with one substitution, SEQ ID NO:65 with one or two substitutions.

[0024] In some embodiments, the binding site of the antibodies of the invention to PD-L1 comprises a heavy chain variable domain comprising the following sequences: SEQ ID NO:5, SEQ ID NO:16, and SEQ ID NO:21, and a light chain variable domain comprising the following sequences: SEQ ID NO:35, SEQ ID NO:49, and SEQ ID NO:65.

[0025] In some embodiments, the binding site of the antibody of the present invention to CD47 is Fab, sc It may be an Fv, scFab or an isolated VH or VHH monodomain. In some embodiments, the binding portion of the antibody of the invention to PD-L1 is a Fab, scFv, scFab, or an isolated VH or VHH monodomain.

[0026] In some embodiments, the antibodies of the invention are characterized by stimulating antibody-dependent cellular cytotoxicity, macrophage-mediated phagocytosis, and / or T-cell mediated cytotoxicity in a percentage of cells bearing CD47 and / or PD-L1 antigens on their surface.

[0027] In some embodiments, the antibodies of the present invention are characterized in that they comprise an Fc portion that contains at least one mutation or modification that increases antibody-dependent cell-mediated cytotoxicity (ADCC) when compared to the same antibody that does not contain that mutation or modification.

[0028] In some embodiments, the antibodies of the present invention are intended to be used as pharmaceuticals for the treatment of cancer. In one aspect, the invention relates to a nucleic acid encoding any of the above antibodies.

[0029] In some embodiments, the nucleic acid of the invention is DNA. In one aspect, the present invention relates to an expression vector comprising the above-mentioned nucleic acid. In one aspect, the invention relates to a method for obtaining a host cell for preparing any of the above antibodies, comprising transformation of the cell with a vector of the invention.

[0030] In one aspect, the invention relates to a host cell for obtaining any of the above antibodies, comprising the above nucleic acid. In one aspect, the invention relates to a method for obtaining any of the above antibodies, comprising culturing a host cell in a culture medium under conditions sufficient to obtain a specified antibody, and then, if necessary, isolating and purifying the antibody obtained.

[0031] In one aspect, the invention relates to a pharmaceutical composition for the prevention or treatment of a disease or disorder mediated by PD-L1 and CD47, comprising any of the above-mentioned antibodies, in combination with one or several pharma- ceutically acceptable excipients.

[0032] In some embodiments, the pharmaceutical compositions of the present invention are for use in treating or treating cancer including head and neck squamous cell carcinoma (HNSCC), cervical cancer, cancer of unknown primary, glioblastoma, esophageal cancer, bladder cancer, TNBC (triple negative breast cancer), CRC (colorectal cancer), hepatocellular carcinoma, melanoma, NSCLC (non-small cell lung cancer), renal cancer, ovarian cancer, MSI It is intended for the prevention or treatment of a disease or disorder mediated by PD-L1 and CD47 selected from the group of CRC (colorectal cancer with microsatellite instability), leukemia (acute leukemia or myeloblastic leukemia), lymphoma, multiple myeloma, breast cancer, prostate cancer, sarcoma, hepatocellular carcinoma, Hodgkin's lymphoma, T and B cell acute lymphoblastic leukemia, small cell lung cancer, acute myeloblastic leukemia, refractory non-Hodgkin's B cell lymphoma, follicular lymphoma, marginal zone B cell lymphoma, diffuse large B cell lymphoma, pancreatic cancer, and high-risk myelodysplastic syndromes.

[0033] In one aspect, the invention relates to a method for treating a disease or disorder mediated by PD-L1 and CD47, comprising administering to a subject in need of such treatment a therapeutically effective amount of any of the above-described antibodies or pharmaceutical compositions of the invention.

[0034] In some embodiments of the methods for treatment of the present invention, the disease or disorder is head and neck squamous cell carcinoma (HNSCC), cervical cancer, cancer of unknown primary, glioblastoma, esophageal cancer, bladder cancer, TN Selected from the group of BC (triple negative breast cancer), CRC (colorectal cancer), hepatocellular carcinoma, melanoma, NSCLC (non-small cell lung cancer), renal cancer, ovarian cancer, MSI CRC (colorectal cancer with microsatellite instability), leukemia (acute leukemia or myeloblastic leukemia), lymphoma, multiple myeloma, breast cancer, prostate cancer, bladder cancer, sarcoma, hepatocellular carcinoma, glioblastoma, Hodgkin's lymphoma, T and B cell acute lymphoblastic leukemia, small cell lung cancer, acute myeloblastic leukemia, refractory non-Hodgkin's B cell lymphoma, follicular lymphoma, marginal zone B cell lymphoma, diffuse large B cell lymphoma, pancreatic cancer, ovarian cancer, and high-risk myelodysplastic syndrome.

[0035] In one aspect, the invention relates to a method for inhibiting the biological activity of PD-L1 and / or CD47 in a subject in need of such inhibition, comprising administering an effective amount of any of the above-described antibodies.

[0036] In one aspect, the invention relates to the use of any of the above antibodies or pharmaceutical compositions for the treatment of a disease or disorder mediated by PD-L1 and CD47 in a subject in need of such treatment.

[0037] In some embodiments of the use of the antibodies of the present invention, the disease or disorder is selected from the group consisting of head and neck squamous cell carcinoma (HNSCC), cervical cancer, cancer of unknown primary, glioblastoma, esophageal cancer, bladder cancer, TNBC (triple negative breast cancer), CRC (colorectal cancer), hepatocellular carcinoma, melanoma, NSCLC (non-small cell lung cancer), renal cancer, ovarian cancer, MSI, Selected from the group of CRC (colorectal cancer with microsatellite instability), leukemia (acute leukemia or myeloblastic leukemia), lymphoma, multiple myeloma, breast cancer, prostate cancer, bladder cancer, sarcoma, hepatocellular carcinoma, glioblastoma, Hodgkin's lymphoma, T and B cell acute lymphoblastic leukemia, small cell lung cancer, acute myeloblastic leukemia, refractory non-Hodgkin's B cell lymphoma, follicular lymphoma, marginal zone B cell lymphoma, diffuse large B cell lymphoma, pancreatic cancer, ovarian cancer, and high-risk myelodysplastic syndrome. [Brief description of the drawings]

[0038] [Figure 1] Plasmid map for transient production of human CD47-Fc in mammalian cell CHO-K1 culture. [Diagram 2] SDS-gel electrophoresis under non-reducing conditions of preparations of human CD47-Fc. [Diagram 3] SDS-gel electrophoresis under reducing conditions of control anti-CD47 antibody B6H12 product preparation. [Figure 4] SDS-gel electrophoresis under non-reducing conditions of a control anti-CD47 antibody B6H12 product preparation. [Diagram 5] Diagram of ELISA of polyclonal phages carrying VHH antibody fragments that specifically interact with human CD47 antigen. [Figure 6] Schematic diagram of the domain structure of anti-PD-L1 / anti-CD47 bispecific antibodies, where A is based on the anti-CD47 scFv fragment and B is based on the anti-CD47 VHH fragment, while the PD-L1 binding portion is represented by the Fab fragment. [Figure 7] SDS-gel electrophoresis under non-reducing conditions of an anti-PD-L1 / anti-CD47 preparation of anti-CD47 scFv fragment-based bispecific antibody. [Figure 8] SDS-gel electrophoresis under reducing conditions of anti-PD-L1 / anti-CD47 bispecific antibody preparations based on anti-CD47 VHH fragments. [Figure 9] Dependence of cytotoxic effect on the concentration of the studied anti-PD-L1 / anti-CD47 bispecific antibodies. [Figure 10] Dependence of cytotoxic effect on the concentration of the studied anti-PD-L1 / anti-CD47 bispecific antibodies. [Figure 11] Efficacy of phagocytosis of MDA-MB-231 cell line by human macrophages in the presence of anti-PD-L1 / anti-CD47 bispecific antibody. [Figure 12] Dependence of the level of fluorescence on the concentration of anti-PD-L1 / anti-CD47 bispecific antibody. [Figure 13]Dependence of the level of fluorescence on the concentration of anti-PD-L1 / anti-CD47 bispecific antibody. [Figure 14] Dependence of the level of fluorescence on the concentration of anti-PD-L1 / anti-CD47 bispecific antibody. [Figure 15] Dependence of the level of fluorescence on the concentration of anti-PD-L1 / anti-CD47 bispecific antibody. [Figure 16] Anti-PD-L1 activity of anti-PD-L1 / anti-CD47 bispecific antibodies. The vertical axis shows the ratio of luminescence from wells containing the aHTH-CD47 / PD-L1 antibodies tested to the luminescence from wells without added antibody. [Figure 17] Gel filtration profile to assess aggregation homogeneity of anti-PD-L1 / anti-CD47 bispecific antibodies. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0039] Definitions and general methods Unless otherwise defined herein, scientific and technical terms used in connection with the present invention will have the meanings that are commonly understood by those of ordinary skill in the art.

[0040] Further, unless otherwise required by context, singular terms shall include the plural and plural terms shall include the singular. Typically, the classification and methods of cell culture, molecular biology, immunology, microbiology, genetics, analytical chemistry, synthetic organic chemistry, medical and pharmaceutical chemistry, and protein and nucleic acid hybridization and chemistry described herein are well known and widely used by those of skill in the art. Enzymatic reactions and purification methods are performed according to manufacturer's instructions as common in the art or as described herein.

[0041] Antibody Related Definitions PD-L1 (Programmed Death Ligand 1), also known as Cluster of Differentiation 274 (CD274) or B7 Homolog 1 (B7-H1), is a 40 kDa type 1 transmembrane protein. PD-L1 consists of three domains: an extracellular domain (220), a transmembrane domain (21) and an intracellular domain (31), designated as IgV and C-type domains. The molecule plays an important role in suppressing the immune system during pregnancy, during transplantation of foreign tissues, and in certain diseases, such as hepatitis. Under normal conditions, in response to self-antigens, a certain amount of antigen-specific CD8+ T effector cells accumulate in lymph nodes and spleen, and to prevent autoimmune processes, PD-1 / PD-L1 or B7-1 / PD-L1 complexes are formed, resulting in the transmission of inhibitory signals that reduce the proliferation of these CD8+ T cells in lymph nodes. Thus, PD-1 / PD-L1 interaction is one of the key factors in the development of immune tolerance.

[0042] CD47 is a multi-transmembrane receptor belonging to the immunoglobulin superfamily, which interacts with SIRPα (signal regulatory protein α) on macrophages, thereby inhibiting phagocytosis. Cancer cells in which this pathway is active avoid phagocytosis. Therefore, therapeutic effects against CD47 are widely used in various cancers. Antibodies against CD47 may or may not have the ability to block the interaction between CD47 and SIRPα.

[0043] The term "binding molecule" includes antibodies and immunoglobulins. The term "antibody" or "immunoglobulin" or "monoclonal antibody" or "bispecific antibody" "Heterospecific antibodies" or "multispecific antibodies" (Ig), as used herein, include complete / full-length antibodies and any antigen-binding fragments (i.e., "antigen-binding portions"). Further, for example, the term "antibody" or "immunoglobulin" or "monoclonal antibody" includes any combination of antigen-binding fragments with one or more valencies and one or more specificities, as well as constant regions of immunoglobulins, and such terms may have a similar meaning to the term "bispecific antibodies" or "multispecific antibodies". Further, for example, the term "antibody" or "immunoglobulin" or "monoclonal antibody" includes any combination of antigen-binding fragments and constant regions of immunoglobulins, covalently or non-covalently bound to any polypeptide of any nature. Furthermore, the term "antibody" refers to a glycoprotein, or antigen-binding portion, comprising at least two heavy (H) chains and two light (L) chains interconnected by, for example, disulfide bonds. Each heavy chain comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. There are five known types of mammalian Ig heavy chains, designated by the Greek letters: α, δ, ε, γ, and μ. The type of heavy chain present defines the antibody class: these chains are found in IgA, IgD, IgE, IgG, and IgM antibodies, respectively. The different heavy chains differ in size and composition; α and γ contain approximately 450 amino acids, while μ and ε consist of approximately 550 amino acids. Each heavy chain contains two regions: a constant region and a variable region. The constant region is identical in all antibodies of the same isotype, but differs in antibodies of different isotypes. The heavy chains γ, α, and δ contain a constant region, made up of three constant domains, CH1, CH2, and CH3 (in a row), as well as a hinge region to add flexibility (Woof J., Burton D., Nat Rev Immunol 4, 2004, cc.89-99); the heavy chains μ and ε have a constant region composed of four constant domains, CH1, CH2, CH3 and CH4. In mammals, only two types of light chains are known, designated lambda (λ) and kappa (κ). Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The approximate length of the light chain is 211-217 amino acids. Preferably, the light chain is a kappa (κ) light chain, and the constant domain CL is preferably C kappa (κ).

[0044] An "antibody" of the present invention may be of any class (e.g., IgA, IgD, IgE, IgG, and IgM, preferably IgG) or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2, preferably IgG1).

[0045] The VL and VH regions may be further divided into hypervariable regions, termed complementarity determining regions (CDRs), interspersed between more conserved regions, termed framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, located from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of the antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.

[0046] The term "antigen-binding portion" or "antigen-binding fragment" of an antibody (or simply "antibody portion" or "antibody fragment"), as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody include: (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab')2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) an Fd fragment consisting of the VH and CH1 domains; (iv) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (v) a dAb fragment consisting of the VH / VHH domains (Ward et al. (1989) Nature 1999). 341:544-546); and (vi) extracted complementarity determining regions (CDRs). Furthermore, the two regions of the Fv fragment, VL and VH, are encoded by different genes and may be linked using recombinant methods with a synthetic linker that allows the VL and VH regions to pair to form a monovalent molecule and accept a single protein chain (known as single-chain Fv (scFv); see, for example, Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single-chain molecules are also assumed to be included within the term "antigen-binding portion" of an antibody. Such antibody fragments are obtained using conventional methods known to those skilled in the art, and these fragments are screened in the same manner as intact antibodies.

[0047] Preferably, the CDRs of the antigen binding regions or full length antibody antigen binding regions of the invention are derived from mouse, llama or human donor libraries or are essentially human in origin, with specific amino acid residues modified, e.g. replaced with different amino acid residues, to optimize certain antibody properties, e.g. KD, koff, IC50, EC50, ED50. Preferably, the framework regions of the antibodies of the invention are of human origin or substantially human origin (at least 80, 85, 90, 95, 96, 97, 98 or 99% human origin).

[0048] In other embodiments, the antigen-binding portions of the invention may be derived from other non-human species, including, but not limited to, mouse, llama, rabbit, rat or hamster. Alternatively, the antigen-binding region may be derived from a human species.

[0049] The term "variable domain" refers to the fact that certain regions of the variable domain are highly variable in sequence among antibodies. The V domain mediates antigen binding and determines the specificity of a particular antibody for its particular antigen. However, the variability is distributed unevenly over the 110 amino acid variable domain sites. Instead, the V region consists of invariant segments of 15-30 amino acids called framework regions (FRs) separated by shorter regions of hypervariability called "hypervariable regions" or CDRs. Each of the variable domains of native heavy and light chains each contains four FRs, which are primarily in a beta-sheet configuration and are connected by three hypervariable regions that form loops that connect, and in some cases form part of, the beta-sheet structure. The hypervariable regions in each chain are held together in close proximity by the FRs and, together with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of the antibody. The constant domains are not directly involved in the binding of the antibody to the antigen, but exhibit various effector functions, such as the participation of the antibody in antibody-dependent cellular cytotoxicity (ADCC).

[0050] The term "hypervariable region" as used herein refers to the amino acid residues of an antibody which are involved in antigen binding. Hypervariable regions generally comprise amino acid residues from the "complementarity determining regions" or "CDRs" and / or residues from the "hypervariable loops".

[0051] In certain cases, it may also be desirable to modify one or more CDR amino acid residues to improve binding affinity to the target epitope. This is known as "affinity maturation" and may optionally be performed in conjunction with humanization, for example in situations where humanization of an antibody leads to a decrease in binding specificity or affinity and back mutation alone is not sufficient to improve binding specificity or affinity. A variety of affinity maturation methods are known in the art, such as in vitro scanning saturation mutagenesis as described by Burks et al., Proc Natl Acad Sci USA, 94:412-17 (1997), and Wu et al., Proc Natl Acad Sci USA 95:6037 6042(199 8) has proposed a stepwise in vitro affinity maturation method.

[0052] "Framework Regions" (FR) are those residues of the variable domains that are distinct from the CDR residues. Each variable domain typically has four FRs identified as FR1, FR2, FR3 and FR4. When the CDRs are defined according to Kabat, the FR light chain residues are located at approximately residues 1-23 (LCFR1), 35-49 (LCFR2), 57-88 (LCFR3), and 98-107 (LCFR4), and the heavy chain FR residues are located in the heavy chain in the region of approximately residues 1-30 (HCFR1), 36-49 (HCFR2), 66-94 (HCFR3), and 103-113 (HCFR4). When the CDRs contain amino acid residues from the hypervariable loops, the FR light chain residues are located at approximately residues 1-25 (LCFR1), 33-49 (LCFR2), 53-90 (LCFR3), and 97-107 (LCFR4) in the light chain, and the heavy chain FR residues are located at approximately residues 1-25 (HCFR1), 33-52 (HCFR2), 56-95 (HCFR3), and 102-113 (HCFR4) in the heavy chain. In some cases where the CDRs contain amino acids from both the CDRs and those of the hypervariable loops as defined by Kabat, the FR residues will be adjusted accordingly. For example, if CDRH1 contains amino acids H26-H35, the FR1 residues of the heavy chain are positions 1-25 and the FR2 residues are positions 36-49.

[0053] The antibody of the present invention that "binds" to a target antigen is an antibody that binds to the antigen with sufficient affinity, so that it may be used as a diagnostic and / or therapeutic agent in targeting proteins or cells or tissues that express the antigen, and cross-reacts to a small extent with other proteins. In such embodiments, the degree of antibody binding to non-target proteins is less than 10% of that of antibody binding to specific target proteins, according to analytical methods: fluorescence activated cell sorting (FACS), radioimmunoassay (RIA) or ELISA. With respect to the binding of an antibody to a target molecule, the term "specific binding" or "specifically binds" or "is specific" to a particular polypeptide or epitope on a particular polypeptide target means binding that is significantly (measurably) different from non-specific interactions (e.g., in the case of bH1-44 or bH1-81, non-specific interactions are binding to bovine serum albumin, casein, fetal bovine serum, or neutravidin).

[0054] For example, specific binding may be measured by determining the binding of a molecule compared to the binding of a control molecule. For example, specific binding may be determined by competition with another molecule similar to the target, for example with excess unlabeled target. In this case, specific binding is indicated if the binding of the labeled target to the probe is competitively inhibited by excess unlabeled target. As used herein, the term "specific binding" or "specifically binds" or "specific" for a particular polypeptide or epitope on a particular polypeptide target may be characterized by a molecule having a Kd for the target of at least about 200nM, or at least about 150nM, or at least about 100nM, or at least about 60nM, or at least about 50nM, or at least about 40nM, or at least about 30nM, or at least about 20nM, or at least about 10nM, or at least about 8nM, or at least about 6nM, or at least about 4nM, or at least about 2nM, or at least about 1nM, or more. In one embodiment, the term "specific binding" refers to binding where a molecule binds to a particular polypeptide or an epitope on a particular polypeptide without substantial binding to any other polypeptides or epitopes on polypeptides.

[0055] The term "Ka," as used herein, is intended to refer to the off-rate of a particular antibody-antigen interaction, while the term "Kd" refers to the off-rate of a particular antibody-antigen interaction. "Binding affinity" generally refers to the affinity between a molecule (e.g., an antibody) and its binding partner ( "Affinity" refers to the strength of cumulative non-covalent interactions between a single binding site of a molecule (e.g., an antigen). Unless otherwise indicated, "binding affinity" refers to the intrinsic (characteristic, true) binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its binding partner Y can usually be represented by a dissociation constant (Kd). Preferably, the Kd value is approximately 200nM, 150nM, 100nM, 60nM, 50nM, 40nM, 30nM, 20nM, 10nM, 8nM, 6nM, 4nM, 2nM, 1nM, or less. Affinity may be measured by common methods known in the art, including those described herein. Low affinity antibodies generally tend to bind slowly to antigens and dissociate easily, while high affinity antibodies generally tend to bind more quickly to antigens and remain bound longer. A variety of methods for measuring binding affinity are known in the art, and for the purposes of the present invention, any of these methods may be used.

[0056] In one embodiment of the present invention, "Kd" or "Kd value" is measured at 25°C with immobilized chip CM5 antigen in ∼10 response units (RU) using BIAcore TM The IgG antibody was measured by surface plasmon resonance assay using a BIAcore®-2000 or BIAcore®-3000 (BIAcore, Inc., Piscataway, NJ). Briefly, IgG antibody was measured using a carboxymethylated dextran biosensor chip (CM5, BIAcore, The Fab Fab 1000 (Inc.) is activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the manufacturer's instructions. The antigen is diluted to a concentration of 5 μg / ml (~0.2 μM) in 10 mM sodium acetate, pH 4.8, and then injected at a flow rate of 5 μl / min to achieve approximately 10 relative units (RU) of binding protein. After antigen administration, 1 M ethanolamine solution is injected to block unreacted groups. For kinetic measurements, twofold serial dilutions of Fab (e.g., 0.78 nM-500 nM) are injected in 0.05% Tween 20 (PBST) at 25°C and a flow rate of approximately 25 μl / min. On-rates (kon) and off-rates (koff) are calculated using a simple one-to-one Langmuir binding model (BIAcore evaluation software version 3.2) by simultaneously fitting the association and dissociation sensorgrams. The equilibrium dissociation constant (Kd) is calculated as the ratio koff / kon. See, e.g., Chen, Y. et al. (1999) J. Mol. Biol. 293:865-881. According to the surface plasmon resonance method described above, if the dissociation rate is 10 6 M -1 s -1 This may be determined by fluorescence quenching, measuring the increase or decrease in intensity of the fluorescence emission (excitation = 295 nm; emission (illumination) = 340 nM, 16 nm band) at 25° C. Antibody antigen solutions (Fab form) at a concentration of 20 nM in PBS, pH 7.2, in the presence of increasing concentrations of antigen were measured using a spectrophotometer, for example a stopped-flow spectrophotometer (Aviv Instruments) or a spectrophotometer SLM-Aminco (Thermo Spectronic) series 8000 with a stirred cuvette.

