Antibodies specific for CD47 and PD-L1

Abstract

The present invention relates to the field of bioengineering, and specifically to antibodies or antigen-binding fragments thereof and uses thereof. 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 a given antibody or antigen-binding fragment thereof, expression vectors, methods for producing antibodies, and uses of the above antibodies and compositions in cancer treatment.

Description

Field of the Invention

[0001] The present invention relates to the field of biotechnology, in particular to antibodies or antigen-binding fragments thereof, and uses thereof. 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 the antibodies or antigen-binding fragments thereof, expression vectors, methods for obtaining antibodies, and uses of the antibodies and compositions in cancer treatment. Background of the Invention

[0003] Providing two separate signals to T cells is a broad model of lymphocyte activation with antigen presenting cells (APC) for the rest of the T lymphocytes. This model fully provides the distinction between self and non-self and immune tolerance. After recognition of foreign antigenic peptides presented in the context of the major histocompatibility complex (MHC), the primary signal or antigen-specific signal is transmitted by the T cell receptor (TCR). The second signal or co-stimulatory signal is delivered to T cells by co-stimulatory molecules expressed on antigen presenting cells (APCs), and induces T cell stimulation clonal expansion, cytokine secretion and effector function. In the absence of co-stimulation, T cells may become immune to antigenic stimulation, they cause an effective immune response, and this can further lead to exhaustion or resistance to foreign antigens.

[0004] In the two-signal model, T cells receive two signals: positive and negative secondary co-stimulatory signals. The regulation of this positive and negative signal is crucial to maximize the host's protective immune response while maintaining immune tolerance and preventing autoimmunity. Negative secondary signals appear to be necessary for the induction of T cell tolerance, while positive signals stimulate T cell activation. Although the simple two-signal model still provides a valid explanation regarding naive lymphocytes, the immune response is a dynamic process and co-stimulatory signals to antigen-exposed T cells can also be provided. From a therapeutic point of view, the mechanism of co-stimulation is of interest because manipulation of co-stimulatory signals has been shown to provide a means to enhance or terminate immune responses. Recently, T cell dysfunction or anergy has been found to occur simultaneously with the induction and sustained expression of an inhibitory receptor, polypeptide 1 programmed cell death (PD-1). As a result, therapeutic targeting of PD-1, as well as other molecules that transmit signals through interaction with PD-1, such as programmed death ligand 1 (PD-L1) or programmed death ligand 2 (PD-L2), is an area of ​​intense interest.

[0005] PD-L1 is overexpressed in a variety of malignancies and is often associated with a poor prognosis. Interestingly, in contrast to T lymphocytes in normal tissues and peripheral blood T lymphocytes, the majority of tumor-infiltrating T lymphocytes predominantly express PD-1, indicating that upregulation of PD-1 on tumor-reactive T cells can contribute to an impaired immune response. This may be due to the utilization of the PD-L1 signaling pathway mediated by tumor cells expressing PD-L1 and interacting with PD-1-expressing T cells, with an overall reduction in T cell activation and evasion of immune surveillance. Therefore, inhibition of the PD-L1 / PD-1 interaction can enhance CD8+ T cell-mediated tumor killing.

[0006] Therapeutic targeting of PD-1 and other molecules (e.g., PD-L1 and PD-L2) that transmit signals by interacting with PD-1 is an area of ​​intense interest. Inhibition of PD-L1 signals has been proposed as a means of increasing T cell immunity (e.g., anti-tumor immunity) for the treatment of cancer and infection (including both acute and chronic infections). Inhibitors that block the PD-L1 / PD-1 interaction are known, inter alia, according to WO2001014557, WO2002086083, WO2007005874, WO2010036959, WO2010077634, and WO2011066389. However, the best therapeutic agent targeting this pathway has not yet been commercialized, and this is a significant unmet medical need.

[0007] CD47 is a cell surface glycoprotein that binds to SIRPα (alias SHPS-1) and SIRPγ on the corresponding cells. This interaction leads to negative regulation of immune cell function or can mediate cell adhesion and migration. The use of CD47 as a biological agent in the treatment of autoimmune disorders has been proposed (WO 1999 / 040940). In contrast, there is little data on the possible use of CD47 ligands (such as SIRPα) for similar therapeutic purposes. One explanation is the ubiquitous expression of CD47, which may interfere with 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 the Fc domain of an immunoglobulin can prevent the migration of skin-derived dendritic cells (DC) to draining lymph nodes in mice and thereby (at least partially) attenuate contact hypersensitivity responses in mice. The migration and function of DCs are important for immune or inflammatory responses. Under painful conditions, these exacerbated DC responses can lead to the maintenance of the disease. Interfering with the migration of pathogenic DCs from tissues to lymphoid organs is an attractive opportunity to prevent the vicious cycle that drives autoimmune or inflammatory diseases.

[0008] CD47, also known as integrin-associated protein (IAP), ovarian cancer antigen OA3, Rh-related antigen, and MER6, is a transmembrane receptor that penetrates the membrane several times and belongs to the immunoglobulin superfamily. The expression and / or activity of CD47 has been observed in many diseases and conditions. Accordingly, there is a need for therapies that target CD47. In addition, due to the expression of CD47 on platelets, there is also a need for therapies (e.g., antibodies) that target CD47 that do not cause significant levels of platelet depletion, hemagglutination, red blood cell depletion, and / or anemia when administered to a subject.

[0009] Known antibodies that inhibit the interaction between CD47 and SIRPα ligands have been described in the following sources: Applications WO2014123580, WO2013119714, WO2015191861, WO2011143624, WO / 2014 / 093678, WO2017053423.

[0010] Also known are various sources describing multispecific antibodies, 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 possibility of producing and effectively using multispecific antibodies that specifically bind to CD47 and PD-L1 has not yet been described.

[0011] In conjunction with the foregoing, the generation of new antibodies that effectively bind to CD47 and PD-L1 is relevant. SUMMARY OF THE INVENTION

[0013] The present invention relates to binding molecules, for example, antibodies that bind to CD47 and PD-L1. Such antibodies can 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 comprises one binding site for CD47 and at least one binding site for PD-L1.

[0015] In some embodiments, an antibody of the invention is a full-length antibody or an antigen-binding fragment thereof.

[0016] In some embodiments, the antibodies of the invention include one or two binding sites for PD-L1.

[0017] In some embodiments, the binding site of the antibodies of the invention for CD47 inhibits the interaction between the CD47 receptor and the SIRPα ligand, and / or the binding site for PD-L1 inhibits the interaction between PD-L1 and the PD-1 receptor.

[0018] In some embodiments, the binding site of an antibody of the present invention for CD47 comprises a heavy chain variable domain, the heavy chain variable domain comprising CDR1, CDR2, and CDR3 sequences, wherein CDR1 is a sequence at least 80% homologous to a sequence selected from SEQ ID NOs: 1-4, i.e., CDR1 is a sequence selected from SEQ ID NOs: 1-4, or a sequence selected from SEQ ID NOs: 1-4 with 1 or 2 substitutions, wherein CDR2 is a sequence at least 80% homologous to a sequence selected from SEQ ID NOs: 6-15, i.e., CDR2 is a sequence selected from SEQ ID NOs: 6-15, or a sequence selected from SEQ ID NOs: 6-15 with 1, 2, 3, 4 or 5 substitutions, wherein CDR3 is a sequence at least 80% homologous to a sequence selected from SEQ ID NOs: 17-20, i.e., CDR3 is a sequence selected from SEQ ID NOs: 17-20, or a sequence selected from SEQ ID NOs: 17-20 with 1, 2 or 3 substitutions.

[0019] In some embodiments, the CD47 binding site of an antibody of the present invention comprises a heavy chain variable domain, which comprises a CDR1, CDR2, and CDR3 sequence, wherein CDR1 is a sequence selected from SEQ ID NOs: 1-4, wherein CDR2 is a sequence selected from SEQ ID NOs: 6-15, and wherein CDR3 is a sequence selected from SEQ ID NOs: 17-20.

[0020] In some embodiments, the CD47 binding site of the antibody of the present invention comprises the heavy chain variable domain of claim 4, and a light chain variable domain comprising CDR1, CDR2, and CDR3 sequences, wherein CDR1 is a sequence at least 80% homologous to a sequence selected from SEQ ID NOs: 22-34, i.e., CDR1 is a sequence selected from SEQ ID NOs: 22-34, or a sequence selected from SEQ ID NOs: 22-34 with 1 or 2 substitutions, wherein CDR2 is a sequence at least 80% homologous to a sequence selected from SEQ ID NOs: 36-48, i.e., CDR2 is a sequence selected from SEQ ID NOs: 36-48, or a sequence selected from SEQ ID NOs: 36-48 with 1, 2 or 3 substitutions, wherein CDR3 is a sequence at least 80% homologous to a sequence selected from SEQ ID NOs: 50-64, i.e., CDR3 is a sequence of SEQ ID NOs: 50-64, or a sequence selected from SEQ ID NOs: 50-64 with 1 or 2 substitutions. –64 sequence.

[0021] In some embodiments, the binding site of the antibody of the present invention for CD47 comprises the 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 SEQ ID NOs: 22-34, CDR2 is a sequence selected from SEQ ID NOs: 36-48, and CDR3 is a sequence selected from SEQ ID NOs: 50-64.

[0022] In some embodiments, the binding site of an antibody of the present invention for CD47 comprises a heavy chain variable domain comprising a sequence at least 90% homologous to a sequence selected from SEQ ID NOs: 66-88 and a light chain variable domain comprising a sequence at least 90% homologous to a sequence selected from SEQ ID NOs: 89-106.

[0023] In some embodiments, the binding site of an antibody of the present invention for CD47 comprises a heavy chain variable domain comprising a sequence selected from SEQ ID NOs: 66-88 and a light chain variable domain comprising a sequence selected from SEQ ID NOs: 89-106.