[0057] The term "koff" refers to the dissociation rate constant of a particular interaction between a binding molecule and an antigen. TM The system may be used to measure the koff dissociation rate constant by biolayer interferometry.

[0058] Additionally, at 25°C, ~10 relative units (response units, RU), a BIAcore assay was performed using a chip containing immobilized CM5 antigen. TM The "association rate" ("on rate") or "kon" of the present invention may be measured by using the above-described surface plasmon resonance assay with a BIAcore®-2000 or BIAcore®-3000 (BIAcore, Inc., Piscataway, NJ). Briefly, a carboxymethylated dextran biosensor chip (CM5, BIAcore, Inc.) is activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the manufacturer's instructions. Antigen is dissolved in 10 mM acetic acid and then diluted with 10 mM acetic acid. The antibody is diluted with sodium acetate, pH 4.8, to a concentration of 5 μg / ml (~0.2 μM) and then injected at a flow rate of 5 μl / min to achieve approximately 10 relative units (RU) of bound protein. After challenge, 1 M ethanolamine solution is injected to block unreacted groups.

[0059] Unless otherwise specified, the terms "biologically active" and "biological activity" and "biological properties" with respect to a polypeptide of the present invention means having the ability to bind to biological molecules.

[0060] The expression "biological molecule" refers to nucleic acids, proteins, carbohydrates, lipids, and combinations thereof. In one embodiment of the invention, the biological molecule is naturally occurring. Antibody fragments, such as Fab and F(ab')2 fragments, may be obtained from whole antibodies using conventional techniques, such as papain or pepsin hydrolysis of whole antibodies. Additionally, antibodies, antibody portions, and immunoadhesion molecules may be obtained using standard recombinant DNA techniques, such as those described herein.

[0061] The term "recombinant antibody" is intended to refer to an antibody that is expressed in a cell or cell line that contains a nucleotide sequence(s) encoding an antibody, where said nucleotide sequence(s) is not naturally associated with the cell.

[0062] The term "variant antibody" as used herein refers to an antibody having an amino acid sequence that differs from that of its "parent" antibody due to the addition, deletion and / or substitution of one or more amino acid residues when compared to the sequence of the parent antibody. In preferred embodiments of the invention, the variant antibody contains at least one or more (e.g., 1 to 12, e.g., 2, 3, 4, 5, 6, 7, 8 or 9, 10, 11 or 12; in some embodiments, the variant antibody contains from 1 to about 10) additions, deletions, and / or substitutions of amino acids when compared to the parent antibody. In some embodiments, such additions, deletions and / or substitutions are made in the CDRs of the variant antibody. Identity and homology with respect to the sequence of a variant antibody is defined herein as the percentage of amino acid residues in the variant antibody sequence that are identical to those of the parent antibody after aligning the sequences and introducing gaps if necessary to achieve the maximum percentage of sequence identity. A variant antibody retains the ability to bind to the same antigen, and preferably to the same epitope, as the parent antibody; and in some embodiments, at least one property or biological activity is superior to that of the parent antibody. For example, a variant antibody may have, for example, a greater binding affinity, a longer half-life, a lower IC50, or an enhanced ability to inhibit antigen biological activity, when compared to the parent antibody. Of particular interest in this document are variant antibodies that exhibit biological activity at least 2-fold (preferably at least 5-fold, 10-fold, or 20-fold) higher than the biological activity of the parent antibody.

[0063] The term "bispecific antibody" refers to an antibody that contains an antigen-binding domain or domains that are capable of specifically binding to two different epitopes on one biological molecule or that are capable of specifically binding to epitopes on two different biological molecules. Bispecific antibodies are also referred to herein as having "dual specificity" or as being "bispecific" antibodies.

[0064] In a broad sense, the term "chimeric antibody" refers to an antibody that contains one or more regions of one antibody and one or more regions of one or several other antibodies, typically an antibody that is partly human and partly non-human, i.e. partly derived from a non-human animal, such as mouse, rat or similar vermin, or from the Camelidae family, such as llamas and alpacas. Human anti-antibody immune response, Chimeric antibodies are generally preferred over non-human antibodies, for example to reduce the risk of human anti-mouse antibody immune responses in the case of murine antibodies. A typical example of a chimeric antibody is one in which the variable region sequences are murine sequences, while the constant region sequences are human. In the case of chimeric antibodies, the non-human portions may be further modified to humanize the antibody.

[0065] The term "humanization" refers to the fact that when an antibody has a completely or partially non-human origin, for example a mouse or llama antibody obtained by immunizing a mouse or llama with an antigen of interest, or a chimeric antibody based on such a mouse or llama antibody, it is possible to replace certain amino acids, particularly in the framework regions and constant domains of the heavy and light chains, in order to avoid or minimize immune reactions in humans. The specificity of the antibody interaction with the target antigen is mainly through the amino acid residues located in the six heavy and light chain CDRs. For this reason, the amino acid sequences within the CDRs are much more diverse between individual antibodies than those outside the CDRs. Since the CDR sequences of said sites are involved in the majority of the antibody-antigen interactions, recombinant antibodies that mimic the properties of a specific natural antibody, or more generally, a specific antibody that contains a given amino acid sequence, may be expressed, for example, by constructing an expression vector that expresses the CDR sequences of plots of a specific antibody and the framework sequences of another antibody. As a result, it is possible to "humanize" a non-human antibody and to a large extent retain the binding specificity and affinity of the original antibody. Although it is not possible to accurately predict the immunogenicity, and therefore the human anti-antibody response, of a particular antibody, non-human antibodies are typically more immunogenic than human antibodies. Chimeric antibodies, in which foreign (e.g. vermin or camelid) constant regions are replaced by sequences of human origin, generally show less immunogenicity than antibodies of completely foreign origin, and in therapeutic antibodies there is a trend to use humanized or fully human antibodies. Chimeric or other antibodies of non-human origin may therefore be humanized to reduce the risk of human anti-antibody responses.

[0066] For chimeric antibodies, humanization typically involves modification of the framework regions of the variable region sequences. Amino acid residues that are part of the complementarity determining regions (CDRs) are not modified for humanization in most cases, but in some cases modification may be desirable to modify individual amino acid residues of the CDRs, for example to remove glycosylation sites, deamidation sites, aspartic acid isomerization sites, or undesirable cysteine ​​or methionine residues. N-linked glycosylation occurs by attachment of an oligosaccharide chain to an asparagine residue in the tripeptide sequence Asn-X-Ser or Asn-X-Thr, where X may be any amino acid except Pro. Removal of N-glycosylation sites may be achieved by mutating either the Asn or Ser / Thr residue with another residue, preferably by conservative substitution. Deamidation of asparagine and glutamine residues may occur depending on factors such as pH and surface exposure. Asparagine residues are particularly susceptible to deamidation, especially when present in Asn-Gly sequences, and to a lesser extent in other dipeptide sequences, such as Asn-Ala. In the presence of such deamidated regions, such as Asn-Gly in sequences of CDR regions, it may be preferable to remove the region, generally by a conservative substitution that removes one of the involved residues.

[0067] Many methods for humanizing antibody sequences are known in the art. One commonly used method is CDR site grafting. CDR grafting may be based on the Kabat CDR definition, but the final version (Magdelaine-Beuzelin et al., Crit Rev. Oncol Hematol. 64:210 225 (2007)) suggests that the IMGT® (international ImMunoGeneTics information system®, www.imgt.org) definition may better improve humanization results (Lefranc et al., Dev. Comp Immunol. 27:55-77 (2003). In some cases, CDR grafting can reduce the binding specificity and affinity, and therefore the biological activity, of the CDR-grafted non-human antibody when compared to the parent antibody from which the CDRs were derived. Back mutations (sometimes referred to as "framework region restoration") may be used at selected positions of the CDR-grafted antibody, typically in the framework regions, to restore the binding specificity and affinity of the parent antibody. Information available in the literature and antibody databases may be used to determine positions for possible back mutations. Amino acid residues that are candidates for back mutations are usually located on the surface of the antibody molecule, while buried or less surface-exposed residues will usually not be altered. An alternative humanization method to CDR site grafting and back mutations is surface alteration, in which the non-exposed residues of the non-human origin are retained, while surface-exposed residues are altered to human residues.

[0068] There are two techniques for producing fully human antibodies: using in vitro harvested phage libraries or by immunization of humanized animals (mouse, rat, etc.). Phage display is the first and most widely used in vitro antibody screening technique. In 1985, Smith found that foreign DNA sequences can be cloned into the filamentous bacteriophage M13 and expressed on the surface of the phage particle as fusion proteins (Smith GP: Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface. Science 1985, 228:1315-1317.). Thus, fusion proteins of interest can be selected based on their ability to bind to other proteins. This discovery, combined with PCR amplification techniques, allows the cDNA repertoire of immunoglobulin genes to be cloned to generate diverse phage libraries containing variable domains, which can be used to rapidly screen target-specific monoclonal antibodies. The phage library repertoire reflects the B cell antibody repertoire of each human or animal whose blood was used to generate the library. In 1995, two papers reported the generation of genetically engineered mice expressing a fully human antibody repertoire comparable to that produced by hybridoma technology (Lonberg N, Taylor LD, Harding FA, Trounstine M, Higgins KM, Schramm SR, Kuo CC, Mashayekh R, Wymore K, McCabe JG, et al.: Antigen-specific human antibodies from mice comprising four distinct genetic modifications. Nature 1994, 368:856-859). In these animals, their own endogenous heavy and kappa light immunoglobulin chain genes were purposefully disrupted and then transgenes that were segments of human heavy and kappa light chain genes were introduced.It was found that the mouse immune system was capable of using the human gene repertoire to produce high-specificity and high-affinity antibodies against a wider variety of antigens. The transgenic mice expressed B cell receptors that were essentially hybrids of mouse and human components (human immunoglobulins, mouse Igα, Igβ and other signaling molecules), yet their B cells developed and matured normally.

[0069] In certain cases, it may also be preferable to modify one or more CDR amino acid residues to improve binding affinity to the target epitope. This is known as "affinity maturation" and may optionally be carried out in conjunction with humanization, for example in situations where humanization of an antibody leads to a decrease in binding specificity or affinity and back mutation alone is not sufficient to improve binding specificity or affinity. A variety of affinity maturation methods are known in the art, for example Burks et al. , Proc Natl Acad Sci USA, 94:412-17 (1997), and stepwise in vitro affinity maturation by Wu et al., Proc Natl Acad Sci USA 95:6037 6042 (1998).

[0070] The term "monoclonal antibody" or "mAb" refers to an antibody synthesized and isolated by a distinct clonal population of cells. The clonal population may be a clonal population of immortalized cells. In some embodiments, the immortalized cells in the clonal population are hybrid cells, hybridomas, typically produced by fusion of individual B lymphocytes from an immunized animal with individual cells from a lymphocytic tumor. Hybridomas are an engineered type of cell and do not exist in nature.

[0071] A "native antibody" is usually a heterotetrameric glycoprotein with a molecular weight of approximately 150,000 daltons, consisting of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced interchain disulfide bridges. Each heavy chain has a variable domain (VH) at one end followed by several constant domains. Each light chain has a variable domain (VL) at one end and a constant domain at the other end. The constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain. Certain amino acid residues are believed to form an interface between the light and heavy chain variable domains.

[0072] The term "isolated" as used herein to describe various antibodies refers to an antibody that has been identified and separated and / or regenerated from the cell or cell culture in which it was expressed. Impurities (contaminating components) from the natural environment are substances that would interfere with diagnostic or therapeutic uses of the polypeptide and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In a preferred embodiment, the antibody is purified (1) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequencer (Edman sequencer), or (2) to a degree of homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie Brilliant Blue or, preferably, silver stain. An isolated antibody includes an antibody in situ in a recombinant cell, since at least one component of the polypeptide's natural environment will not be present. An isolated polypeptide is typically obtained by at least one purification step.

[0073] An "isolated" nucleic acid molecule is one that is identified and separated from at least one nucleic acid molecule impurity associated with the antibody nucleic acid in its natural source. An isolated nucleic acid molecule is different from the type or set found under natural conditions. Thus, an isolated nucleic acid molecule is different from the nucleic acid molecule present in a cell under natural conditions. However, an isolated nucleic acid molecule includes the nucleic acid located in the cell in which the antibody is normally expressed, for example, when the nucleic acid molecule has a chromosomal location that is different from its location in the cell under natural conditions.

[0074] The term "epitope" as used herein refers to that portion (determinant) of an antigen that specifically binds to a binding molecule (e.g., an antibody or related molecule, e.g., a bispecific binding molecule). Epitopic determinants usually consist of chemically active surface groupings of a molecule, such as amino acids or carbohydrate or sugar side chains, and typically include specific three dimensional structural characteristics, as well as specific charge characteristics. Epitopes may be either "linear" or "conformational". In a linear epitope, all of the points of interaction between the protein (e.g., an antigen) and the interacting molecule (e.g., an antibody) occur linearly along the primary amino acid sequence of the protein. In a conformational epitope, the interaction points are across amino acid residues on the protein that are separated from each other in the primary amino acid sequence. When a desired epitope of an antigen has been determined, an antibody against this epitope may be generated using techniques well known in the art. Furthermore, the generation and characterization of the antibody or other binding molecule may reveal information about the desired epitope. Based on this information, one may then competitively screen binding molecules that bind to the same or similar epitopes, for example, by performing a competition study to find binding molecules that compete for binding to the antigen.

[0075] The term "peptide linker" is intended herein to mean any peptide capable of linking domains, having a length depending on the domains to be linked together and containing any amino acid sequence. Preferably, the peptide linker has a length of more than 5 amino acids and consists of any set of amino acids selected from G, A, S, P, E, T, D, K.

[0076] The term "in vitro" refers to a biological object, process, or reaction outside the body that is modeled under artificial conditions. For example, cells grown in vitro shall be understood as cells grown in an environment outside the body, such as in a test tube, culture vial, or microtiter plate.

[0077] The term “IC 50 "(50% inhibitory concentration)" refers to the drug concentration that inhibits a measurable activity or response, e.g., the growth / proliferation of cells, such as tumor cells, by 50%. Using an appropriate dose-response curve and special statistical software for curve fitting, the IC 50 The value may be calculated.

[0078] The term GI 50 (50% growth inhibition) refers to the concentration of a drug that inhibits the growth of cells, such as tumor cells, by 50%. The term "ED50" (EC50) (50% effective dose / concentration) refers to the concentration of a drug that produces 50% of its biological effect, which may include cytotoxicity.

[0079] The term "effector functions" of an antibody refers to biological activities that can be attributed to the Fc region of an antibody (either a native Fc region sequence or an Fc region amino acid variant) or vary with antibody isotype. Examples of antibody effector functions include: q These include: binding and complement dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor, BCR) and B cell activation.

[0080] "Antibody-dependent cellular cytotoxicity" or "ADCC" refers to a cell-mediated reaction in which non-specific cytotoxic cells expressing Fc receptors (FcR), such as natural killer (NK) cells, neutrophils, and macrophages, recognize bound antibodies on target cells and subsequently cause lysis of the target cells. The primary cells mediating ADCC, NK cells, express only FcγRIII, whereas monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9: 457-92 (1991). To assess the ADCC activity of a molecule of interest, an in vitro ADCC assay, such as that described in U.S. Pat. No. 5,500,362 or 5,821,337, may be performed. Applicable effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively, or in addition, ADCC activity of the molecule of interest may be assessed in vivo, for example in an animal model such as that disclosed in Clynes et al. PNAS (USA) 95:652-656 (1998).

[0081] "Human effector cells" are leukocytes that express one or more FcRs and perform effector functions. Preferably, the cells express at least FcγRIII and perform ADCC effector functions. Examples of human leukocytes that mediate ADCC include peripheral blood mononuclear cells (PBMCs), natural killer (NK) cells, monocytes, cytotoxic T cells and neutrophils; PBMCs and NK cells are preferred. Effector cells may be isolated from their native source, for example from blood or PBMCs, as described herein.

[0082] The term "Fc receptor" or "FcR" is used to describe a receptor that binds to the Fc region of an antibody. A preferred FcR is a native sequence human FcR. Additionally, a preferred FcR is one that binds IgG antibodies (gamma receptors), and includes receptors of the FcγRI, FcγRII (FcγRIIa and FcγRIIb), and FcγRIII (FcγRIIIa and FcγRIIIb) subclasses, including allelic variants and alternatively spliced ​​forms of these receptors. FcγRI exhibits high affinity for IgG, while FcγRII and FcγRIII exhibit low affinity. FcγRIIa and FcγRIIIa are activating FcγRs expressed on monocytes / macrophages and monocytes / macrophages / natural killer cells, respectively, and can induce cytotoxicity of human target cells. Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. Inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain (see review in Daeron, Annu. Rev. Immunol. 15: 203-234 (1997)). FcRs are reviewed in Ravetch and Kinet, Annu. Rev. Immunol 9: 457-92 (1991). Other FcRs, including those that will be identified in the future, are included in the term "FcR" herein. The term also includes the neonatal receptor FcRn, which is involved in the transfer of maternal IgG to the fetus.

[0083] "Complement-dependent cytotoxicity" or "CDC" refers to the ability of a molecule to lyse a target in the presence of complement. The complement activation pathway is initiated by the binding of the first component of the complement system (C1q) to a molecule (e.g., an antibody) complexed with a cognate antigen. To assess complement activation, a CDC assay, for example as described in Gazzano-Santoro et al., J. Immunol. Methods 202: 163 (1996), may be performed.

[0084] The term "identity" or "homology" is taken to mean the percentage of amino acid residues in a candidate sequence that are identical to those of the corresponding sequence being compared, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent identity for the entire sequence, and without considering any conservative substitutions as part of the sequence identity. Any N- or C-terminal extensions or insertions will not be considered to reduce the identity or homology. Methods and computer programs for alignment are well known in the art. Sequence identity may be measured using sequence analysis software (e.g., Sequence Analysis Software Package, Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Ave., Madison, WI 53705). This software matches similar sequences by assigning a degree of homology to various substitutions, deletions (deletions), and other modifications.

[0085] The term "homologous" with respect to the polypeptide sequence of an antibody should be considered to be an antibody exhibiting at least 70%, preferably 80%, more preferably 90%, and most preferably 95% sequence identity to the polypeptide sequence. To the sequence, nucleotide sequences showing at least 85%, preferably 90%, more preferably 95% and most preferably 97% sequence identity should be considered.

[0086] Proposed modification(s) of the amino acid sequence of the antibody described in this publication. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of the antibody are prepared by introducing appropriate nucleotide changes into the antibody nucleic acid or by peptide synthesis. Such modifications include, for example, deletion, and / or insertion and / or substitution of residues within the amino acid sequence of the antibody. Any combination of deletion, insertion and substitution is made to arrive at the final construct, provided that the final construct possesses the desired properties. Amino acid changes may also modify post-translational processing of the antibody, for example, changing the number or position of glycosylation sites.

[0087] Variants of antibody amino acid sequence modification using amino acid substitution. Such variants are substitutions of at least one amino acid residue in antibody molecule with a different residue. The most interesting sites for substitution mutagenesis include hypervariable regions or CDRs, but FR or Fc modifications are also contemplated. Conservative substitutions are shown in Table A under "preferred substitutions". If such substitutions lead to changes in biological activity, further significant changes, referred to as "exemplary substitutions" in Table A, or changes further described below when describing classes of amino acids, may be introduced and the products screened.

[0088] [Table 1]

[0089] The terms "nucleic acid", "nucleic sequence", "nucleic acid sequence", "polynucleotide", "oligonucleotide", "polynucleotide sequence" and "nucleotide sequence" are used interchangeably in this description and refer to a precise sequence of nucleotides, modified or not, determining a fragment or region of a nucleic acid, containing or not containing non-naturally occurring nucleotides, and being either double-stranded DNA or RNA, single-stranded DNA or RNA, or a transcription product of said DNA.

[0090] It must also be included herein that the present invention is not related to the nucleotide sequence in its natural chromosomal environment, i.e. in its natural state. The sequences of the present invention are isolated and / or purified, i.e. they are sampled directly or indirectly, for example by copying, and their environment is at least partially modified. Thus, the isolated nucleic acid obtained by recombinant genetics, for example by a host cell, or obtained by chemical synthesis must also be mentioned herein.

[0091] A reference to a nucleotide sequence includes its complement unless otherwise specified. Thus, a reference to a nucleic acid having a particular sequence should be understood to include its complementary strand, along with its complementary sequence.

[0092] The expression "control sequences" refers to DNA sequences necessary for the expression of a functionally related coding sequence in a particular host organism. Control sequences suitable for prokaryotes include, for example, promoters, optionally operator sequences, and ribosome binding sites. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

[0093] A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader sequence is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; and a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading frame. However, enhancers need not be contiguous.

[0094] The term "vector" as used herein refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. In some embodiments, a vector is a plasmid, i.e., a circular double-stranded piece of DNA into which additional DNA segments can be ligated. In some embodiments, a vector is a viral vector, where additional DNA segments can be ligated into the viral genome. In some embodiments, a vector is capable of autonomous replication in a host cell into which it is introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). In further embodiments, a vector (e.g., non-episomal mammalian vectors) can be integrated into the genome of the host cell upon introduction into the host cell, and thereby replicated along with the host genes. In addition, certain vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors").

[0095] The term "recombinant host cell" (or simply "host cell") is intended herein to refer to a cell into which a recombinant expression vector has been introduced. The present invention relates to host cells, which may include, for example, the vectors of the present invention described above. The present invention also relates to host cells comprising a nucleotide sequence encoding a heavy chain or an antigen-binding portion thereof, or a light chain encoding nucleotide sequence or an antigen-binding portion thereof, or both, of, for example, the first binding domain and / or the second binding domain of a binding molecule of the present invention. It should be understood that "recombinant host cell" and "host cell" are intended to refer not only to the particular subject cell, but also to the progeny of such a cell. Such progeny may not in fact be identical to the parent cell, since modifications may occur in successive generations, either due to mutation or environmental influences, but such cells are still included within the scope of the term "host cell" herein.

[0096] The term "excipient" is used herein to describe any ingredient that is distinct from the compound(s) of the invention. A "pharmaceutical composition" refers to a pharmaceutical composition comprising an antibody of the invention and pharma- ceutically acceptable and pharmacologically compatible fillers, solvents, diluents, carriers, adjuvants, distributing and sensing agents, delivery agents, such as preservatives, stabilizing agents, bulking agents, disintegrating agents, and the like. It refers to a composition comprising at least one of the components selected from the group including agents, moisturizers, emulsifiers, suspending agents, thickeners, sweeteners, flavoring agents, fragrances, antibacterial agents, fungicides, lubricants, and sustained delivery control agents, the selection and appropriate ratio of which depends on the type and method of administration and dosing. Examples of suitable suspending agents are ethoxylated isostearyl alcohol, polyoxyethene, sorbitol and sorbitol ethers, crystalline cellulose, aluminum metahydroxide, bentonite, agar and tragacanth, and also mixtures thereof. Various antibacterial and antifungal agents, such as parabens, chlorobutanol, sorbic acid, and similar compounds, may provide protection against the action of microorganisms. The composition may also contain isotonic agents, such as sugars, polyols, sodium chloride, etc. The action of the composition may be prolonged by agents that delay the absorption of the active ingredient, such as aluminum monostearate and gelatin. Examples of suitable carriers, solvents, diluents and delivery agents include water for injection, ethanol, polyalcohols and their mixtures, natural oils (e.g. olive oil) and organic esters (e.g. ethyl oleate). Examples of fillers are lactose, milk sugar, sodium citrate, calcium carbonate, calcium phosphate, etc. Examples of disintegrants and dispersants are starch, alginic acid and its salts, silicates. Examples of suitable lubricants are magnesium stearate, sodium lauryl sulfate, talc and high molecular weight polyethylene glycol. Pharmaceutical compositions for oral, sublingual, transdermal, intraocular, intramuscular, intravenous, subcutaneous, topical or rectal administration of active ingredient alone or in combination with another active compound may be administered to humans and animals in standard dosage forms, mixed with traditional pharmaceutical carriers. Suitable standard administration forms include oral forms, such as tablets, gelatin capsules, pills, powders, granules, chewing gum and oral solutions or suspensions; sublingual and transbuccal administration forms; aerosols; implants; topical, transdermal, subcutaneous, intramuscular, intravenous, intranasal or intraocular forms and rectal administration forms.

[0097] "Medicinal products" are chemical compounds (or mixtures of compounds such as pharmaceutical compositions) in tablets, capsules, solutions, ointments and other ready-to-use forms intended for the restoration, improvement or modification of physiological functions in humans and animals, and for the treatment and prevention of diseases, for diagnosis, anesthetics, contraception, cosmetology and other purposes.

[0098] The term "CD47 and PD-L1 mediated disease or disorder" refers to any disease or disorder associated with CD47 and PD-L1, either directly or indirectly, including the etiology, development, progression, persistence or pathology of the disease or disorder. "Treate", "treating" and "treatment" refer to a method of alleviating or abrogating at least one of a biological disorder and / or its associated symptoms. As used herein, "alleviating" a disease, disorder or condition means reducing the severity and / or frequency of occurrence of the symptoms of the disease, disorder or condition. Additionally, as used herein, reference to "treatment" includes reference to curative, palliative and prophylactic treatment.

[0099] In one aspect, the subject or patient of the treatment is a mammal, preferably a human subject. The subject may be either male or female of any age. The term "disorder" refers to any condition that would benefit from treatment with the compounds of the present invention. The definition of this term includes chronic and acute disorders or diseases, including pathological conditions that predispose a mammal to the development of this disorder. The preferred disorder to be treated according to the present invention is cancer.

[0100] The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by uncontrolled growth / proliferation of cells. The definition includes both benign and malignant cancerous diseases. Examples of cancerous diseases include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. Such cancerous diseases include, but are not limited to, cancers, including cancers of the genus, leukemia, and lymphoma. More specific examples include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma and lung squamous cell carcinoma, peritoneal cancer, hepatocellular carcinoma, gastric cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney or renal cancer, prostate cancer, vaginal cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, melanoma, and various head and neck cancers.

[0101] The terms "immune response", "autoimmune response" and "autoimmune inflammation" refer to the action of, for example, lymphocytes, antigen-presenting cells, phagocytic cells, granulocytes and soluble macromolecules produced by said cells or hepatocytes, including antibodies, cytokines and complement, which are produced as a result of the selective damage, destruction or elimination of invasive pathogens, pathogen-infected cells or tissues, cancer cells or, in the case of autoimmune or pathological inflammation, normal cells or tissues from the body.

[0102] A "therapeutically effective amount" is intended to refer to that amount of the therapeutic agent being administered that will relieve to some extent one or more of the symptoms of the disorder being treated. The term "chronic" use refers to continuous (continuous) use of an agent(s) in contrast to acute (short-term) routes of administration, such that the initial therapeutic effect (activity) is maintained for an extended period of time.

[0103] "Intermittent" use refers to treatment that is not administered continuously without interruption, but rather is periodic in nature. As used herein, the words "comprise," "have," "include," or variations such as "comprises," "comprising," "has," "having," "includes," or "including" and all grammatical variations thereof, are understood to indicate the inclusion of a reference integer or group of integers, but are not intended to exclude any other integers or groups of integers.

[0104] Detailed Description of the Invention antibody The present invention relates to antibodies that bind to CD47 and PD-L1.

[0105] In one embodiment of the invention, the antibody is a full-length antibody or an antigen-binding fragment thereof. In one embodiment of the invention, the antibody of the invention comprises one or two binding sites for PD-L1.

[0106] In one embodiment of the invention, the binding site for CD47 inhibits the interaction between the CD47 receptor and a SIRPα ligand, and / or the binding site for PD-L1 inhibits the interaction between PD-L1 and the PD-1 receptor.

[0107] In one embodiment, the present invention provides an antibody that binds to CD47 and PD-L1 and: (a) a CDR1 comprising an amino acid sequence that is at least 80% or 90% homologous or identical to a sequence selected from the group of SEQ ID NOs: 1 to 4 below, i.e., CDR1 is a sequence selected from the group of SEQ ID NOs: 1 to 4 below, or a sequence selected from the group of SEQ ID NOs: 1 to 4 below with one or two substitutions; (b) a CDR2 comprising an amino acid sequence that is at least 80%, 84%, 86%, 88%, 92% or 96% homologous or identical to a sequence selected from the group of SEQ ID NOs: 6 to 15 below, i.e., CDR2 is the sequence of SEQ ID NOs: 6 to 15 or a sequence selected from the group of SEQ ID NOs: 6 to 15 below with one, two, three, four or five substitutions; (c) At least 80%, 85%, 86%, 90%, 93% or 95% homologous or identical to a sequence selected from the group consisting of SEQ ID NOs: 17 to 20, i.e., CDR CDR3 comprising an amino acid sequence, wherein CDR3 is a sequence selected from the group of SEQ ID NOs: 17 to 20 below, or a sequence selected from the group of SEQ ID NOs: 17 to 20 below with one, two or three substitutions. The present invention relates to an antibody that comprises a binding site for CD47, comprising a heavy chain variable region comprising:

[0108] In one embodiment, the present invention provides an antibody that binds to CD47 and PD-L1 and: (d) a CDR1 comprising an amino acid sequence identical to a sequence selected from the group consisting of SEQ ID NOs: 1 to 4; (e) a CDR2 comprising an amino acid sequence identical to a sequence selected from the group consisting of SEQ ID NOs: 6 to 15; (f) a CDR3 comprising an amino acid sequence identical to a sequence selected from the group consisting of SEQ ID NOs: 17 to 20 below; The present invention relates to an antibody that comprises a binding site for CD47, comprising a heavy chain variable region comprising:

[0109] In one embodiment, the present invention provides an antibody that binds to CD47 and PD-L1 and: (a) (i) a CDR1 comprising an amino acid sequence that is at least 80% or 90% homologous or identical to a sequence selected from the group of SEQ ID NOs: 1 to 4, i.e., CDR1 is a sequence selected from the group of SEQ ID NOs: 1 to 4, or a sequence selected from the group of SEQ ID NOs: 1 to 4 with one or two substitutions; (ii) a CDR2 comprising an amino acid sequence that is at least 80%, 84%, 86%, 88%, 92% or 96% homologous or identical to a sequence selected from the group of SEQ ID NOs: 6 to 15, i.e., CDR2 is a sequence of SEQ ID NOs: 6 to 15 or a sequence selected from the group of SEQ ID NOs: 6 to 15 with one, two, three, four or five substitutions; (iii) a CDR3 comprising an amino acid sequence that is at least 80%, 85%, 86%, 90%, 93% or 95% homologous or identical to a sequence selected from the group of SEQ ID NOs: 17 to 20, i.e., the CDR3 is a sequence selected from the group of SEQ ID NOs: 17 to 20 or a sequence selected from the group of SEQ ID NOs: 17 to 20 with one, two or three substitutions. a heavy chain variable region comprising (b) (i) a CDR1 comprising an amino acid sequence that is at least 80% or 90% homologous or identical to a sequence selected from the group of SEQ ID NOs: 22 to 34, i.e., CDR1 is a sequence selected from the group of SEQ ID NOs: 22 to 34, or a sequence selected from the group of SEQ ID NOs: 22 to 34 with one or two substitutions; (ii) a CDR2 comprising an amino acid sequence that is at least 80%, 87% or 94% homologous or identical to a sequence selected from the group of SEQ ID NOs: 36 to 48, i.e., CDR2 is a sequence selected from the group of SEQ ID NOs: 36 to 48 or a sequence selected from the group of SEQ ID NOs: 36 to 48 with one, two or three substitutions; (iii) a CDR3 comprising an amino acid sequence that is at least 80% or 90% homologous or identical to a sequence selected from the group of SEQ ID NOs: 50 to 64, i.e., the CDR3 is a sequence selected from the group of SEQ ID NOs: 50 to 64, or a sequence selected from the group of SEQ ID NOs: 50 to 64 with one or two substitutions; A light chain variable region comprising The present invention relates to an antibody which comprises a binding site for CD47, comprising

[0110] In one embodiment, the present invention provides an antibody that binds to CD47 and PD-L1 and: (a) (i) CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 4; (ii) CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 6 to 15; (iii) CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 17 to 20 and a heavy chain variable region comprising (b) (i) CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 22 to 34; (ii) CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 36 to 48; (iii) CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 50 to 64 A light chain variable region comprising The present invention relates to an antibody which comprises a binding site for CD47, comprising

[0111] In one embodiment, the present invention provides an antibody that binds to CD47 and PD-L1 and: (a) a heavy chain variable region comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous or identical to a sequence selected from the group consisting of SEQ ID NOs: 66 to 88; and (b) a light chain variable region comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homologous or identical to a sequence selected from the group consisting of SEQ ID NOs: 89 to 106; The present invention relates to an antibody which comprises a binding site for CD47, comprising

[0112] In one embodiment, the present invention provides an antibody that binds to CD47 and PD-L1 and: (a) a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 66 to 88; and (b) a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 89 to 106; The present invention relates to an antibody which comprises a binding site for CD47, comprising

[0113] In one embodiment, the present invention provides an antibody that binds to CD47 and PD-L1 and: (a) (i) a CDR1 comprising an amino acid sequence that is at least 80% homologous or identical to the sequence of SEQ ID NO:5, i.e., CDR1 is the sequence of SEQ ID NO:5 or is the sequence of SEQ ID NO:5 with one substitution; (ii) a CDR2 comprising an amino acid sequence that is at least 80%, 86%, or 92% homologous or identical to the sequence of SEQ ID NO: 16, i.e., CDR2 is the sequence of SEQ ID NO: 16 or is the sequence of SEQ ID NO: 16 with one, two or three substitutions; (iii) a CDR3 comprising an amino acid sequence that is at least 80% or 90% homologous or identical to the sequence of SEQ ID NO: 21, i.e., the CDR3 is the sequence of SEQ ID NO: 21 or is the sequence of SEQ ID NO: 21 with one or two substitutions. and a light chain variable region comprising (b) (i) a CDR1 comprising an amino acid sequence that is at least 80%, 86%, or 83% homologous or identical to the sequence of SEQ ID NO: 35, i.e., CDR1 is the sequence of SEQ ID NO: 35 or is the sequence of SEQ ID NO: 35 with one, two or three substitutions; (ii) a CDR2 comprising an amino acid sequence that is at least 80% homologous or identical to the sequence of SEQ ID NO: 49, i.e., CDR2 is the sequence of SEQ ID NO: 49 or is the sequence of SEQ ID NO: 49 with one substitution; (iii) a CDR3 comprising an amino acid sequence that is at least 80% or 90% homologous or identical to the sequence of SEQ ID NO: 65, i.e., the CDR3 is the sequence of SEQ ID NO: 65 or is the sequence of SEQ ID NO: 65 with one or two substitutions. A variable region containing The present invention relates to an antibody that comprises a binding site for PD-L1, comprising:

[0114] In one embodiment, the present invention provides an antibody that binds to CD47 and PD-L1 and: (a) (i) CDR1 comprising the amino acid sequence of SEQ ID NO:5; (ii) CDR2 comprising the amino acid sequence of SEQ ID NO: 16; (iii) CDR3 comprising the amino acid sequence of SEQ ID NO: 21 and a heavy chain variable region comprising (b) (i) CDR1 comprising the amino acid sequence of SEQ ID NO: 35; (ii) CDR2 comprising the amino acid sequence of SEQ ID NO: 49; (iii) CDR3 comprising the amino acid sequence of SEQ ID NO: 65 A light chain variable region comprising The present invention relates to an antibody that comprises a binding site for PD-L1, comprising:

[0115] In one embodiment, the invention relates to an antibody that binds to CD47 and PD-L1 and is characterized in that the binding site for CD47 is a Fab, scFv, scFab, or an isolated VH or VHH monodomain.

[0116] In one embodiment, the invention relates to an antibody that binds to CD47 and PD-L1 and is characterized in that the binding site for PD-L1 is a Fab, scFv, scFab, or an isolated VH or VHH monodomain.

[0117] In one embodiment, the invention relates to antibodies characterized by binding to CD47 and PD-L1 and stimulating antibody-dependent cellular cytotoxicity, macrophage-mediated phagocytosis, complement-dependent cytotoxicity, and / or T-cell-mediated cytotoxicity against cells coated with the CD47 and / or PD-L1 antigens.

[0118] In one embodiment, the invention relates to an antibody that binds to CD47 and PD-L1 and is characterized as comprising an Fc fragment that contains at least one mutation or modification that has increased antibody-dependent cell-mediated cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC) compared to the same antibody without that mutation or modification.

[0119] nucleic acid molecule The present invention also relates to nucleic acid molecules and sequences encoding the anti-CD47 / PD-L1 antibodies of the invention described herein. In some embodiments, multiple nucleic acid molecules encode the first and second domains of the amino acid sequence of the anti-CD47 / PD-L1 antibody. In some embodiments, where the first and / or second domains comprise a heavy chain and a light chain, different nucleic acids encode the heavy and light chain amino acid sequences. In other embodiments, the same nucleic acid molecule encodes the heavy and light chain sequences. In certain embodiments, the nucleic acid molecule may encode any combination of the amino acid sequences of the first and second domains (e.g., heavy and light chain sequences). In certain embodiments, the nucleic acid molecule may encode the amino acid sequence of the first binding domain and the light chain amino acid sequence of the second binding domain, optionally including any sequence of a peptide linker linking them. Reference to a nucleotide sequence includes its complement unless otherwise indicated. Thus, reference to a nucleic acid having a specific sequence should be understood to include its complementary strand along with its complementary sequence. The term "polynucleotide," as used herein, refers to a polymeric form of nucleotides, either ribonucleotides or deoxyribonucleotides or a modified form of either type of nucleotide, of at least 10 bases in length. The term includes single- and double-stranded forms.

[0120] In any of the above aspects, the nucleic acid molecule may be isolated. The nucleic acid molecules of the invention can be isolated from any source that produces anti-CD47 / PD-L1 antibodies. In certain embodiments, the nucleic acid molecules of the present invention may be synthesized rather than isolated.

[0121] In some embodiments, the nucleic acid molecules of the invention may comprise a nucleotide sequence encoding a VH domain from the first or second domain of an antibody of the invention linked in frame to a nucleotide sequence encoding a heavy chain constant domain from any source.Similarly, the nucleic acid molecules of the invention may comprise a nucleotide sequence encoding a VL domain from the first or second domain of an antibody of the invention linked in frame to a nucleotide sequence encoding a light chain constant domain from any source.

[0122] In a further aspect of the invention, the nucleic acid encoding the heavy (VH) and / or light (VL) variable domains of the first or second binding domain may be "converted" into a full-length antibody gene. In one embodiment, the nucleic acid molecule encoding the VH or VL domain is converted into a full-length antibody gene by insertion into an expression vector already encoding a heavy chain constant (CH) or light chain constant (CL) domain, respectively, such that the VH segment is operably linked to a CH segment(s) in the vector and / or the VL segment is operably linked to a CL segment in the vector. In another embodiment, the nucleic acid molecule encoding the VH and / or VL domain is converted into a full-length antibody gene by linking, e.g., linking, the nucleic acid molecule encoding the VH and / or VL domain to the nucleic acid molecule encoding the CH and / or CL domain using standard molecular biology techniques. These may then be expressed in a cell into which the full-length heavy and / or light chain encoding nucleic acid molecule has been introduced.

[0123] The nucleic acid molecules may be used to express large amounts of recombinant anti-CD47 / PD-L1 antibodies, and may be used to produce human antibodies, humanized antibodies, chimeric antibodies, bispecific antibodies, single chain antibodies, immunoadhesins, diabodies, mutated antibodies, and antibody derivatives, as described herein.

[0124] vector In another aspect, the invention relates to vectors suitable for the expression of any of the nucleotide sequences described herein.

[0125] The present invention relates to vectors comprising a nucleic acid molecule encoding any amino acid sequence of an anti-CD47 / PD-L1 antibody or portion thereof (e.g. the heavy chain sequence of the first binding domain and / or the heavy and / or light chain sequences of the second binding domain) as described herein. The present invention further provides vectors comprising the nucleic acid molecule encoding fusion proteins, modified antibodies, antibody fragments.

[0126] In another embodiment, the nucleic acid molecules and vectors may be used to generate mutated anti-CD47 / PD-L1 antibodies. The antibodies may be mutated in the variable domains of the heavy and / or light chains of the first binding domain and / or the heavy and / or light chains of the second binding domain, e.g., to alter the binding characteristics of the antibody. For example, mutations may be made in one or more CDRs to increase the K D may be increased or decreased, k off The binding specificity of the antibody for FcRn may be increased or decreased, or the binding specificity of the antibody for FcRn may be altered. In another embodiment, one or more mutations are made at amino acid residues known to be altered compared to germline in the antibody corresponding to the first or second binding domain of the anti-CD47 / PD-L1 antibody of the invention. Such mutations may be made in the CDR or framework regions of the variable domain, or in the constant domain. In a preferred embodiment, mutations are made in the variable domain. In another embodiment, one or more mutations are made at amino acid residues known to be altered compared to germline in the CDR or framework regions of the variable domain of an antibody of the invention.

[0127] In some embodiments, the anti-CD47 / PD-L1 antibodies of the present invention are expressed by inserting DNA partially or completely encoding the sequence of the first or second binding domain (e.g., the light and heavy chain sequences in the case where the binding domain comprises light and heavy chain sequences), obtained as described above, in an expression vector, such that the gene is operably linked to the necessary expression control sequences, e.g., transcriptional and translational control sequences. Expression vectors include plasmids, retroviruses, adenoviruses, adeno-associated viruses (AAV), plant viruses, such as cauliflower mosaic virus, tobacco mosaic virus, cosmids, YACs, EBV-derived episomes, and the like. The DNA molecule may be ligated into a vector such that the transcriptional and translational control sequences in the vector perform their intended function of controlling the transcription and translation of the DNA. The expression vector and expression control sequences may be selected to be compatible with the host cell used. The DNA molecules partially or completely encoding the sequence of the first and second binding domain (e.g., the heavy and light chain sequences in the case where the binding domain comprises heavy and light chain sequences) may be introduced into the respective vectors. In one embodiment, any combination of the DNA molecules is introduced into the same expression vector. The DNA molecules may be introduced into the expression vector by standard methods (e.g., ligation of complementary restriction sites on the antibody gene fragment and the vector, or blunt end ligation if no restriction sites are present).

[0128] A suitable vector is one that encodes a functionally complete human CH or CL immunoglobulin sequence with appropriate restriction site manipulation so that any VH or VL sequence can be easily inserted and expressed as described above. The HC and LC coding genes in such vectors may contain intron sequences that stabilize the corresponding mRNA, thereby resulting in an increase in the overall antibody protein yield. The intron sequences are flanked by splice donor and splice acceptor sites that determine where RNA splicing occurs. The location of the intron sequence may be in either the variable or constant region of the antibody chain, or in both the variable and constant regions when multiple introns are used. Polyadenylation and transcription termination may occur at the native chromosomal site downstream of the coding region. The recombinant expression vector may also encode a signal peptide that facilitates secretion of the antibody chain from the host cell. The antibody chain gene may be cloned into the vector such that the signal peptide is linked in frame to the amino terminus of the immunoglobulin chain. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (ie, a signal peptide from a non-immunoglobulin protein).