[0024] In some embodiments, the binding site of an antibody of the present invention for PD-L1 comprises a heavy chain variable domain and a light chain variable domain, the heavy chain variable domain comprising a sequence at least 80% homologous to the following sequences: SEQ ID NO: 5, SEQ ID NO: 16 and SEQ ID NO: 21, i.e., an amino acid sequence comprising SEQ ID NO: 5, 16 and 21, or SEQ ID NO: 5 with 1 substitution, SEQ ID NO: 16 with 1, 2 or 3 substitutions, or SEQ ID NO: 21 with 1, 2 or 3 substitutions, and the light chain variable domain comprising a sequence at least 80% homologous to the following sequences: SEQ ID NO: 35, SEQ ID NO: 49 and SEQ ID NO: 65, i.e., an amino acid sequence comprising SEQ ID NO: 35, SEQ ID NO: 49 and SEQ ID NO: 65, i.e., an amino acid sequence comprising SEQ ID NO: 35, 49 and 65, or SEQ ID NO: 35 with 1, 2 or 3 substitutions, SEQ ID NO: 49 with 1 substitution, or SEQ ID NO: 65 with 1 or 2 substitutions.

[0025] In some embodiments, the binding site of the antibody of the present invention against PD-L1 includes a heavy chain variable domain and a light chain variable domain, the heavy chain variable domain comprising the following sequences: SEQ ID NO: 5, SEQ ID NO: 16 and SEQ ID NO: 21, and the light chain variable domain comprising the following sequences: SEQ ID NO: 35, SEQ ID NO: 49 and SEQ ID NO: 65.

[0026] In some embodiments, the binding site of an antibody of the invention against CD47 is a Fab, scFv, scFab, or an isolated VH or VHH single domain.

[0027] In some embodiments, the binding site of an antibody of the invention against PD-L1 is a Fab, scFv, scFab, or an isolated VH or VHH single domain.

[0028] In some embodiments, an antibody of the invention is characterized in that it stimulates antibody-dependent cellular cytotoxicity, macrophage-mediated phagocytosis and / or T cell-mediated cytotoxicity, ratios of cells bearing CD47 and / or PD-L1 antigens on their surface.

[0029] In some embodiments, the antibody of the invention is characterized in that it comprises an Fc part comprising at least one mutation or modification that increases antibody-dependent cellular cytotoxicity (ADCC) compared to the same antibody without the mutation or modification.

[0030] In some embodiments, the antibodies of the invention are contemplated for use as medicaments for cancer treatment.

[0031] In one aspect, the invention relates to nucleic acids encoding any of the above antibodies.

[0032] In some embodiments, the nucleic acid of the invention is DNA.

[0033] In one aspect, the present invention relates to an expression vector comprising the above-mentioned nucleic acid.

[0034] In one aspect, the present invention relates to a method for obtaining a host cell for producing any of the above antibodies, which comprises transforming the cell with the vector of the present invention.

[0035] In one aspect, the present invention relates to a host cell for obtaining any of the above antibodies, which contains the above nucleic acid.

[0036] In one aspect, the present invention relates to a method for obtaining any of the above antibodies, comprising: culturing host cells in a culture medium under conditions sufficient to obtain the specified antibody, and then isolating and purifying the obtained antibody if necessary.

[0037] In one aspect, the invention relates to a pharmaceutical composition for preventing or treating a disease or condition mediated by PD-L1 and CD47, comprising any of the above antibodies in combination with one or more pharmaceutically acceptable excipients.

[0038] In some embodiments, the pharmaceutical composition of the present invention is intended for preventing or treating a disease or condition mediated by PD-L1 and CD47, the disease or condition being selected from (HNSCC) head and neck squamous cell carcinoma, 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), kidney cancer, ovarian cancer, MSI CRC (colorectal cancer with microsatellite instability), leukemia (acute leukemia or myeloblastic leukemia), lymphoma, multiple myeloma, breast cancer, prostate cancer, sarcoma, hepatocellular carcinoma, Hodgkin lymphoma, T-cell and B-cell acute lymphoblastic leukemia, small cell lung cancer, acute myeloblastic leukemia, refractory non-Hodgkin B-cell lymphoma, follicular lymphoma, marginal zone B-cell lymphoma, diffuse large B-cell lymphoma, pancreatic cancer and high-risk myelodysplastic syndrome.

[0039] In one aspect, the invention relates to a method for treating a disease or disorder mediated by PD-L1 and CD47, comprising administering any of the above antibodies or pharmaceutical compositions of the invention in a therapeutically effective amount to a subject in need of such treatment.

[0040] In some embodiments of the treatment methods according to the present invention, wherein the disease or condition is selected from (HNSCC) head and neck squamous cell carcinoma, cervical cancer, cancer of unknown primary site, 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, MSICRC (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 lymphoma, T-cell and B-cell acute lymphoblastic leukemia, small cell lung cancer, acute myeloblastic leukemia, refractory non-Hodgkin B-cell lymphoma, follicular lymphoma, marginal zone B-cell lymphoma, diffuse large B-cell lymphoma, pancreatic cancer, ovarian cancer and high-risk myelodysplastic syndrome.

[0041] 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.

[0042] In one aspect, the invention relates to the use of any of the above-described antibodies or the above-described pharmaceutical compositions for treating a subject having a disease or disorder mediated by PD-L1 and CD47 in need of such treatment.

[0043] In some embodiments of the use of the antibodies according to the present invention, the disease or disorder is selected from (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), kidney 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 lymphoma, T-cell and B-cell acute lymphoblastic leukemia, small cell lung cancer, acute myeloblastic leukemia, refractory non-Hodgkin 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

[0045] Figure 1 . Plasmid map for transient production of human CD47-Fc in CHO-K1 culture of mammalian cells.

[0046] Figure 2 . SDS-gel electrophoresis under non-reducing conditions in the preparation of human CD47-Fc.

[0047] Figure 3 . SDS-gel electrophoresis under reducing conditions for the preparation of the control anti-CD47 antibody B6H12 product.

[0048] Figure 4 . SDS-gel electrophoresis under non-reducing conditions in the preparation of the control anti-CD47 antibody B6H12 product.

[0049] Figure 5 . Schematic representation of ELISA of polyclonal phage carrying VHH antibody fragments that specifically interact with the human CD47 antigen.

[0050] Figure 6 . Schematic representation of the domain structure of the anti-PD-L1 / anti-CD47 bispecific antibody, where A is based on the anti-CD47 scFv fragment and B is based on the anti-CD47 VHH fragment. Meanwhile, the PD-L1 binding part is represented by the Fab fragment.

[0051] Figure 7. SDS-gel electrophoresis under non-reducing conditions of anti-PD-L1 / anti-CD47 preparations of bispecific antibodies based on anti-CD47 scFv fragments.

[0052] Figure 8 . SDS-gel electrophoresis under reducing conditions of preparations of anti-PD-L1 / anti-CD47 bispecific antibodies based on anti-CD47 VHH fragments.

[0053] Fig. 9 . Dependence of the cytotoxic effect on the concentration of the anti-PD-L1 / anti-CD47 bispecific antibodies studied.

[0054] Fig.10 . Dependence of the cytotoxic effect on the concentration of the anti-PD-L1 / anti-CD47 bispecific antibodies studied.

[0055] Fig.11 . Efficacy of phagocytosis of the MDA-MB-231 cell line by human macrophages in the presence of an anti-PD-L1 / anti-CD47 bispecific antibody.

[0056] Fig.12 . Dependence of fluorescence level on the concentration of anti-PD-L1 / anti-CD47 bispecific antibody.

[0057] Fig.13 . Dependence of fluorescence level on the concentration of anti-PD-L1 / anti-CD47 bispecific antibody.

[0058] Fig.14 . Dependence of fluorescence level on the concentration of anti-PD-L1 / anti-CD47 bispecific antibody.

[0059] Fig.15 . Dependence of fluorescence level on the concentration of anti-PD-L1 / anti-CD47 bispecific antibody.

[0060] Fig.16 . Anti-PD-L1 activity of anti-PD-L1 / CD47 bispecific antibodies. The vertical axis shows the luminescence ratio of wells with tested aHTH-CD47 / PD-L1 antibodies to wells without added antibodies.

[0061] Fig.17 . Gel filtration profile for evaluation of aggregation homogeneity of anti-PD-L1 / anti-CD47 bispecific antibodies.

[0062] Invention Disclosure

[0063] Definition and general approach

[0064] Unless otherwise defined herein, scientific and technical terms used in connection with the present invention have the meanings that are commonly understood by those skilled in the art.

[0065] Further, unless the context otherwise requires, singular terms shall include the plural, and plural terms shall include the singular. Generally, the classifications and methods of cell culture, molecular biology, immunology, microbiology, genetics, analytical chemistry, synthetic organic chemistry, medical and pharmaceutical chemistry, and hybridization and chemistry of proteins and nucleic acids described herein are well known and widely used by those skilled in the art. Enzyme reactions and purification methods are performed according to the manufacturer's instructions as commonly used in the art or as described herein.

[0066] Definitions related to antibodies

[0067] 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 represented by Ig V-type and C-type domains (220), a transmembrane domain (21), and an intracellular domain (31). It plays an important role in suppressing the immune system during pregnancy, during foreign tissue transplantation, and in certain diseases such as hepatitis. Under normal circumstances, in response to self-antigens, a certain number of antigen-specific CD8+ T effector cells accumulate in the lymph nodes and spleen in order to prevent the autoimmune process, forming PD-1 / PD-L1 or B7-1 / PD-L1 complexes, resulting in the transmission of inhibitory signals and reducing the proliferation of these CD8+ T cells in the lymph nodes. Therefore, the PD-1 / PD-L interaction is one of the key factors in the development of immune tolerance.

[0068] CD47 is a multi-spanning transmembrane receptor belonging to the immunoglobulin superfamily that interacts with SIRPα (signal regulatory protein α) on macrophages, thereby suppressing phagocytosis. Cancer cells, where this pathway is active, avoid phagocytosis. Therefore, therapeutic effects on CD47 are widely used in various cancers. Antibodies against CD47 may have the ability to block the interaction between CD47 and SIRPα, but they may not have this ability.

[0069] The term "binding molecule" includes antibodies and immunoglobulins.