[0129] In addition to the antibody chain genes, the recombinant vector expression of the present invention may carry a control sequence that controls the expression of the antibody chain genes in a host cell. Those skilled in the art will understand that the design of the expression vector, including the selection of the control sequence, may depend on the choice of the host cell to be transformed, the desired protein expression level, and the like. Preferred control sequences for mammalian expression host cells include viral elements that ensure high levels of protein expression in mammalian cells, such as retroviral LTRs, cytomegalovirus (CMV) (e.g., CMV promoter / enhancer), simian virus 40 (SV40) (e.g., SV40 promoter / enhancer), adenovirus (e.g., major late promoter adenovirus (AdMLP)), polyoma virus derived promoters and / or enhancers, as well as strong mammalian promoters, such as native immunoglobulin promoters or actin promoters. For further description of viral control elements and sequences thereof, see, e.g., U.S. Patent Nos. 5,168,062, 4,510,245, and 4,968,615. Methods for expressing binding molecules, such as antibodies, in plants include promoters and vectors, as well as plant transformation. Methods for expressing polypeptides in bacterial or fungal cells, such as yeast cells, are also well known in the art.

[0130] In addition to the antibody chain genes and control sequences, the recombinant expression vector of the present invention may carry additional sequences, such as sequences that control replication of the vector in a host cell (e.g., origins of replication) and selectable marker genes. The selectable marker gene facilitates selection of a host cell into which the vector has been introduced (see, e.g., U.S. Pat. Nos. 4,399,216, 4,634,665 and 5,179,017). For example, typically the selectable marker gene confers resistance to pharmaceutical agents, such as G418, hygromycin or methotrexate, on the host cell into which the vector has been introduced. For example, selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr- host cells during methotrexate selection / amplification), the neo gene (for G418 selection), and the glutamate synthetase gene.

[0131] The term "expression control sequences" is intended herein to refer to polynucleotide sequences necessary to affect the expression and processing of a coding sequence to which it is linked. Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals, such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (e.g., Kozak consensus sequences); sequences that enhance protein stability; and sequences that enhance protein secretion, if desired. The nature of such control sequences will vary depending on the host organism; in prokaryotes, such control sequences generally include a ribosome binding site, promoter, and transcription termination sequences; in eukaryotes, such control sequences include a promoter and transcription termination sequences. The term "control sequences" includes at least all components whose presence is essential for expression and processing, and may also include additional components whose presence is beneficial, such as leading sequences and fusion cell sequences.

[0132] host cell A further aspect of the present invention relates to a method for producing an antibody against CD47 and PD-L1 of the present invention. One embodiment of the present invention relates to a method for producing an antibody as defined herein, comprising the steps of introducing / preparing a recombinant host cell capable of expressing the antibody, culturing said host cell under conditions suitable for the expression / production of the antibody, and isolating the antibody obtained. Antibodies against CD47 and PD-L1 obtained by such expression in such recombinant host cells are referred to herein as "recombinant antibodies". The present invention also relates to the progeny of cells derived from such host cells, and to the antibodies against CD47 and PD-L1 obtained similarly.

[0133] Nucleic acid molecules encoding the anti-CD47 / PD-L1 antibodies of the present invention and vectors containing these nucleic acid molecules may be used to transfect suitable mammalian or cells thereof, plant or cells thereof, bacterial or yeast host cells. Transformation may be by any known technique for introducing polynucleotides into host cells. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art and include dextran-mediated transfection, cationic polymer-nucleic acid complex transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, encapsulation of polynucleotide(s) in liposomes, and direct microinjection of DNA into the nucleus. Additionally, nucleic acid molecules may be introduced into mammalian cells by viral vectors. Methods for transfecting cells are well known in the art. See, for example, U.S. Pat. No. 4,399,216; U.S. Pat. No. 4,421,331; See, for example, Nos. 912,040, 4,740,461 and 4,959,455. Methods for transforming plant cells are well known in the art, including, for example, Agrobacterium-mediated transformation, biolistic transformation, direct injection, electroporation and viral transformation. Methods for transforming bacterial and yeast cells are also well known in the art.

[0134] Mammalian cell lines used as hosts for transformation are well known in the art and include many immortalized cell lines available. These include, for example, Chinese hamster ovary (CHO) cells, NS0 cells, SP2 cells, HEK-293T cells, FreeStyle 293 cells (Invitrogen), NIH-3T3 cells, HeLa cells, baby hamster kidney (BHK) cells, African green monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., HepG2), A549 cells, and many other cell lines. A cell line is selected by determining which cell line has high expression levels and provides the necessary characteristics for the protein to be produced. Other cell lines that can be used are insect cell lines, such as Sf9 or Sf21 cells. When a recombinant expression vector encoding an antibody against CD47 and PD-L1 is introduced into a mammalian host cell, the antibody is produced by culturing the host cell for a period of time sufficient to allow expression of the antibody in the host cell, or more preferably, secretion of the antibody into the medium in which the host cell is grown. Antibodies against CD47 and PD-L1 may be isolated from the nutrient medium using standard protein purification methods. Plant host cells include, for example, Nicotiana, Arabidopsis, duckweed, corn, wheat, potato, etc. Bacterial host cells include E. coli and Streptomyces species. Yeast host cells include Schizosaccharomyces pombe, Saccharomyces cerevisiae, and Pichia pastoris.

[0135] Furthermore, many known techniques may be used to enhance the production levels of the antibodies against CD47 and PD-L1 of the present invention from the production cell line. For example, the glutamine synthetase gene expression system (GS system) is a common approach to enhance expression under certain conditions. The GS system is discussed in whole or in part in connection with EP Nos. 0216846, 0256055, 0323997 and 0338841.

[0136] Antibodies to CD47 and PD-L1 expressed by different cell lines or transgenic animals will likely have different glycosylation profiles when compared to each other, however, all antibodies to CD47 and PD-L1 encoded by the nucleic acid molecules described herein or comprising the amino acid sequences provided herein, regardless of glycosylation of the binding molecule, and generally regardless of the presence or absence of post-translational modifications, are part of the present invention.

[0137] Preparation of antibodies The present invention also relates to methods and processes for producing antibodies against CD47 and PD-L1, and antigen-binding fragments thereof.

[0138] Monoclonal antibodies Monoclonal antibodies may be prepared using the hybridoma method first described by Kohler et al. Nature 256, 1975, p. 495, or may be prepared using recombinant DNA methods (US 4,816,567).

[0139] Capable of specifically binding to the protein used for immunization when using hybridoma-based methods To induce the formation of lymphocytes that produce or are capable of producing antibodies, a mouse or other suitable host animal, such as a hamster, is immunized according to the methods described above. According to another embodiment, lymphocytes may be produced by in vitro immunization. After immunization, the lymphocytes are fused with a myeloma cell line using a suitable fusing agent, such as polyethylene glycol, to produce hybridoma cells.

[0140] The hybridoma cells thus obtained are seeded and grown in an appropriate medium, which preferably contains one or more substances that inhibit the growth or survival of the unfused parental myeloma cells. For example, if the parental myeloma cells do not contain hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the medium for the hybridomas must usually contain hypoxanthine, aminopterin and thymidine (HAT medium), substances that inhibit the growth of HGPRT-deficient cells.

[0141] Preferred myeloma cell lines are mouse myeloma cell lines, such as those based on murine tumor cells MORS-21 and MPC-11 available from the Salk Institute Cell Distribution Center, San Diego, Calif., USA, and cell lines SP-2 or X63-Ag8-653 available from the American Type Culture Collection, Rockville, Md., USA. The use of human-mouse myeloma and mouse-human heteromyeloma cell lines to produce monoclonal antibodies has also been described (Kozbor, J. Immunol, 133, 1984, p. 3001).

[0142] Preferably, the binding specificity of monoclonal antibodies obtained from the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).

[0143] The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis described in Munson et al., Anal. Biochem., 107:220 (1980).

[0144] After identifying hybridoma cells that produce antibodies of the desired specificity, affinity, and / or activity, the clones may be subcloned using limiting dilution methods and grown by standard methods. Suitable media for this purpose include, for example, D-MEM or RPMI-1640 medium. In addition, hybridoma cells may be grown in vivo as ascites tumors in animals, for example, by injecting the cells intraperitoneally (ip) into mice.

[0145] The monoclonal antibodies secreted by the subclones may be separated from the culture medium, ascites fluid, or serum by conventional antibody purification techniques, such as affinity chromatography (e.g., with Protein A or Protein G-Sepharose) or ion exchange chromatography, hydroxylapatite chromatography, gel electrophoresis, dialysis, etc.

[0146] DNA encoding the monoclonal antibodies is readily isolated and sequenced using conventional techniques (e.g., by using oligonucleotide probes capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). Hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA may be placed into an expression vector which can then be transfected into host cells, such as E. coli cells, monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells which would not otherwise produce the antibody protein, to produce the recombinant host cell. Obtaining synthesis of monoclonal antibodies in host cells. Overview of recombinant expression in bacteria of DNA encoding antibodies.

[0147] In further embodiments, monoclonal antibodies or antibody fragments may be isolated from antibody phage libraries generated using the techniques described in McCafferty et al., Nature, 348:552-554 (1990). Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991) describe the isolation of murine and human antibodies, respectively, using phage libraries. Subsequent publications have described the production of high affinity (nM range) human antibodies by chain shuffling (Marks et al., Bio / Technology, 10:779-783 (1992)), as well as combinatorial infection and in situ hybridization as strategies for constructing very large phage libraries. in vivo recombination (Waterhouse et al., Nucl. Acids. Res. 21:2265-2266 (1993)). Thus, these techniques are promising alternatives to traditional monoclonal antibody hybridoma techniques for the isolation of monoclonal antibodies.

[0148] For example, to produce chimeric or fusion antibody polypeptides, DNA encoding an antibody may be modified, for example, by substituting heavy and light chain (CH and CL) constant region sequences for homologous murine sequences (US 4816567 and Morrison et al., Proc. Natl. Acad. Sci. USA: 81:6851 (1984)), or by covalently linking an immunoglobulin coding sequence to all or part of the coding sequence of a non-immunoglobulin polypeptide (heterologous polypeptide). Non-immunoglobulin polypeptide sequences may be substituted for the constant regions of an antibody, or these may be substituted for the variable domains of the antigen-binding center of the antibody to generate chimeric bivalent antibodies containing one antigen-binding site with specificity for an antigen and another antigen-binding site with specificity for a different antigen.

[0149] Humanized antibodies Methods for producing "humanized" non-human animal antibodies are well known in the art. Preferably, a humanized antibody has one or more integral amino acid residues introduced from a source that is non-human. These non-human amino acid residues are often referred to as "imported" residues because they are typically taken from an "import" variable region. Essentially, humanization may be performed according to the method of Winter and co-authors (Jones et al., Nature, 321:522-525 (1986)) by substituting hypervariable region sequences with the corresponding sequences of a human antibody. Such "humanized" antibodies are thus chimeric antibodies (US 4816567) in which less than substantially intact human variable regions have been substituted by the corresponding sequences from a non-human species. In effect, humanized antibodies are typically human antibodies in which some hypervariable region residues and / or some FR residues are substituted by residues from the analogous regions in rodent antibodies.

[0150] The choice of human variable regions, both light and heavy, used in producing a humanized antibody is very important to reduce antigenicity and HAMA reactions (human anti-mouse antibodies) if the antibody is intended for human therapeutic use. According to the so-called "best-fit" method, the sequence of the variable region of a rodent antibody is screened against the entire library of known human variable domain sequences. The human V domain sequence closest to the rodent one is identified, and the human framework region (FR) therein, suitable for use in the humanized antibody, is selected (Sims et al., J. Immunol. 151:2296 (1993)). Another method involves the selection of specific framework regions (FRs) from the consensus sequences of a particular subgroup of light or heavy chains of all human antibodies. The same framework region may be used for several different humanized antibodies (Carter et al., Proc. Natl. Acad. Sci. USA: 89:4285 (1992)).

[0151] It is also important to humanize antibodies while retaining high binding affinity to the antigen and other important biological properties. To this end, according to a preferred method, humanized antibodies are prepared by analysis of the parental sequences and various humanized products using conceptual three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available that illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these images allows analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., analysis of residues that influence the ability of the candidate immunoglobulin to bind to an antigen. In this manner, FR residues may be selected and combined with the recipient and import sequences to achieve the desired antibody property, e.g., increased affinity for the target antigen(s). In general, the hypervariable region residues are directly and most substantially involved in influencing antigen binding.

[0152] The humanized antibody may be an antibody fragment, such as a Fab, that is optionally conjugated to one or more cytotoxic agent(s) to generate an immunoconjugate. Alternatively, the humanized antibody may be a full-length antibody, such as a full-length IgG1 antibody.

[0153] Phage display library-based human antibodies and methodologies As an alternative to humanization, human antibodies may be produced. For example, it is now possible to produce transgenic animals (e.g., mice) that can produce the full range of human antibodies after immunization without endogenous immunoglobulin production. For example, it has been described that the homozygous deletion of antibody heavy chain joining region (JH) genes in chimeric and germline mutant mice results in complete inhibition of endogenous antibody production. Transfer of human germline immunoglobulin gene arrays into these germline mutant mice results in the production of human antibodies after antigen exposure (US 5545806, 5569825, 5591669 (all GenPharm); 5545807; and WO 97 / 17852).

[0154] Alternatively, human antibodies and antibody fragments may be produced in vitro from immunoglobulin variable (V) region gene repertoires derived from the bodies of immunized donors using phage display technology (McCafferty et al., Nature, 348:552-554 (1990)). According to this technology, antibody V region genes are cloned in frame with either the major or minor coat protein gene of a filamentous bacteriophage, such as M13 or fd, and displayed as functional antibody fragments on the surface of the phage particle. Because the filamentous particle contains a single-stranded DNA copy of the phage genome, selections based on the functional properties of the antibody also result in selection of the gene encoding the antibody exhibiting said properties. Thus, the phage mimics some of the properties of B cells. Phage display may be performed in a variety of formats. Several sources of V gene segments may be used for phage display. Clackson et al., Nature, 352:624-628 (1991) isolated a diverse array of anti-oxazolone antibodies from a small random combinatorial library of V genes derived from the spleens of immunized mice. Essentially following the techniques described in Marks et al., J. Mol. Biol. 222:581-597 (1991), one can construct a repertoire of V genes from immunized human donors and isolate antibodies to a diverse array of antigens, including self-antigens.

[0155] As discussed above, human antibodies may also be generated by in vitro activated B cells (see US Pat. Nos. 5,567,610 and 5,229,275). antibody fragment In certain circumstances it is advantageous to use antibody fragments rather than whole antibodies: the small size of fragments contributes to their rapid clearance and may contribute to better penetration into dense tumors.

[0156] A variety of techniques have been developed for the production of antibody fragments. Traditionally, these fragments were obtained through proteolytic digestion of intact antibodies. However, now these fragments may be obtained directly by recombinant host cells. Fab, Fv and ScFv antibody fragments may be expressed in and secreted from E. coli, thereby facilitating the production of large amounts of these fragments. Antibody fragments may be isolated from the antibody phage libraries described above. According to another embodiment, Fab'-SH fragments may be directly isolated from E. coli and chemically coupled to form F(ab')2 fragments (Carter et al., Bio / Technology 10:163-167 (1992)). According to another approach, F(ab')2 fragments may be directly isolated from recombinant host cell cultures. Fab and F(ab')2 fragments with increased in vivo half-life while retaining epitope binding receptor residues are described in US 5,869,046. Other techniques for obtaining antibody fragments should be apparent to those skilled in the art. In other embodiments, the antibody of choice is a single chain Fc fragment (scFv) (see WO 93 / 16185; US 5571894 and US 5587458). Fv and scFv are the only species that do not contain constant regions and contain intact binding sites; as a result, they are suitable for reducing non-specific binding during in vivo use. Fusion proteins carrying scFvs can be designed to result in fusion of effector proteins at either the N- or C-terminus of the scFv. The antibody fragment can also be a "linear antibody" as described, for example, in US 5641870. Such linear antibody fragments can be monospecific or bispecific.

[0157] multispecific antibodies A multispecific antibody is an antibody that has binding specificity for at least two different epitopes. For example, a bispecific antibody may bind to two different epitopes of a protein. Other multispecific antibodies may combine binding sites for CD47 and PD-L1 in combination with binding sites for another protein. Bispecific antibodies may be obtained as full length antibodies or antibody fragments (e.g., F(ab')2 fragments of bispecific antibodies).

[0158] Methods for producing multispecific antibodies are known in the art. For example, the traditional production of full-length bispecific antibodies is based on the co-expression of two immunoglobulin heavy / light chain pairs, where the two chains have different specificities. Because the immunoglobulin heavy and light chains are random in type, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. Purification of the correct molecule is usually performed by several steps of affinity chromatography, which is quite cumbersome and the product yield is low. A similar process is described in WO 93 / 08829.

[0159] According to a different approach, antibody variable domains with the desired binding specificities (antigen binding sites of binding) are fused to immunoglobulin constant domain sequences. Preferably, the fusion is with an Ig heavy chain constant region, comprising at least part of the hinge, C H 2, and C H Preferably, the first heavy-chain constant region (C ) contains the site necessary for light-chain binding. H DNA encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain, are inserted into a variety of expression vectors and expressed in an appropriate Co-transfect into host cell.This provides greater flexibility in selecting the mutual ratio of the three polypeptide fragments in the embodiment when using three polypeptide chains in unequal ratio in construction to provide optimal yield.However, if the expression of at least two polypeptide chains in equal ratio produces high yield, or if the ratio does not have a significant effect, it is possible to insert the coding sequence for two or all three polypeptide chains in a single expression vector.

[0160] In a preferred embodiment of this approach, the bispecific antibody is a hybrid immunoglobulin heavy chain in the first arm providing a first binding specificity, and a hybrid immunoglobulin heavy / light chain pair in the second arm providing a second binding specificity. It has been found that this asymmetric structure facilitates the separation of the desired bispecific molecule from undesired immunoglobulin chain combinations, since the immunoglobulin light chain is present in only half of the bispecific molecule, facilitating the separation. This approach is disclosed in WO94 / 04690. For more details on bispecific antibody production, see, for example, Suresh et al., Methods in Enzymology 121:210 (1986).

[0161] According to another approach described in US 5731168, the interface between a pair of antibody molecules may be engineered to maximize the percentage of heterodimers obtained from recombinant cell culture. H The antibody comprises at least a portion of the three regions. According to this method, one or more small amino acids with side chains are replaced from the interface of the first antibody molecule with those with larger side chains (e.g., tyrosine or tryptophan). Compensatory "cavities" of identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule by replacing amino acids containing large side chains with amino acids containing smaller side chains (e.g., alanine or threonine). This provides a mechanism for increasing the yield of heterodimers relative to other unwanted end-products.

[0162] Bispecific antibodies include cross-linked or "heteroconjugate" antibodies. For example, one of the antibodies in the heteroconjugate may be coupled to avidin and the other to biotin. For example, such antibodies may be used to target immune system cells to unwanted cells (US 46769809) and to treat HIV infection (WO 91 / 00360, WO 92 / 200373, and EP 03089). Heteroconjugate antibodies may be produced using any conventional cross-linking method. Suitable cross-linking agents are well known in the art and are disclosed in US 4676980, along with a variety of cross-linking techniques.

[0163] Methods for obtaining bispecific antibodies from antibody fragments have also been described in the literature. For example, bispecific antibodies may be obtained by chemical coupling. Brennan et al., Science 229:81 (1985) describe a method whereby intact antibodies are proteolytically cleaved to produce F(ab')2. These fragments are reduced in the presence of dithiol complexing agents, such as sodium arsenite, to stabilize vicinal dithiols and prevent the formation of intermolecular disulfide bonds. The Fab' fragments produced are then converted to thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB derivatives is then reconverted to a Fab'-thiol by reduction with mercaptoethylamine and mixed with an equimolar amount of another Fab'-TNB derivative to obtain the bispecific antibody. The bispecific antibody produced may be used as an agent for the selective immobilization of enzymes.

[0164] Recent progress has facilitated the direct recovery of Fab'-SH fragments from E. coli, which may be chemically coupled to produce bispecific antibodies. Shalaby et al., J. Exp. Med. 175:217-225 (1992) described a fully humanized The production of F(ab')2 bispecific antibody molecules is described. Each Fab' was secreted separately from E. coli and subjected to direct chemical coupling in vitro to form the bispecific antibody. The resulting bispecific antibody is capable of binding to cells overexpressing the ErbB2 receptor and normal human T cells, as well as inducing the lytic activity of human cytotoxic lymphocytes against human breast tumor targets.

[0165] A variety of techniques have also been described for obtaining and isolating bispecific antibody fragments directly from recombinant cell culture. For example, bispecific antibodies have been produced using leucine zippers (Kostelny et al., J. Immunol. 148(5):1547-1553 (1992)). Leucine zipper peptides from Fos and Jun proteins were linked to the Fab' of two different antibodies by gene fusion. The antibody homodimers were reduced at the hinge region to form monomers and then reoxidized to obtain the antibody heterodimers. This method may also be used to obtain homodimeric antibodies. The "double antibody" technology described by Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993) is an alternative mechanism for producing bispecific antibody fragments. The fragments contain a VH region linked to a VL region by a linker that is too short to allow pairing between the two domains on the same chain. Thus, the VH and VL domains of one fragment must pair with the complementary VL and VH domains of another fragment, thereby forming two antigen-binding sites. Another strategy for producing bispecific antibody fragments using single-chain (Fv)-(sFv) dimers has also been described (see Gruber et al., J. Immunol., 152:5368 (1994)).

[0166] The present invention also provides for antibodies with more than two valencies, for example trispecific antibodies may be generated. Multivalent antibodies Multivalent antibodies may be internalized (and / or catabolized) more rapidly than bivalent antibodies by cells expressing the antigen to which the antibody binds. The antibodies of the present invention may be multivalent antibodies (other than IgM class) with three or more antigen binding sites (e.g., tetravalent antibodies), which may be readily obtained by recombinant expression of nucleic acids encoding antibody polypeptide chains. The multivalent antibody may comprise a dimerization domain and three or more antigen binding sites. A preferred dimerization domain comprises (or consists of) an Fc fragment or a hinge region. In this scenario, the antibody will comprise an Fc fragment and three or more antigen binding sites at the N-terminus of the Fc fragment. A preferred multivalent antibody herein comprises (or consists of) three to about eight, but preferably four, antigen binding sites. A multivalent antibody comprises at least one polypeptide chain (and preferably two polypeptide chains), where the polypeptide chain(s) comprises two or more variable regions. For example, the polypeptide chain(s) may comprise VD1-(X1)n-VD2-(X2)n-Fc, where VD1 refers to the first variable region, VD2 refers to the second variable region, Fc refers to one polypeptide chain of an Fc fragment, X1 and X2 refer to amino acids or polypeptides, and n is 0 or 1. For example, the polypeptide chain(s) may comprise the following chain: VH-CH1-flexible linker-VH-CH1-Fc fragment; or VH-CH1-VH-CH1-Fc fragment. The multivalent antibody herein preferably further comprises at least two (and preferably four) light chain variable region polypeptides. The multivalent antibody herein may comprise, for example, about two to about eight light chain variable region polypeptides. In the context of the present invention, the light chain variable region polypeptide comprises a light chain variable region and, optionally, further comprises a CL region.