[0070] As used herein, the term "antibody" or "immunoglobulin" or "monoclonal antibody" or "bispecific antibody" or "multispecific antibody" (Ig) includes complete / full-length antibodies and any antigen-binding fragment (i.e., "antigen-binding portion"). In addition, for example, the term "antibody" or "immunoglobulin" or "monoclonal antibody" includes any combination of an antigen-binding fragment having one or more valencies and one or more specificities with a constant region of an immunoglobulin, and may have a similar meaning to the term "bispecific antibody" or "multispecific antibody". In addition, for example, the term "antibody" or "immunoglobulin" or "monoclonal antibody" includes any combination of an antigen-binding fragment and an immunoglobulin constant region, which covalently or non-covalently binds to any polypeptide of any nature. In addition, for example, the term "antibody" refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or an antigen-binding portion. Each heavy chain comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. Five types of mammalian Ig heavy chains are known, represented by Greek letters: α, δ, ε, γ and μ. The type of heavy chain present defines the class of antibodies; 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 about 450 amino acids, while μ and ε consist of about 550 amino acids. Each heavy chain contains two regions, the constant region and the variable region. The constant region is identical in all antibodies of the same isotype, but is different in antibodies of different isotypes. Heavy chains γ, α and δ contain a constant region consisting of three constant domains CH1, CH2 and CH3 (in a line), and a hinge region for increased flexibility (Woof J., Burton D., Nat Rev Immunol 4, 2004, cc.89-99); heavy chains μ and ε have a constant region consisting of four constant domains CH1, CH2, CH3 and CH4. In mammals, only two types of light chains are known, represented by 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 a light chain is 211 to 217 amino acids. Preferably, the light chain is a kappa (κ) light chain, and the constant domain CL is preferably C kappa (κ).

[0071] An “antibody” according to 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).

[0072] The VL and VH regions can be further subdivided into hypervariable regions called complementarity determining regions (CDRs), which are interspersed between more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, located in the following order from the amino terminus to the carboxyl terminus: 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 an antibody can mediate the binding of immunoglobulins 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.

[0073] As used herein, the term "antigen-binding portion" or "antigen-binding fragment" of an antibody (or simply "antibody portion" or "antibody fragment") 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 included 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, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a 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 341:544-546); and (vi) extracted complementarity determining regions (CDRs). In addition, the two regions of the Fv fragment, VL and VH, are encoded by different genes, which can be connected using recombinant methods using synthetic linkers that enable them to accept a single protein chain in which the VL and VH regions are paired to form a monovalent molecule (called 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). It is assumed that such single-chain molecules are also included in 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.

[0074] Preferably, the CDRs of the antigen binding region or the entire antibody antigen binding region of the invention are derived from a mouse, llama or human donor library, or are substantially human in origin, wherein certain amino acid residues are changed, for example, replaced by different amino acid residues, in order to optimize the properties of the specific antibody, such as KD, koff, IC50, EC50, ED50. Preferably, the framework regions of the antibodies of the invention are of human origin or substantially of human origin (at least 80, 85, 90, 95, 96, 97, 98 or 99% of human origin).

[0075] In other embodiments, the antigen binding portion 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.

[0076] The term "variable domain" refers to the fact that certain regions of the variable domain differ greatly in sequence in antibodies. The V domain mediates antigen binding and determines the specificity of a particular antibody for its particular antigen. However, variability is not evenly distributed across the sites of the 110 amino acid variable domain. Instead, the V region consists of an invariant segment called a framework region (FR) of 15-30 amino acids, separated by a shorter region of extreme variability called a "hypervariable region" or CDR. Each variable domain of a natural heavy chain and light chain comprises four FRs, mainly in a β-folded configuration, connected by three hypervariable regions that form loops that connect the β-folded structure and in some cases constitute part of the β-folded structure. The hypervariable regions in each chain are tightly bound together by the FRs and, together with the hypervariable regions in the other chain, contribute to the formation of the antigen binding site of the antibody. The constant domain is not directly involved in the binding of the antibody to the antigen, but rather displays various effector functions, such as antibody participation in antibody-dependent cellular cytotoxicity (ADCC).

[0077] As used herein, the term "hypervariable region" refers to the amino acid residues of an antibody that are responsible for antigen binding. A hypervariable region generally comprises amino acid residues from a "complementarity determining region" or "CDR" and / or those residues from a "hypervariable loop".

[0078] In some cases, it may also be desirable to change one or more CDR amino acid residues in order to improve binding affinity to the target epitope. This is called "affinity maturation" and can be optionally performed in conjunction with humanization, such as in cases where humanization of an antibody results in reduced binding specificity or affinity and the binding specificity or affinity cannot be adequately improved by back mutation alone. Various affinity maturation methods are known in the art, such as the in vitro scanning saturation mutagenesis method described by Burks et al., Proc Natl Acad Sci USA, 94: 412–417 (1997), and the stepwise in vitro affinity maturation proposed by Wu et al., Proc Natl Acad Sci USA 95: 6037 6042 (1998).

[0079] "Framework regions" (FRs) are variable domain residues that are different from the CDR residues. Each variable domain typically has four FRs identified as FR1, FR2, FR3, and FR4. If the CDRs are defined according to Kabat, the FR light chain residues are approximately located at residues 1-23 (LCFR1), 35-49 (LCFR2), 57-88 (LCFR3), and 98-107 (LCFR4), while the heavy chain FR residues are approximately located in the region of residues 1-30 (HCFR1), 36-49 (HCFR2), 66-94 (HCFR3), and 103-113 (HCFR4) in the heavy chain. If the CDRs comprise amino acid residues from a hypervariable loop, the FR light chain residues are approximately located at residues 1-25 (LCFR1), 33-49 (LCFR2), 53-90 (LCFR3), and 97-107 (LCFR4) in the light chain, while the heavy chain FR residues are approximately located at residues 1-25 (HCFR1), 33-52 (HCFR2), 56-95 (HCFR3), and 102-113 (HCFR4) in the heavy chain. In some cases, when the CDRs comprise amino acids from both the CDRs as defined by Kabat and amino acids from a hypervariable loop, the FR residues will be adjusted accordingly. For example, when CDRH1 includes amino acids H26-H35, the heavy chain FR1 residues are at positions 1-25, and the FR2 residues are at positions 36-49.

[0080] An antibody of the invention that "binds" a target antigen is an antibody that binds to the antigen with sufficient affinity so that when targeting a protein or cell or tissue expressing the antigen, the antibody can be used as a diagnostic and / or therapeutic agent and is slightly cross-reactive with other proteins. Depending on the analytical method: fluorescence activated cell sorting (FACS), radioimmunoassay (RIA) or ELISA, in such embodiments, the extent of binding of the antibody to non-target proteins is less than 10% of the binding of the antibody to the specific target protein. With respect to the binding of an antibody to a target molecule, the term "specific binding" or "specific binding to" or "specific for" a specific polypeptide or an epitope on a specific polypeptide target means binding that is significantly (measurably) different from non-specific interactions (e.g., in the case of bH1-44 or bH1-81, the non-specific interaction is binding to bovine serum albumin, casein, fetal bovine serum or neutravidin).

[0081] Specific binding can be measured, for example, by comparing the binding of the assay molecule with the binding of a control molecule. For example, specific binding can be measured by competing with another molecule similar to the target (e.g., an excessive amount of unlabeled target). In this case, if the binding of the labeled target to the probe is competitively inhibited by an excessive amount of unlabeled target, specific binding is indicated. As used herein, the term "specific binding" or "specific binding to" an epitope on a specific polypeptide or a specific polypeptide target or "specific to" an epitope on a specific polypeptide or a specific polypeptide target can be characterized by having a molecule with the following Kd for a target: at least about 200 nM, or at least about 150 nM, or at least about 100 nM, or at least about 60 nM, or at least about 50 nM, or at least about 40 nM, or at least about 30 nM, or at least about 20 nM, or at least about 10 nM, or at least about 8 nM, or at least about 6 nM, or at least about 4 nM, or at least about 2 nM, or at least about 1 nM or greater. In one embodiment, the term "specific binding" refers to binding wherein a molecule binds to a specific polypeptide or epitope on a specific polypeptide and does not substantially bind to any other polypeptide or epitope on a polypeptide.

[0082] As used herein, the term "Ka" refers to the association rate of a particular antibody-antigen interaction, while the term "Kd" is intended to refer to the dissociation rate of a particular antibody-antigen interaction.

[0083] "Binding affinity" generally refers to the intensity of the cumulative non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, "binding affinity" refers to an inherent (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 generally be represented by a dissociation constant (Kd). Preferably, the Kd value is about 200 nM, 150 nM, 100 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 8 nM, 6 nM, 4 nM, 2 nM, 1 nM or less. Affinity can be measured by common methods known in the art (including methods described herein). Low-affinity antibodies generally bind antigens slowly and tend to dissociate easily, while high-affinity antibodies generally bind antigens faster and tend to remain bound longer. Various methods of measuring binding affinity are known in the art, any of which can be used for the purposes of the present invention.

[0084] In one embodiment of the invention, "Kd" or "Kd value" is measured by surface plasmon resonance assay using BIAcore™-2000 or BIAcore®-3000 (BIAcore, Inc., Piscataway, NJ) at 25°C with immobilized chip CM5 antigen at ~10 response units (RU). Briefly, carboxymethylated dextran biosensor chips (CM5, BIAcore Inc.) are activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the manufacturer's instructions. Antigen is diluted to a concentration of 5 μg / ml (~0.2 μM) with 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 bound protein. After antigen application, a 1 M ethanolamine solution is injected to block unreacted groups. For kinetic measurements, two-fold serial dilutions of Fab (e.g., 0.78 nM to 500 nM) were injected in PBS with 0.05% Tween 20 (PBST) at 25°C at a flow rate of approximately 25 μl / min. The association rate (kon) and the off rate (koff) were calculated by simultaneously fitting the association and dissociation sensorgrams using a simple one-to-one Langmuir binding model (BIAcoreEvaluation Software version 3.2). The equilibrium dissociation constant (Kd) was calculated as the ratio koff / kon. See, e.g., Chen, Y. et al., (1999) J. Mol. Biol. 293:865-881. If the association rate exceeds 10 according to the surface plasmon resonance method above, the 6 M −1 s −1 , it can be determined by fluorescence quenching, which measures the increase or decrease in fluorescence emission intensity (excitation = 295 nm; emission (radiation) = 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, are measured using a spectrophotometer, such as a stopped-flow spectrophotometer (Aviv Instruments) or a spectrophotometer SLM-Aminco (Thermo Spectronic) Series 8000 with a cuvette accompanied by stirring.