[0167] Pharmaceutical Compositions In another aspect, the present invention provides a method for the treatment of cancer, comprising administering to a patient a therapeutically effective amount of CD4 as an active ingredient (or as the only active ingredient). In some embodiments, the compositions are intended to ameliorate, prevent, or treat disorders mediated by IgG.

[0168] "Pharmaceutical composition" means a composition comprising an anti-CD47 / PD-L1 antibody of the present invention and at least one component selected from the group consisting of pharma- ceutically acceptable and pharmacologically compatible excipients, such as fillers, solvents, diluents, carriers, adjuvants, dispensing agents, delivery agents, preservatives, stabilizers, emulsifiers, suspending agents, thickening agents, sustained-delivery control agents, the selection and proportions of which depend on the type and route of administration and dosing. The pharmaceutical composition of the present invention and the method for its preparation will be clearly apparent to those skilled in the art. The pharmaceutical composition should preferably be manufactured in accordance with Good Manufacturing Practice (GMP) requirements. The composition may also include buffer compositions, isotonicity agents, stabilizers and solubilizers. Prolonged action of the composition may be achieved by agents that delay absorption of the active pharmaceutical ingredient, such as aluminum monostearate and gelatin. Examples of suitable carriers, solvents, diluents and delivery agents include water, ethanol, polyalcohols and mixtures thereof, oils, and organic esters for injection.

[0169] A "medicament" is a substance or mixture of substances as tablets, capsules, powders, lyophilisates, injections, infusions, ointments and other ready-to-use pharmaceutical compositions intended for the restoration, improvement or modification of physiological functions, for the treatment and prevention of diseases, for diagnosis, anesthesia, contraception, cosmetology, etc. in humans and animals. Any method for administering peptides, proteins or antibodies accepted in the art may be suitably used in connection with the anti-CD47 / PD-L1 antibodies of the invention.

[0170] The term "pharmaceutical acceptable" refers to one or more compatible liquid or solid components suitable for administration in a mammal, preferably a human. The term "excipient" as used herein describes any other component of the present invention. These are inorganic or organic substances used in pharmaceutical manufacturing to impart the necessary physicochemical properties to the pharmaceutical product.

[0171] As used herein, the terms "buffer solution", "buffer composition" and "buffer" refer to a solution that is capable of resisting changes in pH by the action of its acid-base conjugate components and allows the anti-CD47 / PD-L1 antibody drug to resist changes in pH. In general, the pharmaceutical composition preferably has a pH in the range of 4.0 to 8.0. Examples of buffers that may be used include, but are not limited to, acetate, phosphate, citrate, histidine, succinate, and the like buffer solutions.

[0172] The terms "isotonicity agent", "osmolyte" or "osmotic pressure regulator" as used herein refer to excipients that can increase the osmotic pressure of a liquid antibody formulation. An "isotonic" agent is one that has an osmotic pressure equivalent to that of human blood. An isotonic agent typically has an osmotic pressure of about 250-350 mOsm / kg. Isotonicity agents that may be used include, but are not limited to, polyols, sugars and sucrose, amino acids, metal salts such as sodium chloride, and the like.

[0173] "Stabilizer" refers to an excipient, or a mixture of two or more excipients, that provides physical and / or chemical stability to an active agent. Stabilizers include amino acids, such as, but not limited to, arginine, histidine, glycine, lysine, glutamine, proline; surfactants, such as, but not limited to, polysorbate 20 (trade name); Antioxidants, including but not limited to methionine, acetylcysteine, ascorbic acid, monothioglycerol, sulfates, and the like; chelating agents, including but not limited to ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), sodium citrate, and the like.

[0174] A pharmaceutical composition is "stable" if the active agent retains its physical and / or chemical and / or biological stability at a storage temperature, e.g., 2-8°C, during a specified shelf life. Preferably, the active agent retains both physical and chemical stability, as well as biological activity. The shelf life is adjusted based on the results of stability tests under accelerated or natural aging conditions.

[0175] The pharmaceutical composition of the present invention may be manufactured, packaged, or widely sold in the form of a single unit dose or a plurality of single unit doses in the form of a ready-to-use formulation.The term "single unit dose" as used herein refers to a discrete amount of pharmaceutical composition that contains a predetermined amount of active ingredient.The amount of active ingredient is usually equal to the dosage of the active ingredient to be administered to a subject, or a suitable fraction of such a dosage, such as a half or a third of the dosage.

[0176] The pharmaceutical compositions of the present invention are suitable for parenteral administration, typically as sterile formulations intended to be administered into the human body by injection, infusion and implantation, bypassing the gastrointestinal tract and through a breach in the skin or mucosal barrier. For example, parenteral administration includes, among others, subcutaneous, intraperitoneal, intramuscular, intrasternal, intravenous, intraarterial, intrathecal, intraventricular, intraurethral, ​​intracranial, intrasynovial, percutaneous injection or infusion; and kidney dialysis infusion techniques. Regional perfusion is also provided. Preferred embodiments include intravenous and subcutaneous routes. Any method for administering peptides or proteins accepted in the art may be suitably used for the anti-CD47 / PD-L1 antibodies of the present invention.

[0177] Injectable formulations may be prepared, packaged, or sold in dosage form, such as, without limitation, in ampoules, vials, plastic containers, prefilled syringes, autoinjection devices, etc. Formulations for parenteral administration include, among others, suspensions, solutions, emulsions in oily or aqueous bases, pastes, and the like.

[0178] In another embodiment, the present invention provides compositions for parenteral administration, including pharmaceutical compositions provided in dry (i.e., powder or granule) form for reconstitution with a suitable base (e.g., sterile, pathogen-free water) prior to administration. Such formulations may be obtained, for example, by a lyophilization process, known in the art as freeze-drying, and involving freezing the product followed by removal of the solvent from the frozen material.

[0179] The antibodies against CD47 and PD-L1 of the invention may also be administered intranasally or by inhalation, either alone or in admixture with suitable excipients, from an inhalation device, such as a pressurized aerosol container, pump, spray, atomizer or nebulizer, with or without a suitable propellant, or as nasal drops, or in a spray.

[0180] Dosage forms for parenteral administration may be formulated to be immediate or modified release. Modified release formulations include delayed-, sustained-, pulsed-, controlled-, targeted and programmed release.

[0181] Therapeutic Uses of Anti-CD47 / PD-L1 Antibodies of the Invention In one aspect, the anti-CD47 / PD-L1 antibodies of the invention are used to treat a disorder mediated by CD47 and PD-L1, such as a disease or disorder selected from the group including: (HNSCC) head and neck squamous cell carcinoma, cervical cancer, carcinoma of unknown primary, glioblastoma, esophageal cancer, bladder cancer, TNBC (triple negative breast cancer), CRC (colorectal cancer), hepatocellular carcinoma, melanoma, NSCLC (non-small cell lung cancer), renal cancer, ovarian cancer, Hodgkin's lymphoma, MSI CRC (colorectal cancer with microsatellite instability), leukemia (acute leukemia or myeloblastic leukemia), non-Hodgkin's lymphoma, multiple myeloma, myelodysplastic syndrome.

[0182] In one aspect, the subject or patient of treatment is a mammal, preferably a human subject. The subject may be male or female and may be of any age.

[0183] In the case of tumors (e.g., cancer), the antibody or fragment thereof (e.g., an antibody or fragment thereof that specifically binds to CD47 and PD-L1) may reduce the number of cancer cells; reduce the initial tumor size; inhibit (i.e., slow to some extent and preferably stop) cancer cell invasion into peripheral organs; inhibit (i.e., slow to some extent and preferably stop) tumor metastasis; inhibit tumor growth to some extent; and / or alleviate to some extent one or more symptoms associated with the disorder. The antibody or fragment thereof may prevent to some extent the growth of cancer cells and / or kill existing cancer cells, which may be cytostatic and / or cytotoxic. With respect to cancer therapy, in vivo efficacy may be measured, for example, by assessing overall survival (OS), time to tumor growth (TTP), overall tumor response rate (ORR) to treatment, duration of response (DR) and / or quality of life.

[0184] As used herein, the terms “co-administration,” “co-administered,” and “in combination with,” referring to an anti-CD47 / PD-L1 antibody and one or more different therapeutic agents, are expected to mean, refer to, or include the following: 1) simultaneous administration of such a combination of an anti-CD47 / PD-L1 antibody of the invention and a therapeutic agent to a patient in need of treatment, where such components are formulated together in a single dosage form that releases said components to said patient substantially simultaneously; 2) the substantially simultaneous administration of such combinations of anti-CD47 / PD-L1 antibodies of the invention and therapeutic agents to a patient in need of such treatment, where such components are formulated separately in different dosage forms, and the introduction of such components occurs approximately simultaneously in the indicated patient after release of such components approximately simultaneously in the identified patient; 3) sequential administration of such combinations of anti-CD47 / PD-L1 antibodies of the invention and therapeutic agents to a patient in need of treatment, where such components are formulated in separate dosage forms from one another, which are taken sequentially by the patient with a sufficient time interval between each administration, such that upon administration, the components are released to the patient at substantially different times; and 4) Sequential administration of such combinations of antibodies against CD47 and PD-L1 of the present invention and therapeutic agents to a patient in need of treatment, where such components are formulated together in a single dosage form which releases said components in a controlled manner and upon release to said patient at the same and / or different time points, simultaneously, sequentially or conjointly, wherein each part may be administered either by the same or different routes.

[0185] The anti-CD47 / PD-L1 antibodies of the invention may be administered without further therapeutic treatment, i.e., as a stand-alone therapy. Moreover, treatment with the antibodies of the invention may be accompanied by at least one further In some embodiments of the invention, the anti-CD47 / PD-L1 antibody may be administered in combination or formulated together with a different cancer medicine / agent.

[0186] As used herein, the term "cytotoxic agent" refers to a substance that inhibits or prevents the function of cells and / or causes destruction of cells. The term includes radioactive isotopes (e.g., At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P 32 and radioactive isotopes of Lu), chemotherapeutic agents, and toxins of bacterial, fungal, plant or animal origin, including fragments and / or variants thereof, such as small molecule toxins or enzymatically active toxins.

[0187] A "chemotherapeutic agent" is a compound useful in the treatment of cancer. Examples of chemotherapeutic agents include alkylating agents, such as thiotepa and cyclophosphamide (CYTOXAN®); alkyl sulfonates, such as busulfan, improsulfan, and piposulfan; aziridines, such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolmelamine; acetogenins (e.g., bullatacin and bullatacinone); beta-lapachone; lapachol; colchicine; betulinic acid; camptothecin (synthetic analog topotecan (HYCAMTIN®), CPT-11 (ICAMTIN®), and cyclophosphamide (HYCAMTIN®). camptothecin, rinotecan, CAMPTOSAR®, acetylcamptothecin, scopolecin, and 9-aminocamptothecin; bryostatin; kallistatin; CC-1065 (including its adozelesin, carzelesin, and bizelesin synthetic analogs); podophyllotoxin; podophyllic acid; teniposide; cryptophycins (e.g., cryptophycin 1 and cryptophycin 8); dolastatins; duocarmycins (including the synthetic analogs KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictin; spongistatin; nitrogen mustards, e.g., chlorambucil, chlornaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride oxide hydrochloride), melphalan, novembitine, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine and ranimustine; antibiotics, such as enediyne antibiotics (e.g. calicheamicins, e.g. calicheamicin gamma II and calicheamicin omega II (e.g. Agnew, Chem. Intl. Ed. Engl., 33:183-186 (1994)); the dynemicins, including dynemicin A; esperamicin; and the neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores, aclacinomycin, actinomycin, authramicin, azaserine, bleomycin, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (ADRIAMYCIN® morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, doxorubicin HCl liposome injection (DOXOL®), liposomal doxorubicin TLC D-99 (including MYOCET®, PEGylated liposomal doxorubicin (including CAELYX®, and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcelomycin, mitomycins, such as mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptomycin, glycine, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate, gemcitabine (GEMZAR®), tegafur (UFTORAL®), capecitabine (XELODA®), epothilones, and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmophor, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; anti-adrenals such as amino Glutethimide, mitotane, trilostane; folic acid supplements such as floric acid; aceglatone; aldophosphamide glycosides; aminolevulinic acid; eniluracil; amsacrine; bestravcil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfornitine; elliptinium acetate; etoglucide; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocin; mitoguazone; mitoxantrone; mopidamol; nitraerine; pentostatin; phenamet; pirarubicin; rosoxantrone; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, OR); razoxane; rhizoxin; schizofiran; spirogermanium; tenuazonic acid; triazicon; 2,2',2"-trichlorotriethylamine; trichothecenes (e.g., T-2 toxin, veraculin A, roridin A, and anguidine); urethanes; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); thiotepa; taxoids such as paclitaxel (Taxol®), albumin engineered nanoparticle formulations of paclitaxel (ABRAXANE®), and docetaxel (TAXOTERE®); chlorambucil;6-thioguanine; mercaptopurine; methotrexate; platinum agents, such as cisplatin, oxaliplatin, and carboplatin; vincas that prevent tubulin polymerization to form microtubules, including vinblastine (VELBAN®), vincristine (ONCOVIN®), vindesine (ELDISINE®), FILDESIN®, and vinorelbine (NAVELBINE®); etoposide (VP16); ifosfamide; mitoxantrone; leucovorin; novantrone; edatrexate. oxetine; daunomycin; aminopterin; ibandronate; the topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoids, such as retinoic acid, including bexarotene (TARGRETIN®); bisphosphonates, such as asclodronate (e.g., BONEFOS® or OSTAC®), ethilodonate (DIDROCAL®), NE-58095, zoledronic acid / zoledronate (ZOMETA®), alendronate (FOSAMAJX®), troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides, particularly those that inhibit expression of genes in signal transduction pathways involved in abnormal cell proliferation, such as PKC-alpha, Raf, H-Ras, and epidermal growth factor receptor (EGF-R); vaccines, such as the THERATOPE vaccine and gene therapy vaccines, e.g. for example, ALLOVECTIN® vaccine, LEUVECTIN® vaccine, and VAXID® vaccine; topoisomerase 1 inhibitors (e.g., LURTOTECAN®); rmRH (e.g., ABARELIX®); BAY439006 (sorafenib; Bayer); SU-11248 (Pfizer); perifosine, COX-2 inhibitors (e.g., celecoxib or etoricoxib), proteosome inhibitors (e.g., PS341); bortezomib (VELCADE®); CCI-779;Tipifarnib (RI; 1577); orafenib, ABT510; Bcl-2 inhibitors, such as oblimersen sodium (GENASENSE®); pixantrone; EGFR inhibitors (see definition below); tyrosine kinase inhibitors (see definition below); and pharmacologic acceptable acids or derivatives of any of the above; and combinations of two or more of the above, such as CHOP, which is an abbreviation for combination therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone, and FOLFOX, which is an abbreviation for treatment with oxaliplatin in combination with 5-FU and leucovorin (ELOXATIN™).

[0188] Also included in this definition are antihormonal agents, such as antiestrogens with a mixed agonist / antagonist profile, which act to regulate or inhibit hormone action on tumors, including tamoxifen (NOLVADEX®), 4-hydroxytamoxifen, toremifene (FARESTON®), idoxifene, droloxifene, raloxifene (EVTSTA®), trioxyfene, ketoxifene, and selective estrogen receptor modulators (SERMs), For example, SERM3; pure antiestrogens without agonistic properties, such as fulvestrant (FASLODEX®), and EM800 (such agents can also block estrogen receptor (ER) dimerization, inhibit DNA binding, increase ER turnover, and / or suppress ER levels); steroidal aromatase inhibitors, such as formestane and exemestane (AROMASIN®), and nonsteroidal aromatase inhibitors, such as anastrazole (AREVIIDEX®). aromatase inhibitors, including vorozole (RIVISOR®), megestrol acetate (MEGASE®), fadrozole, and other aromatase inhibitors including the imidazoles; luteinizing hormone-releasing hormone agonists, including leuprolide (LUPRON® and ELIGARD®), goserelin, buserelin, and tripterelin; progestins, such as megestrol acetate and medoxomil acetate; sex steroids including roxyprogesterone, estrogens such as diethylstilbestrol and premarin, and androgens / retinoids such as floxymesterone, all-trans retinoic acid and fenretinide; onapristone; antiprogesterones; estrogen receptor downregulators (ERDs); antiandrogens such as flutamide, nilutamide and bicalutamide; testolactone; and pharma- ceutically acceptable salts, acids or derivatives of any of the above; and combinations of two or more of the above.

[0189] Other therapeutic agents which may be used in combination with the anti-CD47 / PD-L1 antibodies of the invention may be inhibitors of growth factor function, for example such inhibitors include growth factor antibodies and growth factor receptor antibodies (e.g. anti-erbB2 antibodies, trastuzumab [Herceptin], anti-EGFR antibodies, panitumumab, anti-erbB1 antibodies, cetuximab [Erbitux, C225], and any of the growth factor or growth factor receptor antibodies disclosed by Stern et al. Critical reviews in oncology / haematology, 2005, Vol. 54, pp11-29); anti-angiogenic agents, such as those which inhibit the effects of vascular endothelial growth factor [e.g. anti-vascular endothelial growth factor antibodies, bevacizumab (Avastin)], anti-vascular endothelial growth factor receptor antibodies, such as anti-KDR antibodies and anti-flt1 antibodies; antisense nucleotides, such as those directed against the targets listed above, for example ISIS 2503, anti-ras antisense or G3139 (Genasense), anti-bcl2 antisense; gene therapy approaches, including approaches to replace abnormal genes, such as abnormal p53 or abnormal BRCA1 or BRCA2, approaches such as those using GDEPT (gene directed enzyme prodrug therapy), cytosine deaminase, thymidine kinase or bacterial nitroreductase enzymes, and and approaches to increase patient resistance to chemotherapy or radiotherapy, e.g. multidrug resistance gene therapy; e.g. alemtuzumab (Campus-1H), treatment with monoclonal antibodies directed against CD52, or treatment with antibodies directed against CD22, ex vivo and in vivo approaches to increase the immunogenicity of patient tumor cells, transfection with cytokines, e.g. interleukin 2, interleukin 4 or granulocyte-macrophage colony-stimulating factor, approaches to reduce T cell anergy, e.g. treatment with monoclonal antibodies that inhibit CTLA-4 function, approaches using transfected immune cells, e.g. dendritic cells transfected with cytokines, approaches using tumor cell lines transfected with cytokines and approaches using anti-idiotypic antibodies, ex vivo approaches to increase the immunogenicity of patient tumor cells, transfection with cytokines, e.g. interleukin 2, interleukin 4 or granulocyte-macrophage colony-stimulating factor, approaches using anti-T cell anergy, e.g. treatment with monoclonal antibodies that inhibit CTLA-4 function, approaches using transfected immune cells, e.g. approaches using dendritic cells transfected with cytokines, approaches using tumor cell lines transfected with cytokines and approaches using anti-idiotypic antibodies, Immunotherapeutic approaches include adoptive T cell transfer, using T cells that have been non-specifically activated in vivo or targeted to a specific antigen of interest; inhibitors of protein degradation, such as proteasome inhibitors, e.g., Velcade (bortezomib); biotherapeutic approaches, such as those that use peptides or proteins (e.g., antibodies or soluble external receptor domain constructs) that either sequester receptor ligands, block ligand binding to the receptor, or reduce receptor signaling (e.g., by increasing receptor degradation or reducing expression levels).

[0190] Dosage and Route of Administration The anti-CD47 / PD-L1 antibodies of the invention must be administered in an amount effective to treat the condition in question, i.e., at dosages and for periods of time necessary to achieve the desired result. A therapeutically effective amount may vary according to factors such as the particular condition being treated, the age, sex, and weight of the patient, and whether the anti-CD47 / PD-L1 antibody is administered as a stand-alone treatment or in combination with one or more additional treatments.

[0191] Dosage regimes may be adjusted to provide an optimal response. For example, a single bolus may be administered, or several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. Particularly useful is the preparation of parenteral compositions in standard dosage forms for ease of administration and uniformity of dosage. Unit dosage forms, as used herein, are intended to refer to physically discrete units suitable as unitary dosages for the patient / subject to be treated; each unit contains a predetermined amount of active compound calculated to produce the desired therapeutic effect, in association with the desired pharmaceutical carrier. The specification of standard dosage forms of the present invention is typically dictated by and directly dependent on (a) the unique characteristics of the chemotherapeutic agent and the particular therapeutic or prophylactic effect to be achieved, and (b) the limitations inherent in the art of compounding such active compounds for the treatment of subjects.

[0192] Thus, those skilled in the art will recognize from the disclosure herein that dosages and dosing regimens will be adjusted according to methods well known in the therapeutic field. That is, the maximum tolerated dose can be easily established, and the effective amount that provides a detectable therapeutic effect on the patient can also be determined, as can the temporal requirements for administering each agent to provide a detectable therapeutic effect on the patient. Thus, in this document, some dosages and dosing regimens are provided as examples, but these examples do not in any way limit the dosages and dosing regimens that may be required for a patient in the practice of the present invention.