[0085] The term "koff" refers to the dissociation rate constant for a specific interaction of a binding molecule and an antigen. The Koff dissociation rate constant can be measured by biolayer interferometry, for example using an Octet™ system.

[0086] The "association rate" ("binding rate") or "kon" according to the present invention can also be measured by using the surface plasmon resonance assay described above using a chip with immobilized CM5 antigen at ~10 relative units (response units, RU) using a BIAcore™-2000 or BIAcore®-3000 (BIAcore, Inc., Piscataway, NJ) at 25°C. Briefly, carboxymethylated dextran biosensor chips (CM5, BIAcore Inc.) were activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the manufacturer's instructions. Antigen was diluted to a concentration of 5 μg / ml (~0.2 μM) with 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 bound protein. After antigen application, a 1 M ethanolamine solution was injected to block unreacted groups.

[0087] Unless otherwise indicated, the terms "biologically active" and "biological activity" and "biological characteristics" in relation to the polypeptides of the present invention mean the ability to bind to a biological molecule.

[0088] The expression "biomolecule" refers to nucleic acids, proteins, carbohydrates, lipids and combinations thereof. In one embodiment of the present invention, the biomolecule exists in nature.

[0089] Antibody fragments, such as Fab and F(ab')2 fragments, can be obtained from intact antibodies using conventional methods, such as papain or pepsin hydrolysis of intact antibodies. In addition, antibodies, antibody portions and immunoadhesion molecules can be obtained using standard recombinant DNA methods, such as those described herein.

[0090] The term "recombinant antibody" is intended to refer to an antibody expressed in a cell or cell line that contains a nucleotide sequence encoding the antibody, wherein the nucleotide sequence is not naturally associated with the cell.

[0091] As used herein, the term "variant antibody" refers to an antibody having an amino acid sequence that is different from the amino acid sequence of its "parent" antibody by adding, deleting and / or replacing one or more amino acid residues compared to the sequence of the parent antibody. In a preferred embodiment of the present invention, the variant antibody comprises at least one or more (e.g., one to twelve, e.g., two, three, four, five, six, seven, eight or nine, ten, eleven or twelve; in some embodiments, the variant antibody comprises one to about ten) amino acid additions, deletions and / or substitutions. In some embodiments, such additions, deletions and / or substitutions are made in the CDRs of the variant antibody. The identity or homology of the sequence of the variant antibody is defined herein as the percentage of amino acid residues in the variant antibody sequence that are equivalent to the amino acid residues of the parent antibody after aligning the sequences and, if necessary, introducing gaps to achieve the maximum percentage of sequence identity. The variant antibody retains the ability to bind to the same antigen and preferred epitopes bound by the parent antibody; and in some embodiments, at least one property or biological activity is superior to those of the parent antibody. For example, compared to the parent antibody, the variant antibody may have, for example, a more significant binding affinity, a longer half-life, a lower IC50 or an enhanced ability to inhibit the biological activity of an antigen. Of particular interest in this document are variant antibodies whose biological activity is at least 2 times (preferably at least 5 times, 10 times or 20 times) that of the parent antibody.

[0092] The term "bispecific antibody" means an antibody containing an antigen binding domain that can specifically bind to two different epitopes on one biomolecule or can specifically bind to epitopes on two different biomolecules. Bispecific antibodies are also referred to herein as having "dual specificity" or "dual specificity" antibodies.

[0093] In a broad sense, the term "chimeric antibody" refers to an antibody that contains one or more regions of an antibody and one or more regions of one or more other antibodies, typically part human and part non-human, i.e., partly derived from non-human animals, such as vermin such as mice, rats, or camelids, such as llamas and alpacas. Chimeric antibodies are generally preferred over non-human antibodies in order to reduce the risk of human anti-antibody immune responses, such as human anti-mouse antibody immune responses in the case of mouse antibodies. Common examples of chimeric antibodies are antibodies in which the variable region sequences are mouse sequences and the constant region sequences are human. In the case of chimeric antibodies, the non-human portion may be further modified to humanize the antibody.

[0094] 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 a target antigen, respectively, or a chimeric antibody based on such an antibody of a mouse or llama, it is possible to replace certain amino acids, particularly in the framework region and constant domain of the heavy and light chains, in order to avoid or minimize an immune response in humans. The specificity of an antibody interacts with the target antigen predominantly through 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 variable between individual antibodies than the amino acid sequences outside the CDRs. Because the CDR sequences of the sites are responsible for most antibody-antigen interactions, recombinant antibodies can be expressed, for example, by constructing an expression vector that expresses the CDR sequences of the mapping specific antibody and the framework sequences of another antibody, and the recombinant antibody simulates the properties of a specific natural antibody or more generally a specific antibody having a given amino acid sequence. As a result, it is possible to "humanize" a non-human antibody and, to a large extent, preserve the binding specificity and affinity of the initial antibody. Although it is impossible to accurately predict the immunogenicity of a particular antibody and thus the human anti-antibody response, non-human antibodies are generally more immunogenic than human antibodies. Chimeric antibodies, in which foreign (e.g., vermin or Camelidae) constant regions have been replaced with sequences of human origin, show generally lower immunogenicity than antibodies of completely foreign origin, and there is a trend to use humanized or fully human antibodies in therapeutic antibodies. Therefore, chimeric antibodies or other antibodies of non-human origin can be humanized to reduce the risk of human anti-antibody response.

[0095] For chimeric antibodies, humanization generally involves modification of the framework region of the variable region sequence. The amino acid residues that are part of the complementary determining region (CDR) are most often modified without the aid of humanization, although in some cases, it may be desirable to modify the individual amino acid residues of the CDR, for example, to remove glycosylation sites, deamidation sites, aspartic acid isomerization sites, or unwanted cysteine ​​or methionine residues. N-linked glycosylation occurs by attaching an oligosaccharide chain to an asparagine residue in a tripeptide sequence Asn-X-Ser or Asn-X-Thr, wherein X can be any amino acid except Pro. Removal of N-glycosylation sites can be achieved by mutating Asn or Ser / Thr residues with another residue, preferably by conservative substitution. Deamidation of asparagine and glutamine residues can occur depending on such factors as pH and surface exposure. Asparagine residues are particularly susceptible to deamidation, especially when they are present in Asn-Gly sequences, and to a lesser extent in other dipeptide sequences such as Asn-Ala. Where such a deamidated region, e.g. Asn-Gly, is present in the sequence of a CDR region, it may often be preferred to remove this region by conservatively replacing one of the residues involved.

[0096] Numerous methods for humanization of antibody sequences are known in the art. A commonly used method is CDR site transplantation. CDR transplantation can be based on Kabat CDR definition, although the latest version (Magdelaine-Beuzelin et al., CritRev.Oncol Hematol. 64: 210 225 (2007)) suggests that the definition of IMGT® (International ImMunoGeneTics Information System®, www.imgt.org) can improve humanization results (see Lefranc et al., Dev. Comp Immunol. 27: 55-77 (2003)). In some cases, compared with the parent antibody from which the CDR is obtained, CDR transplantation can reduce the binding specificity and affinity of the non-human antibody transplanted by CDR, and thus reduce its biological activity. Back mutation (which is sometimes referred to as "framework repair") can be used in the selected position (usually in the framework region) of the CDR transplanted antibody to restore the binding specificity and affinity of the parent antibody. The information available in the literature and antibody database can be used to perform the determination of the position for possible back mutation. The amino acid residues that are candidates for back mutations are usually located on the surface of the antibody molecule, and buried residues or residues with a lower degree of surface exposure are usually not changed. As an alternative to CDR site transplantation and back mutation, the humanization method is surface change, in which non-exposed residues of non-human origin are retained, while residues exposed on the surface are changed to human residues.

[0097] There are two techniques for generating fully human antibodies: using phage libraries collected in vitro or in vivo by immunizing humanized animals (mice, rats, etc.).

[0098] Phage display is the first and most widely used in vitro antibody search technology. In 1985, Smith found that foreign DNA sequences can be cloned into filamentous phage M13, and such cloned sequences can be expressed as fusion proteins on the surface of phage particles (Smith GP: Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface. Science 1985, 228: 1315-1317.). Therefore, it is possible to select the target fusion protein based on its ability to bind other proteins. This discovery is combined with the PCR amplification method, which makes it possible to clone the cDNA reservoir of immunoglobulin genes to produce various phage libraries containing variable domains, which can be used to quickly search for target-specific monoclonal antibodies. The phage library reservoir reflects the B cell antibody reservoir of each person or animal whose blood is used to produce the library. In 1995, two articles reported the production of genetically modified mice expressing a full human antibody library that was comparable to those 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 chain and kappa light chain immunoglobulin genes were intentionally destroyed, followed by the introduction of transgenes, which were segments of human heavy chain and kappa light chain genes. It was demonstrated that human gene repertoires can be used by the mouse immune system to produce highly specific and high affinity antibodies against a wider variety of antigens. Although the transgenic mice expressed a B-cell receptor that was essentially a hybrid of mouse and human components (human immunoglobulins, mouse Igα, Igβ, and other signaling molecules), their B cells developed and matured normally.

[0099] In some cases, it may also be preferred to change one or more CDR amino acid residues in order to improve the binding affinity to the target epitope. This is called "affinity maturation" and can be optionally performed in conjunction with humanization, such as in cases where humanization of an antibody results in reduced binding specificity or affinity and the binding specificity or affinity cannot be sufficiently improved by back mutation alone. Various affinity maturation methods are known in the art, such as the in vitro scanning saturation mutagenesis method described by Burks et al., Proc Natl Acad Sci USA, 94: 412–417 (1997), and the stepwise in vitro affinity maturation method of Wu et al., Proc Natl Acad Sci USA 95: 6037 6042 (1998).

[0100] The term "monoclonal antibody" or "mAb" refers to an antibody that is synthesized and isolated from a separate 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 fusing individual B lymphocytes from an immunized animal with individual cells from a lymphoma. Hybridomas are a type of constructed cell and do not exist in nature.

[0101] "Native antibodies" are usually heterotetrameric glycoproteins with a molecular weight of about 150,000 daltons, consisting of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to the heavy chain by one covalent disulfide bond, while the number of disulfide bonds varies in the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has a variable domain (VH) at one end, followed by multiple constant domains. Each light chain has a variable domain (VL) at one end and a constant domain at its other end. The constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the variable domain of the light chain is aligned with the variable domain of the heavy chain. It is believed that specific amino acid residues form an interface between the light chain and heavy chain variable domains.

[0102] The term "isolated" used in this specification to describe various antibodies refers to antibodies that have been identified and separated and / or regenerated from cells or cell cultures in which the antibodies are expressed. Impurities (contaminant components) from the natural environment are materials that will interfere with the diagnostic or therapeutic use of the polypeptide, and may include enzymes, hormones, and other protein or non-protein 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 using a spinning cup sequencer (Edman sequencer), or (2) to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie Brilliant Blue or preferably silver staining. Isolated antibodies include antibodies in situ within recombinant cells because at least one component of the natural environment of the polypeptide is absent. Isolated polypeptides are typically obtained by at least one purification step.

[0103] An "isolated" nucleic acid molecule is a nucleic acid molecule that is identified and separated from at least one nucleic acid molecule-impurity to which it is associated in the natural source of the antibody nucleic acid. An isolated nucleic acid molecule is different from the form or collection in which it is found under natural conditions. Thus, an isolated nucleic acid molecule is different from a nucleic acid molecule that is present in a cell under natural conditions. However, an isolated nucleic acid molecule includes a nucleic acid molecule that is located in a cell in which the antibody is normally expressed, for example, if the nucleic acid molecule has a chromosomal localization that is different from its localization in the cell under natural conditions.

[0104] As used herein, the term "epitope" refers to a portion (determinant) of an antigen that specifically binds to a binding molecule (e.g., an antibody or related molecule, such as a bispecific binding molecule). Epitope determinants are typically grouped by chemically active surface groups of molecules, such as amino acids or carbohydrate or sugar side chains, and typically contain specific three-dimensional structural features as well as specific charge characteristics. Epitopes can be "linear" or "conformational". In a linear epitope, all interaction points between a protein (e.g., an antigen) and an interacting molecule (e.g., an antibody) exist linearly along the primary amino acid sequence of the protein. In a conformational epitope, the interaction points exist across amino acid residues on the protein that are separated from each other in the primary amino acid sequence. When the desired antigenic epitope is determined, antibodies against the epitope can be generated using techniques well known in the art. In addition, the generation and characterization of antibodies or other binding molecules can clarify information about the desired epitope. Based on this information, binding molecules that bind to the same or similar epitopes can then be competitively screened, for example, by conducting competition studies to find binding molecules that compete for binding to the antigen.

[0105] As used herein, the term "peptide linker" is intended to mean any peptide having the ability to connect domains, whose length depends on the domains to which it is bound to each other, and comprises any amino acid sequence. Preferably, the peptide linker has a length of more than 5 amino acids and is composed of any amino acid set selected from G, A, S, P, E, T, D, K.

[0106] The term "in vitro" refers to biological objects, biological processes or biological reactions outside the body that are modeled under artificial conditions. For example, cells grown in vitro are understood to be cells grown in an environment outside the body, such as in a test tube, culture vial or microtiter plate.

[0107] The term "IC 50 "(Inhibitory concentration 50%)" refers to the concentration of a drug at which a measurable activity or response, e.g., growth / proliferation of cells such as tumor cells, is inhibited by 50%. IC 50 Values ​​can be calculated using appropriate dose-response curves using specialized statistical software for curve fitting.

[0108] The term GI50 (growth inhibition 50%) refers to the concentration of drug at which the proliferation of cells, such as tumor cells, is inhibited by 50%.

[0109] The term "ED50" (EC50) (50% effective dose / concentration) refers to the concentration of a drug that produces 50% of the biological effect (which may include cytotoxicity).

[0110] The term antibody "effector function" refers to biological activities attributable to the Fc region of an antibody (a native Fc region sequence or an Fc region amino acid variant), or that vary with the antibody isotype. Examples of antibody effector functions include: q 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.

[0111] "Antibody-dependent cellular cytotoxicity" or "ADCC" refers to a cell-mediated response in which nonspecific cytotoxic cells expressing Fc receptors (FcRs), such as natural killer (NK) cells, neutrophils, and macrophages, recognize bound antibodies on target cells and subsequently cause lysis of the target cells. NK cells, the primary cells for mediating ADCC, express only FcγRJII, while 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). In order to evaluate the ADCC activity of a molecule of interest, an in vitro ADCC assay, such as that described in U.S. Pat. Nos. 5,500,362 or 5,821,337, can be performed. Applicable effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively or additionally, ADCC activity of the molecule of interest may be assessed in vivo, for example in a animal model such as that disclosed in Clynes et al. PNAS (USA) 95:652-656 (1998).

[0112] "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; of which PBMCs and NK cells are preferred. Effector cells can be isolated from their natural sources, for example, from blood or PBMCs as described herein.

[0113] 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. In addition, a preferred FcR is an FcR that binds to IgG antibodies (gamma receptors), and includes receptors of the FcγRI, FcγRII (FcγRIIa and FcγRIIb) and FcγRIII (FcγRIIIa and FcγRIIIb) subclasses, including allelic variants and alternative splicing forms of these receptors. FcγRI shows high affinity for IgG, while FcγRII and FcγRIII show low affinity. FcγRIIa and FcγRIIIa are activating FcγRs expressed on monocytes / macrophages and monocytes / macrophages / natural killer cells, respectively, and are capable of triggering cytotoxicity of human target cells. The activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. Inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain (see Daëron, Annu. Rev. Immunol. 15:203-234 (1997) for review). FcRs are reviewed in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). Other FcRs, including those to be identified in the future, are encompassed by the term "FcR" herein. The term also includes the neonatal receptor FcRn, which is responsible for the transfer of maternal IgG to the fetus.

[0114] "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 can be performed, e.g., as described in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996).

[0115] The term "identity" or "homology" should be interpreted as meaning that after comparing sequences, and introducing gaps when necessary, to reach the maximum identity percentage about the entire sequence, and without considering any conservative substitutions as part of sequence identity, the percentage of amino acid residues in the candidate sequence that are equivalent to the residues of the corresponding sequence to which it is compared. Extensions and insertions of the N-terminal or C-terminal should not be interpreted as reducing identity or homology. Methods and computer programs for comparison are well known in the art. Sequence analysis software (e.g., Sequence Analysis Software Package, Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Ave., Madison, WI 53705) can be used to measure sequence identity. This software matches similar sequences by assigning a certain degree of homology to various substitutions, deletions (eliminations) and other modifications.

[0116] The term "homologous" with respect to polypeptide sequences of antibodies should be interpreted as antibodies showing at least 70%, preferably 80%, more preferably 90% and most preferably 95% sequence identity with respect to the polypeptide sequence. The term with respect to nucleic acid sequences should be interpreted as nucleotide sequences showing at least 85%, preferably 90%, more preferably 95% and most preferably 97% sequence identity with respect to the nucleic acid sequence.

[0117] Proposed modifications of the amino acid sequence of an antibody are 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 an antibody are prepared by introducing appropriate nucleotide changes into the antibody nucleic acid, or by peptide synthesis. Such modifications include, for example, deletions and / or insertions and / or substitutions of residues within the amino acid sequence of the antibody. Any combination of deletions, insertions and substitutions is performed to obtain the final construct, provided that the final construct has the desired characteristics. Amino acid changes can also alter post-translational processes in the antibody, such as changing the number or position of glycosylation sites.

[0118] Variants that modify the amino acid sequence of an antibody using amino acid substitutions. Such variants replace at least one amino acid residue in an antibody molecule with different residues. The most interesting sites for substitution mutagenesis include hypervariable regions or CDRs, but FR or Fc changes are also contemplated. Conservative substitutions are shown under "preferred substitutions" in Table A. If such substitutions result in changes in biological activity, additional significant changes may be introduced, referred to as "exemplary substitutions" in Table A, or changes further described in the amino acid classes described below, and the product may be screened.

[0119]

[0120] The terms "nucleic acid", "nucleic acid sequence", "nucleic acid sequence", "polynucleotide", "oligonucleotide", "polynucleotide sequence" and "nucleotide sequence" are used interchangeably in this specification and refer to the precise sequence of nucleotides, which is modified or unmodified, determines the fragment or region of the nucleic acid, contains or does not contain non-natural nucleotides, and is double-stranded DNA or RNA, single-stranded DNA or RNA, or the transcription product of said DNA.

[0121] It should also be included here that the present invention does not relate to nucleotide sequences in their natural chromosomal environment, i.e. in the natural state. The sequences of the present invention have been isolated and / or purified, i.e. they have been sampled directly or indirectly (e.g. by copying), and their environment has been at least partially modified. Thus, it should also be mentioned here that isolated nucleic acids obtained by recombinant genetics, for example with the aid of a host cell, or isolated nucleic acids obtained by chemical synthesis.

[0122] Unless otherwise indicated, reference to a nucleotide sequence encompasses its complement. Thus, reference to a nucleic acid having a particular sequence should be understood to encompass a nucleic acid having the complementary strand of its complementary sequence.

[0123] The expression "control sequence" refers to DNA sequences necessary for the expression of a functionally relevant coding sequence in a specific host organism. Control sequences suitable for prokaryotes include, for example, a promoter, optionally an operator sequence and a ribosome binding site. It is known that eukaryotic cells utilize promoters, polyadenylation signals and enhancers.

[0124] A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, if the DNA of a presequence or secretory leader is expressed as a preprotein that participates in the secretion of a polypeptide, it is operably linked to the DNA of a polypeptide; if a promoter or enhancer affects the transcription of the sequence, it is operably linked to a coding sequence; if a ribosome binding site is positioned so as to promote translation, it is operably linked to a coding sequence. Generally, "operably linked" means that the DNA sequences being linked are contiguous, and in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous.

[0125] As used herein, the term "vector" refers to a nucleic acid molecule that can transport another nucleic acid that it has been connected to. In some embodiments, the vector is a plasmid, i.e., another DNA segment can be connected to a circular double-stranded DNA sheet therein. In some embodiments, the vector is a viral vector, in which another DNA segment can be connected to the viral genome. In some embodiments, the vector can be autonomously replicated in the host cell that they introduce therein (e.g., a bacterial vector and an additional mammalian vector with a bacterial replication origin site). In a further embodiment, the vector (e.g., a non-additional mammalian vector) can be integrated into the genome of the host cell after being introduced into the host cell, and thus replicated together with the host gene. In addition, some vectors can instruct the expression of the gene that they are operably connected to. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors").