[0193] It should be noted that dosage values ​​may vary depending on the type and severity of the condition to be alleviated, and may include single or multiple doses. Moreover, for any particular subject, a particular dosage regimen may be adjusted over time according to the individual need and the judgment of the medical professional administering or supervising the administration of the composition. It should be understood that the dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed compositions. Furthermore, dosage regimens involving the compositions of the invention may be based on a variety of factors, including the type of disease, the patient's age, weight, sex, medical condition, severity of the condition, route of administration, and the particular anti-CD47 / PD-L1 antibody used. Thus, dosage regimens may vary widely, but are routinely determinable using standard methods. For example, doses may be adjusted based on pharmacokinetic or pharmacodynamic parameters, which may include clinical effects, e.g., toxic effects, and / or laboratory values. Thus, the present invention includes intrapatient dose escalation, as determined by one of skill in the art. Methods for determining appropriate dosages and regimens are well known in the art and will be understood by one of skill in the art once provided with the ideas disclosed herein.

[0194] Examples of suitable dosing regimes are provided above. Suitable doses of the antibodies against CD47 and PD-L1 of the present invention will be in the range of 0.1 to 200 mg / kg, preferably 0.1 to 100 mg / kg, including about 0.5 to 50 mg / kg, such as about 1 to 20 mg / kg. For example, the antibodies against CD47 and PD-L1 may be administered at a dose of at least 0.25 mg / kg, such as at least 0.5 mg / kg, including at least 1 mg / kg, such as at least 1.5 mg / kg, such as at least 2 mg / kg, including at least 3 mg / kg, including at least 4 mg / kg, such as at least 5 mg / kg; and for example up to 50 mg / kg, up to 30 mg / kg, including up to 20 mg / kg, including up to 15 mg / kg. Typically, repeated at appropriate time intervals, such as once a week, every 2 weeks, every 3 weeks, or every 4 weeks, and as long as deemed appropriate by the responsible physician, who may in some cases increase or decrease the dose, if necessary.

[0195] Manufactured articles (products) and kits The following aspects of the invention are products containing products used to treat cancer, such as HNSCC, cervical cancer, cancer of unknown primary, glioblastoma, esophageal cancer, bladder cancer, TNBC, CRC, hepatocellular carcinoma, melanoma, NSCLC, kidney cancer, ovarian cancer, Hodgkin's lymphoma, MSI CRC, leukemia (acute leukemia or myeloblastic leukemia), non-Hodgkin's lymphoma, multiple myeloma, myelodysplastic syndrome. The product is a container in a package and a label or instruction insert placed on or enclosed within the container. Suitable containers are, for example, cans, vials, syringes, and the like. The container may be made from a variety of materials, such as glass or plastic. The container contains a composition effective for treating a particular condition and may have a sterile inlet channel (e.g., the container may be an intravenous solution bag or vial with a stopper pierceable by a hypodermic needle). At least one active ingredient in the composition is an anti-PD-L1 antibody according to the invention. The label or instructions in the package indicate that the composition is used to treat a particular condition. The label or package instructions in the package should further contain instructions for administration of the antibody composition to a patient.

[0196] The package insert contains typical instructions contained in the package of a marketed therapeutic product, including some information on indications, frequency of administration, dosage, route of administration, contraindications and / or cautions for such therapeutic products. In one embodiment, the package insert indicates that the composition is intended to be used for the treatment of cancer, such as HNSCC, cervical cancer, cancer of unknown primary, glioblastoma, esophageal cancer, bladder cancer, TNBC, CRC, hepatocellular carcinoma, melanoma, NSCLC, renal cancer, ovarian cancer, Hodgkin's lymphoma, MSI CRC, leukemia (acute leukemia or myeloblastic leukemia), non-Hodgkin's lymphoma, multiple myeloma, myelodysplastic syndrome.

[0197] Additionally, the article may further include a second container containing a pharma- ceutically acceptable buffer, such as bacteriostatic water for injection (BSVI), phosphate-buffered saline, Ringer's solution, and dextrose solution. Additionally, the article may also include other products necessary from a commercial and consumer standpoint, particularly other buffers, diluents, filters, needles, and syringes.

[0198] The present invention also relates to kits that may be used for a variety of purposes, such as for the detection of PD-L1 in mammalian tissues, cells, or body fluids. Such kits may be useful for screening related to PD-L1 diseases. The kits include a specific binding agent or antibody of the present invention, and a means for indicating the reaction of the specific binding agent or anti-PD-L1 antibody, if present. In one embodiment, the antibody is a monoclonal antibody. In one embodiment, the antibody that binds to PD-L1 is labeled. In another embodiment, the antibody is an unlabeled primary antibody, and the kit further includes a means for detecting the primary antibody. In one embodiment, the detection means includes a labeled secondary antibody that is an anti-immunoglobulin. The antibody may be labeled with a marker selected from the group consisting of a fluorochrome, an enzyme, a radionuclide, and a radio-opaque material. The kit may be a kit containing an antibody for detecting and quantifying PD-L1 in vitro, for example, by implementing ELISA or Western blotting. Also, as in the case of articles, the kits include a container, and a label or package insert located on or within the container. The container holds a composition comprising at least one anti-PD-L1 antibody according to the invention. Additional containers may include, for example, diluents and buffers, control antibodies. A label or package insert in the package may contain a description of the composition and instructions for its use in vitro or for diagnostic purposes.

[0199] Diagnostic Uses and Compositions The anti-CD47 / PD-L1 antibodies of the invention may also be used in diagnostic processes (e.g., in vitro, ex vivo). For example, the anti-CD47 / PD-L1 antibodies may be used to detect or measure the levels of CD47 and / or PD-L1 in a sample obtained from a patient (e.g., a tissue sample or a body fluid sample, such as inflammatory exudate, blood, serum, interstitial fluid, saliva, or urine). Suitable methods for detection and measurement include immunoassays, such as flow cytometry, enzyme-linked immunosorbent assay (ELISA), chemiluminescence assays, radioimmunoassays, and immunohistology. The invention further includes kits, such as diagnostic kits, comprising the anti-CD47 / PD-L1 antibodies described herein. EXAMPLES

[0200] The following examples are provided for a better understanding of the present invention. These examples are for illustrative purposes only and are not to be construed as limiting the scope of the invention in any manner.

[0201] All publications, patents, and patent applications cited herein are hereby incorporated by reference. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those skilled in the art that certain changes and modifications may be made based on the ideas disclosed in the invention without departing from the spirit and scope of the accompanying variations. Practice of the invention.

[0202] Example 1. Production of recombinant antigens and antibodies in mammalian cell suspension cultures. The sequences of the extracellular domains of human CD47 (Leu19-Val134) and PD-L1 (Phe19-Arg238) (SEQ ID NOs: 107-108) were cloned at the SalI / NotI restriction sites into a plasmid for producing Fc-tagged proteins in mammalian cells (Figure 1). The required amount of plasmid was produced in E. coli cells and then Q Purification was performed using the iagen kit.

[0203] The sequence of the variable domain of an anti-CD47 antibody (B6H12, Stanford University, US20130142786) was cloned into a plasmid for producing IgG1 protein in mammalian cells. The required amount of the plasmid was produced in E. coli cells and purified using a Qiagen kit.

[0204] Antibodies and antigens were produced in an established cell line (CHO-K1) derived from Chinese hamster ovary cells. Suspension cultures were performed in flasks on an orbital shaker using serum-free medium (Life Technologies Corporation) and according to the manufacturer's instructions. For transient expression, linear polyethyleneimine (e.g., PEI MAX, Polysciences) was used to induce 2-fold transfection of the IgG1-specific ... * 10 6 Cells were transfected at a concentration of 1000 μg / ml. The DNA / PEI ratio was 1:3 / 1:10. Five to seven days after transfection, the cell culture was centrifuged at 2000 g for 20 min and filtered through a 0.22 μm filter. Target proteins were isolated from the culture medium by affine HPLC.

[0205] Recombinant Fc protein was isolated and purified from cell culture on a Protein A column for affine HPLC. The clarified culture fluid was passed through a 5 ml HiTrap rProtein A Sepharose FF column (GE Healthcare) equilibrated with phosphate-buffered saline (PBS, pH 7.4). The column was then washed with 5 volumes of PBS to remove non-specific binding components. Bound antigen was eluted with 0.1 M glycine buffer (pH 8). The main protein elution peak was collected and brought to neutral pH with 1 M Tris buffer (pH 8). All steps were performed under a flow rate of 110 cm / h. The protein was then dialyzed into PBS (pH 7.4) using SnakeSkin dialysis tubing technology, filtered (0.22 μm), transferred into tubing, and stored at -70°C.

[0206] The purity of the resulting protein solution was evaluated by non-reducing SDS-PAGE (12% gel). Example 2. Preparation of full length antibodies.

[0207] Cloning was performed by standard techniques. PCR products were generated containing the genes of the heavy and light variable domains of the antibody with primers containing restriction sites. The heavy variable domain was cloned into the vector pEE-Hc IgG1 at the Sal1 / Nhe1 restriction sites. The light variable domain was cloned into the vector pEE-CK at the Sal1 / BsiW1 restriction sites. The resulting gene construct was used for transient production of the protein in CHO-T cell lines. The protein was isolated and purified according to standard methods by affinity chromatography on bacterial protein A as described in Example 1. Electrophoresis was performed on a 12% denaturing PAGE supplemented with mercaptoethanol (Figure 3) and on an 8% denaturing PAGE without mercaptoethanol (Figure 4).

[0208] Example 3. Engineering of the naive human Fab phage library MeganLib™ B lymphocyte total RNA from over 1000 individual human donor blood samples was isolated using the RNeasy mini kit (QIAGEN) according to the suggested protocol. RNA concentration assays were performed using the Nanovue kit (GE Healthcare); the quality of the isolated RNA was tested by 1.5% agarose gel electrophoresis.

[0209] The MMLV RT kit (Evrogen) was used according to the recommended protocol, using MMuLV reverse transcriptase and random hexamer oligonucleotides as primers. A reverse transcription reaction was carried out using the same.

[0210] The reverse transcription product was used as a matrix for a two-step polymerase chain reaction to obtain the genes of the variable domains flanked by restriction sites; the reaction was carried out using an oligonucleotide kit according to the protocol by [J Biol Chem. 1999 Jun 25; 274(26): 18218-30].

[0211] The resulting DNA product (VL-CK-VH) was treated with NheI / Eco91I restriction endonucleases and ligated into the original phagemid pH5. The ligation product was transformed into SS320 E. coli electrocompetent cells according to the protocol [Methods Enzymol. 2000;328:333-63.]. The repertoire of the combinatorial Fab phage display library MeganLibTM consists of 10 11 The transformants were used to prepare Fab phage library products according to the method previously described [J Mol Biol. 1991 Dec 5;222(3):581-97].

[0212] Example 4. Immunization of llamas with human CD47 antigen and generation of a phage display library of llama antibody fragments Animals, Lama Glama, were immunized five consecutive times by subcutaneous administration of antigenic material mixed with an equal volume of complete (first injection) or incomplete (other injections) Freund's adjuvant. Recombinant human CD47 protein (1 mg / injection) from Example 1 was used as antigen. Antigen injections were given at the following intervals: 0, 2, 4, 5, 8 weeks. Starting from the third injection, blood samples (50 ml) were collected five times after each injection. 3.8% sodium citrate was used as anticoagulant (1:9). Blood was diluted 2-fold with sterile saline solution. Then, 30 ml of the diluted blood solution was diluted with 15 ml of Lymphoprep. TM (Axis-Shield, Norway) medium (density 1.077 g / ml) and centrifuged at 800 g for 20 min. Mononuclear cells (lymphocytes and monocytes) were selected from the interphase zone of the plasma / Lymphoprep medium and washed with sterile PBS.

[0213] The obtained titer of serum immunoglobulins against CD47 was evaluated according to standard protocols and found to be at least 1 / 100000, which is sufficient for preparing a library of antibodies.

[0214] Total RNA from mononuclear llama cells was isolated using the RNeasy mini kit according to the protocol (QIAGEN). RNA concentration assays were performed using Nanovue (GE Healthcare); the quality of the isolated RNA was tested by 1.5% agarose gel electrophoresis.

[0215] Reverse transcription reactions were carried out using MMuLV reverse transcriptase and random hexamer primers using the MMLV RT kit (Evrogen) according to the recommended protocol.

[0216] The reverse transcriptase was used as a matrix in a two-step polymerase chain reaction to obtain monodomain VHH, scFv or Fab genes flanked by restriction sites; the reaction was carried out using an oligonucleotide kit and the protocol according to [FASEB J. 2007 Nov;21(13):3490-8]. The obtained VHH gene DNA products were treated with NcoI / NotI restrictase and ligated into the original phagemid pscFv, which is similar in composition to pHEN2 used in [FASEB J. 2007 Nov;21(13):3490-8]. The protocol [Methods Enzymol. 2000;328: 333-63.] The ligation products were transformed into the electrocompetent SS320 cells prepared accordingly. The repertoire of the VHH-based library constructed ranged from 0.5 to 2 * The repertoires of the constructed scFv / Fab-based libraries ranged from 0.5 to 2. * 10E+9 / 1.2~2.5 *There were 10E+9 independent transformants. Phage library products were prepared according to the method previously described [J Mol Biol. 1991 Dec 5;222(3):581-97].

[0217] Example 5. Selection of a phage display library of antibody fragments Specific anti-CD47 phage antibodies were selected from phage Fab, VHH, or scFv display libraries (Examples 3, 4) using magnetic beads and the KingFisher Flex device technology, which can simultaneously run up to 96 different schemes and variants, according to conventional selection methods described in [EMBO J. 1994 Jul 15;13(14):3245-60, Nat Biotechnol. 1996 Mar;14(3):309-14; J Mol Biol.1991 Dec 5;222(3): 581-97].

[0218] Human biotinylated PD-L1 / CD47 antigen (Fc, EPEA) was intentionally immobilized on streptavidin magnetic beads (NEB) at a concentration of 10 μg / ml for the first round, 2 μg / ml for the second round, and 0.4 and 0.2 μg / ml for the third and fourth rounds, respectively. The antigen was incubated with the beads for 1 h at room temperature on a rotator. The beads were then washed with PBS (pH 7.4) and the bead surface was blocked with a solution of 2% skim milk or 1% BSA in PBS (pH 7.4) for 1 h. The human phage library MeganLibTM was incubated for 2 h in PBS (pH 7.4) containing 2% skim milk and non-target antigens containing target antigen tags. * 10 13 The phage were diluted to a concentration of 10 ...

[0219] Unbound phages were removed by washing the magnetic beads several times with a solution of PBS (pH 7.4) containing 0.1% Tween-20. The number of washing cycles was increased from round to round (3 washing cycles in the first round, 9 washing cycles in the second round, and 15 washing cycles in the fourth round). Phages bound to antigens on the magnetic bead surface were eluted from the beads with 100 mM Gly-HCl solution (pH 2.2) under agitation for 15 min, and then neutralized with 1 M Tris-HCl (pH 7.6). E. coli TG1 bacteria were infected with the phages, grown in medium, and then used for the next selection cycle. After 3 or 4 rounds, phagemid DNA was isolated from E. coli TG1 cultures according to the manufacturer's (Qiagen) protocol. Polyclonal phage enzyme immunoassay (ELISA) was used to enrich the library against the target antigen and to evaluate the presence of nonspecifically bound phage particles.

[0220] Example 6. ELISA of polyclonal phages against specific and non-specific antigens. To perform the ELISA, target antigens (CD47 / PD-L1) and non-target antigens (including Fc fusion proteins) were immobilized on high-binding plates (Greiner-Bio). Proteins were added at concentrations of 1 μg / ml and 5 μg / ml, respectively, in 0.1 M NaHCO3 (pH 9.0) and titrated at 2-7 dilution increments, then the sealed plates were incubated overnight at 4°C. All subsequent steps were performed according to standard ELISA protocols using a highly automated Tecan Freedom EVO 200-based robotic platform (Tecan). To block non-specific binding, plate wells were filled with 2% skim milk in PBS (pH 7.4) or 1% PBS (pH 7.4). Blocking buffer containing 0.05% BSA was added. The plates were incubated for 1 h at room temperature. After several washing cycles with phosphate-saline buffer containing Tween 20 (PBST), 50 μl / well of test polyclonal phage was added. After washing, each well was coated (50 μl / well) with anti-M13 HRP-conjugated secondary antibody (Pierce-ThermoScientific) (1:7500) in PBST. After 50 min of incubation at room temperature, the plates were washed three times with PBST. Colorimetric signal was obtained by adding substrate solution (H2O2-0.02% and TMB in CH3COONa pH 5.5) for 10 min; color development was then blocked by adding 1% sulfuric acid (20 μl). Color signal was measured at 450 nm using an appropriate Tecan-Sunrise plate reader (Tecan).

[0221] ELISA of the polyclonal phage products showed significant enrichment after the third and fourth rounds of selection on the target antigen. The library was selected for recloning and further screening, where a signal was observed to be greater than 5-fold at the lowest dilution of the phage library versus the heterologous control antigen.

[0222] Example 7 Recloning of antibody fragment genes into expression plasmids After successive rounds of selection, the antibody variable domain genes were recloned from the phagemid vector into expression plasmids using restriction ligation techniques following standard protocols.

[0223] The resulting pool of clones enriched for VHH monodomains or scFvs specific for CD47 was recloned into the expression plasmid pET-22 (Novagen) under the control of the T7 promoter, carrying myc and His6 tag sequences at the C-terminus of the VHH. The Fab genes of the library containing the enriched sequences against the CD47 antigen were recloned into the expression vector pLL4, under the control of the lac promoter, further containing myc and His6 tag sequences at the C-terminus of the heavy chain CH1 domain.

[0224] The expression vectors containing the antibody fragments were then transformed into E. coli B121(DE3)Gold (Stratagene) for production of the antibody fragments by secretion into the culture medium and comparative analysis of the affinity of the variable antibody fragments from the display library to the antigen by ELISA using the Mabnext flow chart platform.

[0225] Example 8. Analysis of specific binding of scFv or VHH monodomains to human CD47-Fc. Binding of the specific test antibody fragment of Example 4 to human CD47-Fc was measured using ELISA. ELISA well plates (Nunc ImmunoMaxisorp) were covered with 50 μl / well of human CD47-Fc (Biocad) (0.5 μg / ml in 1× coating carbonate buffer), sealed, and incubated overnight at 4° C. All further steps were carried out according to standard ELISA protocols on high-performance automation platforms based on robotic systems such as GenetixQ-pix2xt (Molecular Devices) and Tecan Freedom EVO 200 (Tecan). Non-specific binding was blocked by adding blocking buffer BB (0.5% skim milk in 200 μl PBS). The plates were incubated for 1 hour at room temperature on a shaker. After washing with PBS-Tween, each cell was coated with 100 μl of cell supernatant containing the test antibody fragment. The plate was incubated for 1 hour at room temperature on a shaker; each plate well was then rinsed with PBS-Tw Plates were shaken in a rotary shaker (50 min at room temperature) and then washed five times with PBS-Tween buffer as described above. After washing, mouse anti-MYC IgG clone 9E10 (ThermoFisher Scientific) (50 μl / well) was added in PBS-Tween (1:5000). Plates were shaken in a rotary shaker (50 min at room temperature) and then washed five times with PBS-Tween buffer as described above. After washing, anti-mouse IgG HRP conjugate (ThermoFisher Scientific) (50 μl / well) was added in PBS-Tween (1:10000). Plates were shaken in a rotary shaker (50 min at room temperature) and then washed five times with PBS-Tween buffer as described above. Colorimetric signal was obtained by adding TMB (50 μl / well) until saturation (average 10-12 min); further color development was blocked by adding stop solution (25 μl / well, 1% sulfuric acid). The color signal was measured at 450 nm using a suitable Tecan-Sunrise plate reader (Tecan). Antibody binding was proportional to the signal produced. Clones with a color signal greater than 5 times the background signal were tested in a competitive ELISA assay to identify antagonistic specific antibody fragments that block the interaction between SIRP-Fc ligand (BIOCAD) and human CD47-Fc receptor under conditions similar to those described in [WO2016048188 A8] and with a 4-fold reduced signal compared to the control without the test antibody fragment.

[0226] Screening of 2400 clones yielded 265 scFv and VHH clones that showed a signal more than 5-fold higher than background. The panel of positive clones yielded 27 antagonistic clones capable of blocking the interaction between SIRP-Fc ligand (BIOCAD) and human CD47-Fc receptor. The nucleotide sequences of the positive clone genes were determined by Sanger sequencing on a 3130xl Genetic analyzer (Applied Biosystems). Six exemplary VHH monodomain clones were obtained that differed in sequence by at least one amino acid but each had a CDR region that was at least 90% homologous to the others, thus indicating that they were derived from one parental clone by in vivo maturation in immunized llamas (Table 1).

[0227] Table 1. Sequences of CD47-specific binding VHH monodomains against human CD47.

[0228] [Table 2-1]

[0229] [Table 2-2]

[0230] [Table 2-3]

[0231] Example 9. Analysis of specific binding of Fab to human CD47-Fc. Fab production was carried out according to standard techniques: bacterial cells were transformed with an expression vector containing the Fab gene, and subsequently, during cultivation of the resulting transformants, an inducer that induces transcription of the lac operon was added to the medium, causing expression of the Fab.

[0232] An ELISA was then performed to search for Fabs that bound to human CD47. B6H12 Fab with published sequence (see Example 1) was used as a positive control. To test for specific binding, ELISA well plates (medium binding, Greiner bio one) were covered with 50 μl / well of CD47 Fc-llama (0.2 μg / ml in 1× carbonate buffer), sealed, and incubated overnight at 4° C. Genetix Qpix2xt (Molecular Devices) and Tecan All further steps were carried out according to standard ELISA protocols on a high-performance automated platform based on a robotic system such as Freedom EVO 200 (Tecan). Non-specific binding was blocked by adding blocking buffer BB (0.5% skim milk in 200 μl PBS). The plate was incubated for 1 h at room temperature on a shaker. After washing with PBS-Tween, each cell was coated with 60 μl / well of cell supernatant containing the test antibody fragment. The plate was incubated for 1 h at room temperature; then each plate well was washed three times with PBS-Tween buffer. After washing, each well was coated (50 μl / well) with anti-human Fab HRP-conjugated secondary antibody (Pierce-ThermoScientific) (1:7500) in PBS-Tween. The plate was incubated for 1 h at room temperature and washed three times with PBS-Tween buffer as described above. Further colorimetric signal was obtained by adding TMB (50 μl / well) until saturation (15 min); further color development was blocked by adding stop solution (25 μl / well, 1% sulfuric acid). Color signal was measured at 450 nm using a suitable Tecan-Sunrise plate reader (Tecan). Antibody binding was proportional to the signal produced. Clones with color signal exceeding the signal from the control antibody were tested for non-specific binding by ELISA.