[0126] As used herein, the term "recombinant host cell" (or simply "host cell") is intended to refer to a cell into which a recombinant expression vector has been introduced. The present invention relates to a host cell, which may include, for example, a vector according to the present invention described above. The present invention also relates to a host cell, which comprises, for example, a nucleotide sequence encoding a heavy chain or an antigen binding portion thereof of the first binding domain and / or the second binding domain of a binding molecule of the present invention, a nucleotide sequence encoding a light chain or an antigen binding portion thereof of the first binding domain and / or the second binding domain of a binding molecule of the present invention, or both. It should be understood that "recombinant host cell" and "host cell" are intended to refer not only to a specific subject cell, but also to the progeny of such cells. Because modifications may occur in subsequent generations due to mutations or environmental influences, therefore, in fact, such progeny may not be equivalent to the parental cell, however, such cells are still included within the scope of the term "host cell" as used herein.

[0127] The term "excipient" is used herein to describe any ingredient other than a compound of the invention.

[0128] "Pharmaceutical composition" refers to a composition comprising an antibody of the invention and at least one component selected from the group consisting of pharmaceutically acceptable and pharmacologically compatible fillers, solvents, diluents, carriers, adjuvants, dispensing agents and sensors, delivery agents such as preservatives, stabilizers, fillers, disintegrants, wetting agents, emulsifiers, suspending agents, thickeners, sweeteners, flavoring agents, aromatizers, antibacterial agents, fungicides, lubricants and extended delivery control agents, the selection and appropriate proportion of which depends on the type and mode of administration and the dosage. Examples of suitable suspending agents are ethoxylated isostearyl alcohol, polyethylene oxide, sorbitol and sorbitol ethers, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar and tragacanth gum and mixtures thereof. Protection against the action of microorganisms can be provided by various antibacterial and antifungal agents, such as parabens, chlorobutanol, sorbic acid and similar compounds. The composition may also contain isotonic agents such as sugars, polyols, sodium chloride and the like. The extended effect of the composition can be achieved by slowing down the absorption of the active ingredient, such as aluminum monostearate and gelatin. Examples of suitable carriers, solvents, diluents and delivery agents include water, ethanol, polyols and mixtures thereof, natural oils (such as olive oil) and organic esters (such as ethyl oleate) for injection. Examples of fillers are lactose, milk-sugar, sodium citrate, calcium carbonate, calcium phosphate, etc. Examples of disintegrants and dispensing agents are starch, alginic acid and its salts, silicates. Examples of suitable lubricants are magnesium stearate, sodium lauryl sulfate, talcum and high molecular weight polyethylene glycol. Alone or in combination with another active compound, the pharmaceutical composition for oral, sublingual, transdermal, intraocular, intramuscular, intravenous, subcutaneous, topical or rectal administration of the active ingredient can be applied to humans and animals in a mixture with a conventional pharmaceutical carrier in a standard application form. Suitable standard administration forms include oral forms, such as tablets, gelatin capsules, pills, powders, granules, chewing gum and oral solutions or suspensions; sublingual and buccal administration forms; aerosols; implants; topical, transdermal, subcutaneous, intramuscular, intravenous, intranasal or intraocular forms and rectal administration forms.

[0129] “Medicament” – is a compound (or a mixture of compounds as a pharmaceutical composition) in the form of tablets, capsules, solutions, ointments and other ready-made forms, which is intended for use in restoring, improving or modifying physiological functions in humans and animals, as well as for the treatment and prevention of diseases, for diagnosis, anesthesia, contraception, cosmetology and others.

[0130] The term "by CD47 and PD-L1 "Disease or condition mediated" means a disease or condition CD47 and PD-L1All diseases or conditions directly or indirectly related, including the cause, development, progression, persistence or pathology of the disease or condition. "Treat", "treating" and "treatment" refer to a method of alleviating or eliminating a biological condition and / or at least one of its associated symptoms. As used herein, "alleviating" a disease, condition or illness means reducing the severity and / or frequency of the symptoms of the disease, condition or illness. Further, references herein to "treatment" include references to curative, palliative and preventive treatments.

[0131] In one aspect, the subject or patient treated is a mammal, preferably a human subject. The subject can be male or female of any age.

[0132] The term "disorder" means any condition that would benefit from treatment with the compounds of the invention. The definition of this term includes chronic and acute disorders or diseases, including pathological conditions that predispose mammals to such an insult. A preferred disorder to be treated according to the invention is cancer.

[0133] The terms "cancer" and "cancerous" refer to a physiological condition or describe a physiological condition in a mammal that is generally characterized by unregulated cell growth / proliferation. This definition encompasses both benign and malignant cancerous diseases. Examples of cancerous diseases include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More specific examples of such cancerous diseases include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma and lung squamous 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 cancer or uterine cancer, salivary gland cancer, kidney cancer or renal cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, melanoma, and various head and neck cancers.

[0134] The terms "immune response," "autoimmune response," and "autoimmune inflammation" refer to the actions of, for example, lymphocytes, antigen presenting cells, phagocytes, granulocytes, and soluble macromolecules produced by such cells or hepatocytes, including antibodies, cytokines, and complement produced as a result of selective damage, destruction, or elimination of invading pathogens, cells or tissues infected by pathogens, cancer cells, or, in the case of autoimmunity or pathological inflammation, normal cells or tissues from the human body.

[0135] A "therapeutically effective amount" is intended to mean an amount of the therapeutic agent being administered which relieves to some extent one or more of the symptoms of the condition being treated.

[0136] The term "chronic" use refers to the continuous (ongoing) use of an agent, as opposed to an acute (short-term) route of administration, in order to maintain the initial therapeutic effect (activity) over an extended period of time.

[0137] "Intermittent" use refers to treatment that is not continuous without interruption, but is cyclical in nature.

[0138] As used herein, the words “comprise,” “have,” “include” or variations such as “comprises,” “comprising,” “has,” “having,” “includes,” or “including,” and all grammatical variations thereof, will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. DETAILED DESCRIPTION OF THE INVENTION

[0140] Antibody

[0141] The present invention relates to antibodies that bind to CD47 and PD-L1.

[0142] In one embodiment of the invention, the antibody is a full-length antibody or an antigen-binding fragment thereof.

[0143] In one embodiment of the invention, the antibodies of the invention comprise one or two binding sites for PD-L1.

[0144] In one embodiment of the present invention, the binding site against CD47 inhibits the interaction between the CD47 receptor and the SIRPα ligand, and / or the binding site against PD-L1 inhibits the interaction between PD-L1 and the PD-1 receptor.

[0145] In one embodiment, the invention relates to an antibody that binds to CD47 and PD-L1, and the binding site for CD47 comprises a heavy chain variable region comprising:

[0146] (a) a CDR1 comprising an amino acid sequence that is at least 80% or 90% homologous or identical to a sequence selected from SEQ ID NOs: 1 to 4, i.e., CDR1 is a sequence selected from SEQ ID NOs: 1 to 4, or a sequence selected from SEQ ID NOs: 1 to 4 with 1 or 2 substitutions;

[0147] (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 SEQ ID NOs: 6 to 15, i.e., the CDR2 is a sequence of SEQ ID NOs: 6 to 15, or a sequence selected from SEQ ID NOs: 6 to 15 having 1, 2, 3, 4 or 5 substitutions;

[0148] (c) 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 SEQ ID NOs: 17 to 20, i.e., the CDR3 is a sequence selected from SEQ ID NOs: 17 to 20, or a sequence selected from SEQ ID NOs: 17 to 20 having 1, 2 or 3 substitutions.

[0149] In one embodiment, the invention relates to an antibody that binds to CD47 and PD-L1, and the binding site for CD47 comprises a heavy chain variable region comprising:

[0150] (d) a CDR1 comprising an amino acid sequence identical to a sequence selected from SEQ ID NOs: 1 to 4;

[0151] (e) a CDR2 comprising an amino acid sequence identical to a sequence selected from SEQ ID NOs: 6 to 15;

[0152] (f) a CDR3 comprising an amino acid sequence identical to a sequence selected from SEQ ID NOs: 17-20.

[0153] In one embodiment, the invention relates to an antibody that binds to CD47 and PD-L1 and comprises a binding site for CD47 comprising:

[0154] (a) a heavy chain variable region comprising:

[0155] (i) a CDR1 comprising an amino acid sequence that is at least 80% or 90% homologous or identical to a sequence selected from SEQ ID NOs: 1 to 4, i.e., CDR1 is a sequence selected from SEQ ID NOs: 1 to 4, or a sequence selected from SEQ ID NOs: 1 to 4 with 1 or 2 substitutions;

[0156] (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 SEQ ID NOs: 6 to 15, i.e., the CDR2 is a sequence of SEQ ID NOs: 6 to 15, or a sequence selected from SEQ ID NOs: 6 to 15 having 1, 2, 3, 4 or 5 substitutions;

[0157] (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 SEQ ID NOs: 17 to 20, i.e., the CDR3 is a sequence selected from SEQ ID NOs: 17 to 20, or a sequence selected from SEQ ID NOs: 17 to 20 having 1, 2 or 3 substitutions, and

[0158] (b) a light chain variable region comprising:

[0159] (i) a CDR1 comprising an amino acid sequence that is at least 80% or 90% homologous or identical to a sequence selected from SEQ ID NOs: 22 to 34, i.e., CDR1 is a sequence selected from SEQ ID NOs: 22 to 34, or a sequence selected from SEQ ID NOs: 22 to 34 with 1 or 2 substitutions,

[0160] (ii) a CDR2 comprising an amino acid sequence that is at least 80%, 87% or 94% homologous or identical to a sequence selected from SEQ ID NOs: 36 to 48, i.e., the CDR2 is a sequence selected from SEQ ID NOs: 36 to 48, or a sequence selected from SEQ ID NOs: 36 to 48 having 1, 2 or 3 substitutions,

[0161] (iii) a CDR3 comprising an amino acid sequence that is at least 80% or 90% homologous or identical to a sequence selected from SEQ ID NOs: 50-64, i.e., the CDR3 is a sequence selected from SEQ ID NOs: 50-64, or a sequence selected from SEQ ID NOs: 50-64 with 1 or 2 substitutions.