[0233] Example 10. Analysis of non-specific binding of Fabs to various human antigens. The secondary screen aims at the selection of Fab-producing clones that interact with the full-length CD47 antigen and do not interact with non-specific antigens and also compete with the ligand (CD47) for binding to SIRPa.

[0234] Non-specific binding of the test Fabs to other antigens was analyzed using ELISA. The analysis was performed as described above, but 3DHer3-H6E, INFα2b, PD-L1-Fc-llama (2.5 μg / ml in 1x carbonate buffer) were used as antigens for immobilization. CD47 FE and CD47 Fc llama (0.2 μg / ml in 1x carbonate buffer) were used as specific binding controls. All further steps were performed according to standard ELISA protocols using high-performance automation platforms based on robotic systems such as Genetix Qpix2xt (Molecular Device) and Tecan Freedom EVO 200 (Tecan).

[0235] Preselected anti-human CD47 specific Fabs were tested for their ability to block interaction with the SIRPa receptor using a competitive ELISA. A Fab with published sequence (see Example 1) was used as a positive antagonist control.

[0236] ELISA well plates (high binding, Greiner bio one) were covered with 50 μl / well of SIRPa (0.5 μg / ml in 1× carbonate buffer) and incubated overnight at 4° C. All further steps were carried out according to standard ELISA protocols on high performance automation platforms based on robotic systems such as Genetix Qpix2xt (Molecular Devices) and Tecan Freedom EVO 200 (Tecan). Non-specific binding was blocked by adding blocking buffer BB (0.5% skim milk in 200 μl PBS). Pre-treatment was performed at 4° C. for 1 hour. The plates were incubated at room temperature for 1 hour.

[0237] In parallel, cell supernatants containing test Fab and CD47 Fc llama (final concentration of 1 μg / ml in PBS-Tween) were mixed in a 1:1 ratio in a non-adsorbent plate and incubated for 45 min at room temperature.

[0238] After washing the SIRPa receptor-containing plate to remove the BB, the mixture of Fab and CD47 Fc llama was transferred to the plate and incubated for 45 min at room temperature. Each plate well was then washed three times with PBS-Tween buffer and 50 μl / well of anti-human Fab HRP-conjugated secondary antibody (Pierce-ThermoScientific) was added in PBS-Tween (1:7500). The plate was incubated for 45 min at room temperature and washed three times with PBS-Tween as described above. The colorimetric signal was obtained by adding TMB (50 μl / well) until saturation (average 15 min); further color development was blocked by adding stop solution (25 μl / well, 1% sulfuric acid). The color signal was measured at 450 nm using an appropriate Tecan-Sunrise plate reader (Tecan). Fab binding was inversely proportional to the color signal produced. Clones that showed blocking at the level of the control Fab were considered positive and used for further assays. The variable domain genes of positive clones were sequenced and analyzed according to standard protocols on a PCR-based PCR kit (Microelectronics, Inc., Biosystems).

[0239] Example 11. Comparative screening of anti-CD47 antibody fragments by kinetic dissociation constant koff (kdis). VHH antibody fragments were measured to provide an example of the analysis of kinetic parameters of the interaction of antibody fragments that specifically bind to the CD47 receptor. The dissociation constant (k dis) was used for comparative screening. The affinity constants may be calculated based on the exact protein concentration in the test solution, and candidates were compared with each other by using their dissociation constants, since cell growth medium was used as the protein solution and protein concentration was not measured. The biosensor was pre-rehydrated in water for 1 hour. After activating the biosensor, CD47-Fc was immobilized non-specifically (by NH2 groups) on the biosensor at a concentration of 10 μg / ml in acetate buffer pH 4. The sensor was then immersed in a well containing cell growth medium with specific VHH (from about 1 μg VHH in 1 ml medium), where the complex was allowed to associate. The sensor was then immersed in a buffer solution, where the subsequent steps of dissociation of the complex took place. 1 / 10 of the volume of 10x working buffer was added to the test specimen in E. coli growth medium containing anti-CD47 VHH fragments. The resulting curves were analyzed using Octet Data Analysis (version 7.0) according to standard methods using a 1:1 interaction model.

[0240] The results of koff screening of anti-CD47 VHH candidates are shown in Table 2. Specific binding of all VHH fragments to human CD47 was demonstrated: based on significant kdis, candidate BCD106-02_L.Alecto.VHHSel2_MP1_C7_49 was selected for further study and recloned to obtain a bispecific antibody.

[0241] Table 2. Kinetic dissociation constants of CD47-Fc and VHH

[0242] [Table 3]

[0243] Example 12. Obtaining and producing constructs of asymmetric bispecific anti-CD47 / PD-L1 antibodies. The sequences of all variable domains of the optimized scFv fragments, as well as the genes for the synthesis of wild-type and mutant forms of the variable domains VHH of the candidate BCD106-02-VHH_C7_49 (Table 3), and the sequences of the genes of the light and heavy chain variable domains of the anti-PD-L1 antibody (BCD135, the original human antibody from BIOCAD) were obtained de novo by calculation using a proprietary computer algorithm and PCR synthesis from oligonucleotides obtained on an ASM-2000 (Novosibirsk) synthesizer according to standard protocols [http: / / www.openwetware.org / wiki / DNA_Synthesis_from_Oligos]. The long scFv genes were obtained from the VH and VL genes by using a two-step PCR synthesis from single-stranded DNA molecules. After PCR synthesis, the DNA fragments fractionated on an agarose gel were purified on a QIAquick Gel Extraction Kit (Qiagen) column. The scFv and VHH genes were individually ligated into the plasmid pEE-Fc(knob), while the variable heavy domains of aPD-L1 specific antibodies were individually ligated into the plasmid pEE-Fc(hole). pEE-Fc(knob) contains a human IgG1 Fc with mutations S354C+T366W, and pEE-Fc(hole) contains a human IgG1 Fc with mutations Y349C+T366S+L368A, providing heterodimerization of these Fc moieties with each other with minimal homodimerization. Nat Biotechnol.1998 Jul;16(7):677-81.] together with the aPD-L1 light chain variable domain, also cloned into pEE-Clambda, were co-transiently expressed in CHO-EBNA cells. After ligation-independent cloning by a modified LIC method [Aslanidis C, de Jong PJ. Ligation-independent cloning of PCR products (LIC-PCR). Nucleic Acids Res. 1990;18:6069-6074], the DNA was transformed into E. coli. Constructs containing the correct sequence pEE-Fc(knob)-scFv or pEE-Fc(knob)-VHH were co-transfected with pEE-BCD135-VH-299P-HC-hole and pEE-BCD135-01 4LG VL hzau CL to generate the so-called asymmetric bispecific antibodies. were obtained (see Figure 6). The figure shows a schematic model of asymmetric bispecific antibodies, A-based on anti-CD47 scFv and anti-PD-L1 Fab binding fragment, B-based on anti-CD47 VHH and anti-PD-L1 Fab binding fragment. The resulting gene constructs were used to produce protein in CHO-T cell lines according to Example 2. After purification, the antibodies were very homogeneous in composition, and the production yields of bispecific antibodies ranged from 60 to 260 mg / ml of medium. Figure 7 shows an example of purified bispecific antibodies based on anti-CD47 scFv fragment. Figure 8 shows an example of purified bispecific antibodies based on anti-CD47 VHH fragment. The antibody variant containing the sequence VHH47Opt3 shown in Table 3 produced very low antibody yields and was eliminated from further testing.

[0244] Table 3. Amino acid sequences of the variable domains of wild-type / mutant anti-CD47 VHHs and anti-PD-L1 BCD135 from the BCD106-02-VHH_C7_49 clone. Grey indicates anti-CD47 VHH positions that contain mutations other than those in the wild type.

[0245] [Table 4]

[0246] Example 13. Analysis of the interaction of human PD-L1 / CD47 antigen with anti-PD-L1 / anti-CD47 PD-L1 bispecific antibody on OctetRED96. Analysis of the interaction of the PD-L1 bispecific antibody with human PD-L1 / CD47 antigen was performed on OctetRed96 (Pall-ForteBio). The AR2G biosensor was pre-rehydrated in mQ for 1 h. After activating the biosensor, PD-L1-Fc or CD47-Fc was non-specifically immobilized (by NH2 group) on the biosensor at a concentration of 25μg / ml in acetate buffer pH4. The sensor was then immersed in a well containing anti-PD-L1 / anti-CD47 antibody solution (10μg / ml), where the antibody-antigen complex was allowed to associate. The sensor was then immersed in a buffer solution for the subsequent dissociation step. After subtracting the reference signal, the binding curves were analyzed using Octet data analysis software (version 8.2) according to standard methods and using a 1:1 interaction model.

[0247] The results of the analysis are shown in Table 4. Thus, the antibodies have high affinity, where the affinity for the PD-L1 antigen in this format of antibody has nM values, while the affinity for the CD47 antigen has sub-nM values. Such binding is considered sufficient for the antibody to be able to interact with receptors on target cells for subsequent testing of both in vitro and in vivo therapeutic activity.

[0248] Table 4. Kinetic dissociation constants of anti-CD47 / anti-PD-L1 bispecific antibodies

[0249] [Table 5]

[0250] Example 14. Analysis of the interaction of anti-PD-L1 / anti-CD47 bispecific antibodies with cynomolgus monkey CD47 and PD-L1 receptors on Forte Bio Octet RED 384.

[0251] Experimental studies of antibody affinity to animal CD47 / PD-L1 antigens were performed using Forte The assay was performed on a Forte Bio Octert RED384. Antibodies at a concentration of 20 μg / ml were immobilized on an AR2G sensor (Forte Bio) following standard protocols and manufacturer's instructions. The assay was performed at 30°C using PBS with 0.1% Tween 20 and 0.1% BSA as the working buffer. After baseline recording, the sensor was immersed in a well containing antigen solution (animal CD47 and PD-L1) for 300 seconds, where the complexes were allowed to associate. Complex dissociation in the buffer solution was then detected for 600 seconds.

[0252] After subtracting the reference signal, the binding curves were analyzed using Octet Data Analysis (version 9.0) software according to standard methods and using a 1:1 Global interaction model. The anti-CD47 antibodies specifically bind to the cynomolgus monkey antigens CD47 and PD-L1. Tables 5 and 6.

[0253] Table 5. Kinetic values ​​of antibody interaction with cynomolgus antigen (CD-47).

[0254] [Table 6]

[0255] Table 6. Kinetic values ​​of antibody interaction with cynomolgus antigen (PD-L1).

[0256] [Table 7]

[0257] Example 15. Analysis of non-specific binding of anti-PD-L1 / anti-CD47 bispecific antibodies to a panel of antigens on Forte Bio Octet RED 384. Experimental studies of non-specific binding were performed against a panel of non-specific his-tagged antigens. Anti-hIgG Fc capture (AHC) biosensors pre-rehydrated for 10 min in PBS containing 0.1% Tween-20 and 0.1% BSA as working buffer were used for the measurements.

[0258] The antibody at a concentration of 30 μg / ml was applied to an anti-hIgG Fc capture (AHC) sensor (Fort The sensor was immobilized on a well of 1000 nm PBS (E Bio). Analysis was performed at 30° C. using PBS containing 0.1% Tween 20 and 0.1% BSA as working buffer. After a baseline was prescribed in the buffer solution, the sensor was immersed in a well containing a nonspecific antigen solution for 300 seconds, where the complex was allowed to associate. Complex dissociation in the buffer solution was then detected for 600 seconds.

[0259] Binding curves were analyzed using Octet data analysis (version 9.0) using a 1:1 Global interaction model according to standard methods (after subtraction of the reference signal). The anti-PD-L1 / anti-CD47 bispecific antibody does not bind non-specifically to the antigen panel.

[0260] Example 16. Analysis of the interaction of a panel of FcγRIIIa and anti-PD-L1 / anti-CD47 bispecific antibodies on Forte Bio Octet RED 384. Streptavidin (SAX) biosensors were prehydrated for 30 min in PBS containing 0.1% Tween-20 and 0.1% BSA, and then assayed with Forte Experimental studies of antibody affinity to a panel of Fc binding proteins were performed on Bio Octert RED384.

[0261] Biotinylated Fc-binding Avi-tagged proteins, FcγRIIIa-F158 and FcγRIIIa-V158, at a concentration of 5 μg / ml in FSB kinetic buffer pH 7.4 containing 0.1% Tween-20 and 0.1% BSA were incubated for t RecLoadThe antibody was immobilized on a streptavidin (SAX) sensor at 100 nm for 60 s. After baseline recording, the sensor was immersed in a well containing an antibody solution for 60 s, where the complexes were allowed to associate. Complex dissociation in a buffer solution was then detected for 150 s.

[0262] Binding curves were analyzed using Octet data analysis (version 9.0) according to standard methods using a 2:1 Global interaction model (after subtraction of the reference signal). The anti-PD-L1 / anti-CD47 bispecific antibody specifically binds to the Fc binding proteins FcγRIIIa-F158 and FcγRIIIa-V158. Tables 7 and 8.

[0263] Table 7. Kinetic values ​​of the interaction of antibody BCD-106 (02-001, 03-006, 02-013) with FcγRIIIa-F158.

[0264] [Table 8]

[0265] Table 8: Kinetic values ​​of the interaction of antibody BCD-106 (02-001, 03-006, 02-013) with FcγRIIIa-V158.

[0266] [Table 9]

[0267] Example 17. Analysis of the interaction of anti-PD-L1 / anti-CD47 bispecific antibodies with FcRn on Forte Bio Octet RED 384. Streptavidin (SAX) biosensors were prehydrated for 30 min in PBS containing 0.1% Tween-20 and 0.1% BSA, and then assayed with Forte Experimental studies of antibody affinity to a panel of Fc binding proteins were performed on Bio Octert RED384.

[0268] Biotinylated Avi-tagged FcRn at a concentration of 5 μg / ml in PBS kinetic buffer pH 6 containing 0.1% Tween-20 was incubated for t RecLoad After baseline recording in buffer solution, the sensor was immersed for 60 seconds in a well containing the antibody solution in PBS kinetics buffer pH 6 containing 0.1% Tween-20, where the complexes were allowed to associate. Then, 0.1% Tween-20 and 0.1% Complex dissociation in PBS kinetic buffer, pH 7.4, containing BSA was detected for 150 seconds.

[0269] Binding curves were analyzed using Octet data analysis (version 9.0) according to standard methods using a 2:1 Global interaction model (after subtraction of the reference signal). Anti-PD-L1 / anti-CD47 bispecific antibodies specifically bind to FcRn. Table 9.

[0270] Table 9. Kinetic values ​​of the interaction of antibody BCD-106 (02-001, 03-006, 02-013) with FcRn.

[0271] [Table 10]

[0272] Example 18. Anti-PD-L1 / anti-CD47 bispecific antibody PD-L1 is Analysis of the ability to induce antibody-dependent cellular cytotoxicity against CD47-positive cells. To perform ADCC (antibody-dependent cellular cytotoxicity), the MDA-MB-231 cell line, which expresses PD-L1 / CD47 receptors on its surface, and peripheral blood mononuclear cells (PBMCs) were used.

[0273] Obtaining peripheral blood mononuclear cells PBMCs were obtained by fractionating venous blood cells from healthy donors in a density gradient. After isolation, 2–5x10 6Cells were cultured at a concentration of cells / ml in RPMI-1640 medium containing 10% FBS for 18–24 h at 37°C and 5% CO2.

[0274] Preparation of target cells MDA-MB-231 cells were cultured in DMEM medium containing 10% FBS (fetal bovine serum) at 37°C and 5% CO2. Trypsin was used to remove the cells from the plastic surface and resuspended in DMEM containing 10% FBS. Calcein AM was added to a concentration of 5 μM. After 30 min, the cells were washed twice from excess Calcein AM with DMEM containing 10% FBS. 5 A suspension of target cells at a concentration of cells / ml was prepared in DMEM containing 10% FBS.

[0275] Preparation of test antibody dilutions All test antibodies were diluted in DMEM medium containing 10% FBS to a concentration of 10 μg / ml. A serial dilution series with an increment of 5 was prepared. The concentrations of the test antibodies were: 10000; 2000; 400; 80; 16; 3.2; 0.64; 0.128; 0.0256; 0 (ng / ml).

[0276] Preparation of PBMCs Mononuclear lymphocytes were collected from the vials and centrifuged at 200xg for 5 minutes. 6 A cell suspension at a concentration of cells / ml was prepared in DMEM medium containing 10% FBS.

[0277] Running the ADCC assay 50 μl / well of test antibody was added to wells of a 96-well plate. 100 μl / well of target cell suspension was added to wells containing antibody. Plates were incubated at 37°C and 5% CO2 for 15-20 minutes. 50 μl / well of PBMC suspension was added to wells containing antibody and target cells. 50 μl / well of DMEM medium containing 10% FBS, 100 μl of target cell suspension and 50 μl of PBMC suspension were added to three wells (control for maximum lysis, "KL"). Plates were incubated at 37°C and 5% CO2 for 3.5-4 hours. Thirty minutes before the end of incubation, lysis buffer was added to KL wells.

[0278] After incubation, the plate was centrifuged at 200xg for 10 minutes. The supernatant was transferred to a new 96-well plate. The fluorescence was measured in relative fluorescence at excitation / emission wavelengths of 485 / 538 nm by using a plate fluorometer.

[0279] ADCC efficacy was calculated using the formula:

[0280] [ka]

[0281] During the ceremony, K-Control for spontaneous lysis of target cells in the presence of effector cells (50 μl / well of DMEM medium containing 10% FBS + 100 μl / well of target cells + 50 μl / well of PBMCs) Control for maximum lysis of KL-target cells (50 μl / well of DMEM medium containing 10% FBS + 100 μl / well of target cells + 50 μl / well of PBMC + lysis buffer) It was calculated by:

[0282] The results are shown in Figures 9 and 10. According to the data obtained, all anti-CD47 / PD-L1 antibodies show EC50 values ​​that are comparable to or exceed that of the control monospecific anti-CD47 antibody (clone B6H12).

[0283] Example 19. Comparison of the activity of anti-PD-L1 / anti-CD47 bispecific antibodies to CD47 / PD-L1 in an assay for stimulation of phagocytosis by human macrophage cells To perform ADCP (antibody-dependent cellular phagocytosis), the MDA-MB-231 cell line, which has PD-L1 / CD47 receptors on its surface, and human macrophage cells were used.

[0284] Obtaining human macrophages Peripheral blood mononuclear cells (PBMCs) were isolated from venous blood of healthy donors by density gradient separation. Human blood monocytes were isolated using a kit for isolating a fraction of human CD14 positive human cells (Miltenyi Biotec). 350,000 monocytes / well were cultured in 700 μl of RPMI-1640 medium containing 10% FBS, 100 ng / ml GM-CSF (Peprotech) for 3 days at 37°C and 5% CO2 in wells of a 24-well plate. On the fourth day of culture, the medium was replaced with fresh medium containing 700 μl of RPMI-1640 containing 10% FBS, 100 ng / ml GM-CSF (Peprotech), 50 ng / ml IFNγ (Peprotech) and 10 ng / ml LPS (Sigma) per well, and the cells were cultured for another 3 days at 37°C and 5% CO2.

[0285] Preparation of target cells MDA-MB-231 cells were cultured in DMEM medium containing 10% FBS (fetal bovine serum) at 37°C and 5% CO2. The cells were removed from the plastic surface by trypsin and resuspended in DMEM containing 10% FBS. Calcein AM was added to a concentration of 5 μM. After 30 min, the cells were washed twice from excess Calcein AM with DMEM containing 10% FBS. 5A suspension of target cells at a concentration of cells / ml was prepared in DMEM containing 10% FBS.

[0286] Running the ADCP assay The medium was removed from the plate wells containing macrophages and 500 μl of RPMI-1640 medium containing 10% FBS and 20 μg / ml of test antibody was added to the wells. 500 μl / well of the target cell suspension was added to the wells. The plates were incubated at 37° C. The cells were then incubated at 37 °C for 3 h at 25 °C and 5% CO2. Then, the medium was selected and TrypLE Cells were removed from the plastic surface with Express reagent and stained with fluorescently labeled anti-CD14 antibody. A suspension of stained cells was analyzed on a flow cytofluorometer.

[0287] The ADCP effectiveness is calculated as:

[0288] [ka]

[0289] In the formula, calcein + CD14 + is the number of CD14 positive cells containing calcein dye, and CD14 + is the number of all CD14 positive cells It was calculated by:

[0290] The results are shown in Figure 11. According to the data obtained, a number of anti-CD47 / PD-L1 antibodies showed efficacy in stimulating phagocytosis of the MDA-MB-231 cell line by human macrophages, which is comparable to that of a control monospecific anti-CD47 antibody (clone B6H12).

[0291] Example 20. Comparison of the effects of anti-PD-L1 / anti-CD47 bispecific antibody candidates on human erythrocyte aggregation The ability of the antibodies to induce hemagglutination was analyzed using human red blood cells.

[0292] Preparation of red blood cell suspension Blood samples were collected from healthy donor veins in evacuated heparin tubes. 9 ml of blood was transferred into a 50 ml centrifuge tube. Blood was diluted to 30 ml at room temperature with Ca2+- and Mg2+-free DPBS. The suspension was centrifuged under 800 g for 10 minutes and the supernatant was decanted. The cell washing procedure was repeated twice with Ca2+- and Mg2+-free DPBS and centrifugation. Then, 300 μl of the cell pellet was resuspended in 30 ml of DPBS, resulting in a 1% red blood cell suspension.