[0162] In one embodiment, the invention relates to an antibody that binds to CD47 and PD-L1 and comprises a binding site for CD47 comprising:

[0163] (a) a heavy chain variable region comprising:

[0164] (i) a CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 4,

[0165] (ii) a CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 6 to 15,

[0166] (iii) a CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 17-20, and

[0167] (b) the variable region of the light chain, comprising:

[0168] (i) a CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 22 to 34,

[0169] (ii) a CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 36 to 48,

[0170] (iii) a CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 50-64.

[0171] In one embodiment, the invention relates to an antibody that binds CD47 and PD-L1, and comprises a binding site for CD47 comprising:

[0172] (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 SEQ ID NOs: 66-88, and

[0173] (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 SEQ ID NOs: 89-106.

[0174] In one embodiment, the invention relates to an antibody that binds CD47 and PD-L1, and comprises a binding site for CD47 comprising:

[0175] (a) a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 66-88, and

[0176] (b) a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 89-106.

[0177] In one embodiment, the invention relates to an antibody that binds CD47 and PD-L1, and comprises a binding site for PD-L1, said binding site comprising:

[0178] (a) a light chain variable region comprising:

[0179] (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 the sequence of SEQ ID NO: 5 with 1 substitution;

[0180] (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., the CDR2 is the sequence of SEQ ID NO: 16, or the sequence of SEQ ID NO: 16 with 1, 2 or 3 substitutions;

[0181] (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 the sequence of SEQ ID NO: 21 having 1 or 2 substitutions, and

[0182] (b) a variable region comprising:

[0183] (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., the CDR1 is the sequence of SEQ ID NO: 35, or the sequence of SEQ ID NO: 35 having 1, 2 or 3 substitutions;

[0184] (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., the CDR2 is the sequence of SEQ ID NO: 49, or the sequence of SEQ ID NO: 49 with 1 substitution;

[0185] (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 the sequence of SEQ ID NO: 65 with 1 or 2 substitutions.

[0186] In one embodiment, the invention relates to an antibody that binds CD47 and PD-L1, and comprises a binding site for PD-L1, said binding site comprising:

[0187] (a) a heavy chain variable region comprising:

[0188] (i) a CDR1 comprising the amino acid sequence of SEQ ID NO: 5,

[0189] (ii) a CDR2 comprising the amino acid sequence of SEQ ID NO: 16,

[0190] (iii) a CDR3 comprising the amino acid sequence of SEQ ID NO: 21, and

[0191] (b) a light chain variable region comprising:

[0192] (i) a CDR1 comprising the amino acid sequence of SEQ ID NO: 35,

[0193] (ii) a CDR2 comprising the amino acid sequence of SEQ ID NO: 49,

[0194] (iii) a CDR3 comprising the amino acid sequence of SEQ ID NO: 65.

[0195] In one embodiment, the invention relates to an antibody that binds to CD47 and PD-L1 and is characterized in that the binding site against CD47 is a Fab, scFv, scFab or an isolated VH or VHH single domain.

[0196] In one embodiment, the invention relates to an antibody that binds to CD47 and PD-L1 and is characterized in that the binding site against PD-L1 is a Fab, scFv, scFab or an isolated VH or VHH single domain.

[0197] In one embodiment, the invention relates to an antibody that binds to CD47 and PD-L1 and is characterized in that it stimulates antibody-dependent cellular cytotoxicity, macrophage-mediated phagocytosis, complement-dependent cytotoxicity and / or T-cell-mediated cytotoxicity against cells covered with CD47 and / or PD-L1 antigens.

[0198] In one embodiment, the invention relates to an antibody that binds to CD47 and PD-L1 and is characterized in that it comprises an Fc fragment having 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.

[0199] Nucleic acid molecules

[0200] The present invention also relates to encoding the invention described herein Anti-CD47 / PD-L1 Antibodies In some embodiments, the various nucleic acid molecules encode Anti-CD47 / PD-L1 Antibodies The first domain and the second domain of the amino acid sequence. In some embodiments in which the first domain and / or the second domain include heavy chains and light chains, different nucleic acids encode heavy chain and light chain amino acid sequences. In other embodiments, the same nucleic acid molecule encodes heavy chain and light chain sequences. In certain embodiments, the nucleic acid molecule can encode any combination of the amino acid sequences (e.g., heavy chain and light chain sequences) of the first domain and the second domain. In certain embodiments, the nucleic acid molecule can 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 the peptide linker connecting it. Unless otherwise specified, the reference to the nucleotide sequence covers its complement. Therefore, the reference to the nucleic acid with a specific sequence should be understood as covering the nucleic acid with its complementary chain of its complementary sequence. As used herein, the term "polynucleotide" means a polymeric form of a nucleotide or ribonucleotide or deoxyribonucleotide or a modified form of any type of nucleotide having a length of at least 10 bases. The term includes single-stranded and double-stranded forms.

[0201] In any of the above embodiments, the nucleic acid molecule may be isolated.

[0202] 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 invention can be synthesized rather than isolated.

[0203] In some embodiments, the nucleic acid molecules of the present invention may include a nucleotide sequence encoding a VH domain from the first domain or the second domain of an antibody of the present invention, which is linked in frame to a nucleotide sequence encoding a heavy chain constant domain from any source. Similarly, the nucleic acid molecules of the present invention may include a nucleotide sequence encoding a VL domain from the first region or the second region of an antibody of the present invention, which is linked in frame to a nucleotide sequence encoding a light chain constant domain from any source.

[0204] In a further aspect of the invention, nucleic acid molecules encoding the variable domains of the heavy (VH) and / or light (VL) chains of the first or second binding domains can be "converted" over the entire length of an antibody gene. In one embodiment, nucleic acid molecules encoding VH or VL domains are converted to antibody genes over the entire length by insertion into expression vectors that already encode heavy chain constant (CH) or light chain constant (CL) domains, respectively, such that the VH segment is operably linked to the CH segment within the vector and / or the VL segment is operably linked to the CL segment within the vector. In another embodiment, nucleic acid molecules encoding VH and / or VL domains are converted to antibody genes over the entire length using standard molecular biology techniques by connecting (e.g., joining) nucleic acid molecules encoding VH and / or VL domains to nucleic acid molecules encoding CH and / or CL domains. Nucleic acid molecules encoding full-length heavy and / or light chains can then be expressed in cells into which they have been introduced.

[0205] Nucleic acid molecules can be used to express large quantities of recombinant anti-CD47 / PD-L1 antibodies. As described herein, nucleic acid molecules can also be used to produce human antibodies, humanized antibodies, chimeric antibodies, bispecific antibodies, single-chain antibodies, immunoadhesins, diabodies, mutant antibodies, and antibody derivatives.

[0206] Carrier

[0207] In another aspect, the present invention relates to vectors suitable for expressing any of the nucleotide sequences described herein.

[0208] The present invention relates to a vector comprising a nucleic acid molecule encoding a Anti-CD47 / PD-L1 AntibodyOr any amino acid sequence of a portion thereof (e.g., a heavy chain sequence of the first binding domain and / or a heavy chain and / or light chain sequence of the second binding domain). The present invention further provides a vector comprising a nucleic acid molecule encoding a fusion protein, a modified antibody, or an antibody fragment.

[0209] In another embodiment, nucleic acid molecules and vectors can be used to prepare mutant Anti-CD47 / PD-L1 Antibodies The antibody may be mutated in the variable domain of the heavy chain and / or light chain of the first binding domain and / or the heavy chain and / or light chain of the second binding domain, for example, to alter Antibody For example, mutations can be made in one or more CDRs to increase or decrease Antibody K D , increase or decrease k off , or change the binding specificity of the antibody with respect to FcRn. In another embodiment, in conjunction with the present invention Anti-CD47 / PD-L1 Antibodies In an antibody corresponding to the first binding domain or the second binding domain of the present invention, one or more mutations are made at amino acid residues known to be altered compared to the germline. Such mutations can be made in the CDRs or framework regions of the variable domains, or in the constant domains. In a preferred embodiment, mutations are made in the variable domains. In another embodiment, one or more mutations are made at amino acid residues known to be altered compared to the germline in the CDRs or framework regions of the variable domains of the present invention.

[0210] In some embodiments, the present invention Anti-CD47 / PD-L1 AntibodiesBy inserting the DNA obtained as described above in the expression vector, the gene is operably connected to the necessary expression control sequence such as transcription and translation control sequence and expressed, and the DNA partially or completely encodes the sequence of the first binding domain or the second binding domain (for example, light chain and heavy chain sequence, wherein the binding domain comprises light chain and heavy chain sequence). Expression vectors include plasmids, retroviruses, adenoviruses, adeno-associated viruses (AAV), plant viruses (for example, cauliflower mosaic virus, tobacco mosaic virus), cosmids, YACs, EBV derived episomes, etc. DNA molecules can be connected to the vector so that the transcription and translation control sequences in the vector play their expected functions of regulating DNA transcription and translation. Expression vectors and expression control sequences can be selected to be compatible with the expression host cell used. DNA molecules of the sequence of the first binding domain and the second binding domain (for example, heavy chain and light chain sequence, wherein the binding domain comprises heavy chain and light chain sequence) can be introduced into each vector. In one embodiment, any combination of the DNA molecules is introduced into the same expression vector. The DNA molecules can be introduced into the expression vector by standard methods (eg, ligation of complementary restriction sites on the antibody gene fragment and the vector, or blunt-end ligation if no restriction sites are present).

[0211] Suitable vectors are following vectors, which encode functionally complete human CH or CL immunoglobulin sequences, with appropriate restriction sites that can be easily inserted and expressed as described above by transformation. The genes encoding HC and LC in such vectors may include intron sequences, which lead to enhanced total antibody protein yields by stabilizing the corresponding mRNA. The flanks of the intron sequences are splice donor and splice acceptor sites, which determine where RNA splicing occurs. When multiple introns are used, the position of the intron sequence may be in the variable region or constant region of the antibody chain, or in both the variable region and the constant region. Polyadenylation and transcription termination may occur at the natural chromosome site downstream of the coding region. The recombinant expression vector may also encode a signal peptide, which promotes secretion of the antibody chain from the host cell. The antibody chain gene may be cloned into a vector so that the signal peptide is connected to the amino terminus of the immunoglobulin chain in frame. The signal peptide may be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin).