[0293] Performing the hemagglutination assay Test antibodies were diluted in DPBS to a concentration of 20 μg / ml. 100 μl of antibody dilutions and red blood cell suspensions were mixed in a 96-well round-bottom plate. Plates were incubated for 16 hours at 37° C. in a CO2 incubator. Results were visually documented using an arbitrary 4+ cross scale. Significant positive results are 2+ and above. Results are shown in Table 10.

[0294] Table 10. Hemagglutination in the presence of anti-CD47 / PD-L1 antibodies. "-" indicates the absence of agglutination.

[0295] [Table 11]

[0296] According to the data obtained, none of the anti-CD47 / PD-L1 antibodies induce hemagglutination, whereas the reference anti-CD47 monoclonal antibody (clone B6H12) induces significant agglutination due to the bivalent nature of the antibody and its consequent ability to interact with two CD47 molecules located on different erythrocytes.

[0297] Example 21. Analysis of complement-dependent cytotoxicity (CDC) of bispecific anti-CD47 / PD-L1 antibodies. To perform CDC analysis (complement-dependent cytotoxicity), the MDA-MB-231 cell line, which contains PD-L1 and CD47 receptors on its surface, was used as target cells.

[0298] Preparation of target cells MDA-MB-231 cultures were grown in DMEM medium supplemented with 10% fetal bovine serum (FBS).

[0299] Remove the cells from the plastic surface with trypsin and then culture at 1x10 6 The cells were suspended in DMEM medium containing 0.1% BSA at a concentration of cells / ml. Preparation of test antibody dilutions All test anti-CD47 / PD-L1 antibodies were diluted in DMEM medium containing 0.1% BSA to a concentration of 100μg / ml. A series of serial dilutions with 8 increments were prepared. The concentrations of test antibodies were: 100000; 12500; 1562.5; 195.3; 24.4; 3.05; 0.38; 0.04; 0.005 (ng / ml).

[0300] CDC Testing Human complement was thawed and dissolved 1:4 in DMEM medium containing 0.1% BSA.

[0301] 50 μl / well of each dilution of the test antibody was added to wells of a 96-well plate, with 50 μl / well of medium supplemented with DMEM medium containing 0.1% BSA for each antibody preparation as cell control and 150 μl / well of DMEM medium containing 0.1% BSA as medium control.

[0302] 50 μl / well of a suspension of MDA-MB-231 cells was added to each well containing test antibodies and cell controls. 50 μl / well of diluted complement was added to all wells containing test antibodies and cell controls. Plates were shaken for 2-4 minutes at room temperature on an orbital shaker and placed in a CO2 incubator at 37° C. for 2-3 hours.

[0303] 15 μl of Alamar Blue reagent was added to the wells of the test plate. The plate was shaken for 10-20 minutes at room temperature on an orbital shaker. The plate was further incubated for 18-24 hours in a CO2 incubator.

[0304] The plate was shaken on an orbital shaker at room temperature for 10-20 minutes. Fluorescence was measured using a plate fluorometer at excitation / emission wavelengths of 544 / 590 nm in relative fluorescence units. The obtained fluorescent signal is proportional to the number of viable cells. The results are shown in Figures 12-15.

[0305] According to the data obtained, the tested antibody does not induce complement-dependent cytotoxicity (CDC) of the MDA-MB-231 cell line. Example 22. Analysis of the antagonist activity of anti-PD-L1 / anti-CD47 bispecific antibodies on cell cultures bearing PD-L1 membrane receptors.

[0306] To analyze the antagonistic activity of anti-PD-L1 / anti-CD47 antibodies against the PD-L1 receptor, the ability of the antibodies to reactivate luciferase signaling in Jurkat-PD1-NFAT-Luc receptor cell line during co-culture with PD-L1-producing cells was evaluated. did.

[0307] Preparation of PD-L1-producing cells MDA-MB-231 cultures were grown in DMEM medium supplemented with 10% fetal bovine serum (FBS).

[0308] The cells were removed from the plastic surface by trypsin and cultured for 1 h in DMEM medium containing 10% FBS and 20 ng / ml interferon gamma. * 10 5 The cells were suspended at a concentration of 1000 cells / ml. Then, 200 μl / well of the cell suspension was added to the wells of a white 96-well plate and incubated at 37° C. and 5% CO in a CO incubator for 48 hours.

[0309] Preparation of test antibody dilutions All tested anti-CD47 / PD-L1 antibodies were diluted in RPMI-1640 medium containing 10% FBS to a concentration of 5μg / ml. A series of serial dilutions was prepared in increments of 3. The concentrations of the tested antibodies were: 2500; 833.3; 277.7; 92.5; 30.8; 10.2; 3.4; 1.1 (ng / ml).

[0310] Preparation of Jurkat-PD1-NFAT-Luc cells On the day of the experiment, 2.5 mL of RPMI-1640 medium containing 10% FBS was added. * 10 6 A suspension of Jurkat-PD1-NFAT-Luc cells was prepared at a concentration of cells / ml.

[0311] Preparation of activated antibody solution On the day of the experiment, a 10x mixture of activating antibodies was prepared (4 μg / ml anti-CD3; 4 μg / ml anti-CD28; 16 μg / ml anti-mouse in RPMI-1640 medium containing 10% FBS).

[0312] · Test execution Growth medium was removed from plates containing MDA-MB-231 cells. 40 μl / well of antibody dilutions were added to wells containing cells. 40 μl / well of RPMI 1640 medium containing 10% FBS was added to control wells (cells without test antibody, cells without test and activating antibody) and incubated for 30 minutes at room temperature.

[0313] · Then 40μl / well of Jurkat-PD1-NFAT-Luc cell suspension was added to all wells. Then 10μl / well of a 10x solution of activating antibody was added to all wells except the control well "cells without test and activating antibody". Cells were incubated for 6 hours at 37℃ and 5% CO2 in a CO2 incubator.

[0314] · Luciferase substrate One-Glo Luciferase Assay System (Promega) was introduced into all wells at a 1:1 ratio (90 μl / well). After 5-10 minutes, the luminescence levels were measured using a plate reader.

[0315] According to the results obtained, the test anti-PD-L1 / anti-CD47 bispecific antibody, as well as the control anti-PD-L1 monospecific antibody, are antagonists of the PD-L1-dependent signaling pathway and are therefore also capable of stimulating T cell-dependent cytotoxicity against cells bearing the PD-L1 receptor.

[0316] Example 25. Analysis of homogeneity of anti-PD-L1 / anti-CD47 bispecific antibody products. The homogeneity of the bispecific antibody was analyzed by size-exclusion HPLC (SEC HPLC) with UV detection. Chromatography was performed on an HPLC system (Agilent) on a column Tosoh TSK-Gel G3000SWXL, 7.8mmx30cm, order number 08541, with a precolumn Tosoh TSKgel Guard SWXL, 6.0mmx4.0cm, particle diameter 7μm, order number 08543. Detection was performed at wavelengths of 220 and 280nm. Figure 17 shows an exemplary HPLC profile of the product BCD106-02-013 based on VHH47Opt2 (see Example 12). According to the results of the test, the molecule BCD106-02-013 produces a product that is 93% homogeneous in the monomer aggregate composition, and is applicable for subsequent tests in vitro and in vivo. Without being limited thereto, the present invention includes the following aspects. [Aspect 1] A monoclonal antibody that specifically binds to CD47 and PD-L1, the antibody containing one binding site for CD47 and at least one binding site for PD-L1. [Aspect 2] The antibody according to embodiment 1, characterized in that the antibody is a full length antibody or an antigen-binding fragment thereof. [Aspect 3] 2. The antibody of embodiment 1, wherein the antibody comprises one or two binding sites for PD-L1. [Aspect 4] 2. The antibody of embodiment 1, wherein the binding site for CD47 inhibits the interaction between the CD47 receptor and a SIRPα ligand, and / or the binding site for PD-L1 inhibits the interaction between PD-L1 and the PD-1 receptor. [Aspect 5] The antibody of embodiment 1, wherein the CD47-binding site comprises a heavy chain variable domain comprising CDR1, CDR2, and CDR3 sequences, wherein CDR1 is a sequence that is at least 80% homologous to a sequence selected from the group consisting of SEQ ID NOs: 1 to 4 below, CDR2 is a sequence that is at least 80% homologous to a sequence selected from the group consisting of SEQ ID NOs: 6 to 15 below, and CDR3 is a sequence that is at least 80% homologous to a sequence selected from the group consisting of SEQ ID NOs: 17 to 20 below. [Aspect 6] The antibody of embodiment 1, wherein the CD47-binding site comprises a heavy chain variable domain containing CDR1, CDR2, and CDR3 sequences, wherein CDR1 is a sequence selected from the group consisting of SEQ ID NOs: 1 to 4 below, CDR2 is a sequence selected from the group consisting of SEQ ID NOs: 6 to 15 below, and CDR3 is a sequence selected from the group consisting of SEQ ID NOs: 17 to 20 below. [Aspect 7] The antibody of embodiment 1, wherein the binding site to CD47 comprises the heavy chain variable domain of embodiment 4, and a light chain variable domain comprising CDR1, CDR2 and CDR3 sequences, wherein CDR1 is a sequence that is at least 80% homologous to a sequence selected from the group consisting of SEQ ID NOs: 22 to 34 below, CDR2 is a sequence that is at least 80% homologous to a sequence selected from the group consisting of SEQ ID NOs: 36 to 48 below, and CDR3 is a sequence that is at least 80% homologous to a sequence selected from the group consisting of SEQ ID NOs: 50 to 64 below. [Aspect 8] The antibody of embodiment 1, wherein the CD47-binding site comprises the heavy chain variable domain of embodiment 4, and a light chain variable domain comprising CDR1, CDR2, and CDR3 sequences, wherein CDR1 is a sequence selected from the group consisting of SEQ ID NOs: 22 to 34 below, CDR2 is a sequence selected from the group consisting of SEQ ID NOs: 36 to 48 below, and CDR3 is a sequence selected from the group consisting of SEQ ID NOs: 50 to 64 below. [Aspect 9] 2. The antibody according to embodiment 1, characterized in that the binding site for CD47 comprises a heavy chain variable domain comprising a sequence that is at least 90% homologous to a sequence selected from the group of SEQ ID NOs: 66 to 88 below, and a light chain variable domain comprising a sequence that is at least 90% homologous to a sequence in the group of SEQ ID NOs: 89 to 106 below. [Aspect 10] The antibody according to embodiment 1, characterized in that the CD47-binding site comprises a heavy chain variable domain comprising a sequence selected from the group consisting of SEQ ID NOs: 66 to 88, and a light chain variable domain comprising a sequence selected from the group consisting of SEQ ID NOs: 89 to 106. [Aspect 11] The antibody of embodiment 1, wherein the binding site for PD-L1 is characterized in that it comprises a heavy chain variable domain comprising a sequence that is at least 80% homologous to the following: SEQ ID NO:5, SEQ ID NO:16, and SEQ ID NO:21, and a light chain variable domain comprising a sequence that is at least 80% homologous to the following: SEQ ID NO:35, SEQ ID NO:49, and SEQ ID NO:65. [Aspect 12] 2. The antibody of embodiment 1, wherein the binding site for PD-L1 is characterized by comprising a heavy chain variable domain comprising the following sequences: SEQ ID NO:5, SEQ ID NO:16, and SEQ ID NO:21, and a light chain variable domain comprising the following sequences: SEQ ID NO:35, SEQ ID NO:49, and SEQ ID NO:65. [Aspect 13] 2. The antibody of embodiment 1, characterized in that the binding site for CD47 is a Fab, scFv, scFab or an isolated VH or VHH monodomain. [Aspect 14] 2. The antibody of embodiment 1, wherein the binding site for PD-L1 is a Fab, scFv, scFab, or an isolated VH or VHH monodomain. [Aspect 15] The antibody of embodiment 1, characterized by inducing antibody-dependent cellular cytotoxicity, macrophage-mediated phagocytosis, and / or T-cell mediated cytotoxicity in a percentage of cells bearing CD47 and / or PD-L1 antigens on their surface. [Aspect 16] 2. The antibody of embodiment 1, characterized in that it comprises an Fc fragment comprising at least one mutation or modification that increases antibody-dependent cell-mediated cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC) compared to the same antibody without the mutation or modification. [Aspect 17] 17. The antibody according to any one of aspects 1 to 16 for use as a pharmaceutical for treating cancer. [Aspect 18] A nucleic acid encoding the antibody according to any one of embodiments 1 to 16. [Aspect 19] 20. The nucleic acid of embodiment 18, wherein the nucleic acid is DNA. [Aspect 20] An expression vector comprising the nucleic acid according to any one of aspects 18 to 19. [Aspect 21] A method for obtaining a host cell for producing the antibody according to any one of aspects 1 to 16, the method comprising transforming a cell with a vector according to aspect 20. [Aspect 22] A host cell for obtaining the antibody according to any one of aspects 1 to 16, comprising a nucleic acid according to any one of aspects 18 to 19. [Aspect 23] A method for obtaining an antibody according to any one of aspects 1 to 16, comprising culturing a host cell according to aspect 22 in a culture medium under conditions sufficient to obtain the identified antibody, and then, if necessary, isolating and purifying the antibody obtained. [Aspect 24] 17. A pharmaceutical composition for the prevention or treatment of a disease or disorder mediated by PD-L1 and CD47, the pharmaceutical composition comprising an antibody according to any of aspects 1 to 16, in combination with one or several pharma- ceutically acceptable excipients. [Aspect 25] (HNSCC) Head and neck squamous cell carcinoma, cervical cancer, unknown primary cancer, glioblastoma, esophageal cancer, bladder cancer, TNBC (triple negative breast cancer), CRC (colorectal cancer), hepatocellular carcinoma, melanoma, NSCLC (non-small cell lung cancer), kidney cancer, ovarian cancer, MSI 25. The pharmaceutical composition of embodiment 24, intended for the prevention or treatment of a disease or disorder mediated by PD-L1 and CD47 selected from the group consisting of CRC (colorectal cancer with microsatellite instability), leukemia (acute leukemia or myeloblastic leukemia), lymphoma, multiple myeloma, breast cancer, prostate cancer, sarcoma, hepatocellular carcinoma, Hodgkin's lymphoma, T and B cell acute lymphoblastic leukemia, small cell lung cancer, acute myeloblastic leukemia, refractory non-Hodgkin's B cell lymphoma, follicular lymphoma, marginal zone B cell lymphoma, diffuse large B cell lymphoma, pancreatic cancer, and high-risk myelodysplastic syndrome. [Aspect 26] A method for treating a disease or disorder mediated by PD-L1 and CD47, comprising the step of administering to a subject in need of such treatment a therapeutically effective amount of the antibody of any of embodiments 1 to 16, or the pharmaceutical composition of embodiment 24. [Aspect 27] The disease or disorder is head and neck squamous cell carcinoma (HNSCC), cervical cancer, cancer of unknown primary, glioblastoma, esophageal cancer, bladder cancer, TNBC (triple negative breast cancer), CRC (colorectal cancer), hepatocellular carcinoma, melanoma, NSCLC (non-small cell lung cancer), renal cancer, ovarian cancer, MSI 27. The method of claim 26, wherein the cancer is selected from the group consisting of CRC (colorectal cancer with microsatellite instability), leukemia (acute leukemia or myeloblastic leukemia), lymphoma, multiple myeloma, breast cancer, colorectal cancer, prostate cancer, bladder cancer, sarcoma, hepatocellular carcinoma, glioblastoma, Hodgkin's lymphoma, T and B cell acute lymphoblastic leukemia, small cell lung cancer, acute myeloblastic leukemia, refractory non-Hodgkin's B cell lymphoma, follicular lymphoma, marginal zone B cell lymphoma, diffuse large B cell lymphoma, head and neck squamous cell carcinoma, pancreatic cancer, ovarian cancer, acute myeloblastic leukemia and high-risk myelodysplastic syndrome. [Aspect 28] 17. A method for inhibiting the biological activity of PD-L1 and / or CD47 in a subject in need of inhibition of such activity, the method comprising administering to the subject an effective amount of an antibody according to any of aspects 1 to 16. [Aspect 29] Use of the antibody of any of embodiments 1 to 16, or the pharmaceutical composition of embodiment 24, for the treatment of a disease or disorder mediated by PD-L1 and CD47 in a subject in need of such treatment. [Aspect 30] The disease or disorder is head and neck squamous cell carcinoma (HNSCC), cervical cancer, cancer of unknown primary, glioblastoma, esophageal cancer, bladder cancer, TNBC (triple negative breast cancer), CRC (colorectal cancer), hepatocellular carcinoma, melanoma, NSCLC (non-small cell lung cancer), renal cancer, ovarian cancer, MSI 30. Use of the antibody of embodiment 29, selected from the group consisting of CRC (colorectal cancer with microsatellite instability), leukemia (acute leukemia or myeloblastic leukemia), lymphoma, multiple myeloma, breast cancer, colorectal cancer, prostate cancer, bladder cancer, sarcoma, hepatocellular carcinoma, glioblastoma, Hodgkin's lymphoma, T and B cell acute lymphoblastic leukemia, small cell lung cancer, acute myeloblastic leukemia, refractory non-Hodgkin's B cell lymphoma, follicular lymphoma, marginal zone B cell lymphoma, diffuse large B cell lymphoma, head and neck squamous cell carcinoma, pancreatic cancer, ovarian cancer, acute myeloblastic leukemia and high-risk myelodysplastic syndrome.

Claims

1. A monoclonal antibody that specifically binds to CD47 and PD-L1, the antibody comprising one binding site for CD47 and at least one binding site for PD-L1; The binding site for CD47 comprises a heavy chain variable domain comprising CDR1, CDR2, and CDR3 sequences, wherein: CDR1 is the sequence shown by SEQ ID NO: 2 or SEQ ID NO: 4, CDR2 is the sequence shown by SEQ ID NO: 10 or SEQ ID NO: 15, and CDR3 is the sequence shown by SEQ ID NO: 18 or SEQ ID NO: 20; and, The binding site for PD-L1 is A heavy chain variable domain comprising CDR1, CDR2, and CDR3 sequences, wherein CDR1 is the sequence set forth in SEQ ID NO:5, CDR2 is the sequence set forth in SEQ ID NO:16, and CDR3 is the sequence set forth in SEQ ID NO:21, and a light chain variable domain comprising CDR1, CDR2, and CDR3 sequences, wherein CDR1 is the sequence set forth in SEQ ID NO:35, CDR2 is the sequence set forth in SEQ ID NO:49, and CDR3 is the sequence set forth in SEQ ID NO:65; Includes The antibody, characterized by:

2. The antibody of claim 1, characterized in that the antibody is a full-length antibody or an antigen-binding fragment thereof.

3. The antibody of claim 1, characterized in that the antibody contains one or two binding sites for PD-L1.

4. The antibody of claim 1, wherein the binding site for CD47 inhibits the interaction between the CD47 receptor and a SIRPα ligand, and / or the binding site for PD-L1 inhibits the interaction between PD-L1 and the PD-1 receptor.

5. The binding site for CD47 is (i) a heavy chain variable domain VHH comprising the sequence SEQ ID NO: 88; or (ii) a heavy chain variable domain VHH comprising the sequence SEQ ID NO: 83; or (iii) a heavy chain variable domain VHH comprising the sequence SEQ ID NO: 109; The antibody of claim 1 , comprising:

6. The antibody of claim 1, characterized in that the binding site for CD47 is a Fab, scFv, scFab, or an isolated VH or VHH monodomain.

7. The antibody of claim 1, wherein the binding site for PD-L1 is a Fab, scFv, or scFab.

8. The antibody of claim 1, characterized in that it induces antibody-dependent cellular cytotoxicity, macrophage-mediated phagocytosis, and / or T-cell-mediated cytotoxicity.

9. The antibody of claim 1, characterized in that it comprises an Fc fragment containing at least one mutation or modification that increases antibody-dependent cell-mediated cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC) compared to the same antibody without the mutation or modification.

10. The antibody according to any one of claims 1 to 9 for use as a medicament for the treatment of cancer.

11. A nucleic acid encoding an antibody described in any one of claims 1 to 9.

12. The nucleic acid of claim 11, wherein the nucleic acid is DNA.

13. An expression vector comprising a nucleic acid according to any one of claims 11 to 12.

14. A method for obtaining a host cell for producing an antibody according to any one of claims 1 to 9, comprising transforming a cell with an expression vector according to claim 13.

15. A host cell for obtaining an antibody according to any one of claims 1 to 9, comprising a nucleic acid according to any one of claims 11 to 12.

16. A method for obtaining an antibody according to any one of claims 1 to 9, which comprises culturing a host cell according to claim 15 in a culture medium under conditions sufficient to obtain the specified antibody.

17. 17. The method of claim 16, further comprising isolating and purifying the antibody obtained.

18. A pharmaceutical composition comprising an antibody according to any one of claims 1 to 9 in combination with one or several pharma- ceutically acceptable excipients.

19. The pharmaceutical composition according to claim 18, for the prevention or treatment of a disease or disorder mediated by PD-L1 and CD47.

20. HNSCC (head and neck squamous cell carcinoma), cervical cancer, unknown primary cancer, glioblastoma, esophageal cancer, bladder cancer, TNBC (triple negative breast cancer), CRC (colorectal cancer), hepatocellular carcinoma, melanoma, NSCLC (non-small cell lung cancer), kidney cancer, ovarian cancer, MSI 20. The pharmaceutical composition of claim 19, intended for the prevention or treatment of a disease or disorder mediated by PD-L1 and CD47 selected from the group consisting of CRC (colorectal cancer with microsatellite instability), leukemia (acute leukemia or myeloblastic leukemia), lymphoma, multiple myeloma, breast cancer, prostate cancer, sarcoma, hepatocellular carcinoma, Hodgkin's lymphoma, T and B cell acute lymphoblastic leukemia, small cell lung cancer, acute myeloblastic leukemia, refractory non-Hodgkin's B cell lymphoma, follicular lymphoma, marginal zone B cell lymphoma, diffuse large B cell lymphoma, pancreatic cancer, and high-risk myelodysplastic syndromes.

21. A pharmaceutical composition for inhibiting the biological activity of PD-L1 and / or CD47, comprising an antibody according to any one of claims 1 to 9 in combination with one or several pharma- ceutically acceptable excipients.

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