[0212] In addition to the chain genes of the antibody, the recombinant vector expression of the present invention can also carry regulatory sequences that control the expression of the genes of the antibody chain in the host cell. It will be appreciated by those skilled in the art that the design of the expression vector, including the selection of regulatory sequences, can depend on such factors as the selection of the host cell to be transformed, the expression level of the desired protein, and the like. Preferred control sequences for expression host cells in mammals include viral elements that ensure high levels of protein expression in mammalian cells, such as promoters and / or enhancers derived from: retroviral LTR, 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, and strong mammalian promoters such as natural immunoglobulin promoters or actin promoters. For further description of viral regulatory elements and sequences thereof, see, for example, U.S. Pat. Nos. 5,168,062, 4,510,245, and 4,968,615. Methods for expressing binding molecules such as antibodies in plants, including descriptions of promoters and vectors and transformation of plants, are known in the art. See, for example, U.S. Pat. No. 6,517,529. Methods for expressing polypeptides in bacterial cells or fungal cells such as yeast cells are also well known in the art.

[0213] In addition to the antibody chain genes and regulatory sequences, the recombinant expression vectors of the present invention may also carry additional sequences, such as sequences (e.g., replication origins) and selectable marker genes that regulate the replication of the vector in the host cell. The selectable marker gene promotes the selection of the host cell into which the vector has been introduced (see, e.g., U.S. Patent Nos. 4,399,216, 4,634,665, and 5,179,017). For example, typically, the selectable marker gene confers resistance to medical agents such as G418, hygromycin, or methotrexate to the host cell into which the vector has been introduced. For example, selectable marker genes include dihydrofolate reductase (DHFR) genes (for use in dhfr host cells during methotrexate selection / amplification), neo genes (for G418 selection), and glutamate synthetase genes.

[0214] As used herein, the term "expression control sequence" is intended to refer to such polynucleotide sequences, which are necessary for affecting the expression and processing of the coding sequences to which they are connected. Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; effective RNA processing signals, such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequences); sequences that enhance protein stability; and sequences that enhance protein secretion when necessary. The nature of such control sequences varies depending on the host organism; in prokaryotes, such control sequences generally include promoters and transcription termination sequences of ribosome binding sites; in eukaryotes, generally, such control sequences include promoters and transcription termination sequences. The term "control sequence" includes at least all components whose presence is necessary for expression and processing, and may also include additional components whose presence is beneficial, such as leader sequences and sequences of fusion cells.

[0215] Host cells

[0216] A further aspect of the invention relates to a method for producing antibodies against CD47 and PD-L1 of the invention. One embodiment of the invention relates to a method for producing antibodies as defined herein, comprising introducing / preparing a recombinant host cell capable of expressing an antibody, culturing the host cell under conditions suitable for antibody expression / production, and isolating the obtained antibody. Antibodies against CD47 and PD-L1 obtained by such expression in such recombinant host cells are referred to herein as "recombinant antibodies". The invention also relates to cell progeny from such host cells and antibodies against CD47 and PD-L1 obtained similarly.

[0217] Encoding the Invention Anti-CD47 / PD-L1 AntibodiesNucleic acid molecules and vectors containing these nucleic acid molecules can be used to transfect suitable mammals or cells thereof, plants or cells thereof, bacteria or yeast host cells. Transformation can be performed by any known technique for introducing polynucleotides into host cells. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art, including dextran-mediated transfection, cationic polymer-nucleic acid complex transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, polynucleotide encapsulation in liposomes, and direct microinjection of DNA into the nucleus. In addition, nucleic acid molecules can be introduced into mammalian cells by viral vectors. Methods for transfecting cells are well known in the art. See, for example, U.S. Patent Nos. 4,399,216, 4,912,040, 4,740,461, and 4,959,455. Methods for transforming plant cells are well known in the art, including, for example, Agrobacterium-mediated transformation, bioballistic transformation, direct injection, electroporation, and viral transformation. Methods for transforming bacteria and yeast cells are also well known in the art.

[0218] Mammalian cell lines used as hosts for transformation are well known in the art and include a number of available immortalized cell lines. 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., Hep G2), A549 cells, and many other cell lines. Cell lines are selected by determining which cell lines have high expression levels and provide the necessary characteristics of the produced protein. 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 for 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 the antibody to be expressed in the host cell, or more preferably, the antibody is secreted into the culture medium in which the host cell grows. Antibodies to CD47 and PD-L1 can 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 Escherichia coli and Streptomyces species. Yeast host cells include Schizosaccharomyces pombe, Saccharomyces cerevisiae, and Pichia pastoris.

[0219] In addition, a variety of known techniques can be used to enhance the production level 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 method for enhancing expression under certain conditions. The GS system is discussed in conjunction with ER Nos. 0216846, 0256055, 0323997 and 0338841 in whole or in part.

[0220] Antibodies to CD47 and PD-L1 expressed by different cell lines or in transgenic animals are likely to have different glycosylation profiles 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 are part of the present invention, regardless of the glycosylation of the binding molecules and, in general, regardless of the presence or absence of post-translational modifications.

[0221] Antibody preparation

[0222] The present invention also relates to methods and processes for producing antibodies and antigen-binding fragments thereof against CD47 and PD-L1.

[0223] Monoclonal antibodies

[0224] Monoclonal antibodies may be prepared using the hybridoma method first described by Kohler et al. Nature 256, 1975, p. 495, or may be made using recombinant DNA methods (US 4816567).

[0225] When a hybridoma-based method is used, mice or other suitable host animals, such as hamsters, are immunized according to the above method to cause the formation of lymphocytes that produce or can produce antibodies that can specifically bind to the protein used for immunization. According to another embodiment, lymphocytes can be produced by in vitro immunization. After immunization, lymphocytes are fused with myeloma cell lines using a suitable fusing agent such as polyethylene glycol to produce hybridoma cells.

[0226] The hybridoma cells thus obtained are seeded and grown in an appropriate culture medium that 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 the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas should typically include hypoxanthine, aminopterin, and thymidine (HAT medium), substances that inhibit the growth of HGPRT-deficient cells.

[0227] Preferred myeloma cell lines are mouse myeloma lines, such as those based on the murine tumor cell lines MORS-21 and MPC-11 (which can be obtained from the Salk Institite Cell Disrtibution Center, San Diego, pc. California, USA) and lines SP-2 or X63-Ag8-653 (which can be obtained from the American Type Culture Collection, Rockville, ea. Maryland, USA). The use of human mouse myeloma and mouse-human heteromyeloma cell lines for the production of monoclonal antibodies has also been described (Kozbor, J. Immunol., 133, 1984, p. 3001).

[0228] Preferably, the binding specificity of monoclonal antibodies obtained from hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).

[0229] The binding affinity of the monoclonal antibody can be determined, for example, by Scatchard analysis as described in Munson et al., Anal. Biochem., 107:220 (1980).

[0230] After identifying hybridoma cells that produce antibodies of the desired specificity, affinity and / or activity, clones can be subcloned using limiting dilution methods and grown by standard methods. Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 culture media. In addition, hybridoma cells can be grown in vivo as ascites tumors in animals, for example, by injecting the cells intraperitoneally (ip) into mice.

[0231] The monoclonal antibodies secreted by the subclones can be separated from the culture medium, ascites fluid or serum by conventional antibody purification techniques, such as affinity chromatography (e.g., using protein A- or protein G-Sepharose) or ion exchange chromatography, hydroxyapatite chromatography, gel electrophoresis, dialysis, and the like.

[0232] The DNA encoding the monoclonal antibody is easily separated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that can specifically bind to the genes encoding the heavy and light chains of the mouse antibody). Hybridoma cells serve as a preferred source of such DNA. Once separated, the DNA can be placed in an expression vector, which is then transfected into a host cell to obtain the synthesis of the monoclonal antibody in a recombinant host cell, such as an E. coli cell, a monkey COS cell, a Chinese hamster ovary (CHO) cell, or a myeloma cell, which does not produce antibody protein in the absence of transfection. A review of articles on recombinantly expressing DNA encoding antibodies in bacteria.

[0233] In a further embodiment, monoclonal antibodies or antibody fragments can 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 mouse and human antibodies using phage libraries, respectively. Subsequent publications described the generation of high affinity (nM range) human antibodies by chain shuffling (Marks et al., Bio / Technology, 10:779-783 (1992)) and combinatorial infection and in vivo recombination as strategies for the construction of very large phage libraries (Waterhouse et al., Nucl. Acids. Res. 21:2265-2266 (1993). Therefore, these techniques are viable alternatives to conventional monoclonal antibody hybridoma techniques for isolation of monoclonal antibodies.

[0234] For example, by replacing the homologous mouse sequences with heavy and light chain (CH and CL) constant region sequences (US 4816567 and Morrison et al., Proc. Natl. Acad. Sci. USA: 81: 6851 (1984)), or by covalently linking the immunoglobulin coding sequence to all or part of the coding sequence of a non-immunoglobulin polypeptide (heterologous polypeptide), the DNA encoding the antibody can be modified, for example, to produce a chimeric or fusion antibody polypeptide. The non-immunoglobulin polypeptide sequences can replace the constant region of the antibody, or they can replace the variable domain of the antigen binding center of the antibody to produce a chimeric bivalent antibody comprising one antigen binding site having specificity for an antigen and another antigen binding site having specificity for a different antigen.

[0235] Humanized Antibodies

[0236] Methods for producing "humanized" non-human animal antibodies are well known in the art. Preferably, humanized antibodies have one or more integrated amino acid residues introduced therein from a non-human source. These non-human amino acid residues are often referred to as "import" residues because they are usually taken from the "import" variable region. Humanization can be performed by replacing the hypervariable region sequence with the corresponding sequence of a human antibody, essentially following the method of Winter and co-authors (Jones et al., Nature, 321: 522-525 (1986)). Accordingly, such "humanized" antibodies are chimeric antibodies (US4816567), in which regions substantially smaller than the complete human variable region have been replaced by corresponding sequences from non-human species. In fact, humanized antibodies are typically human antibodies, in which some hypervariable region residues and possibly some FR residues are replaced by residues from similar regions in rodent antibodies.

[0237] When antibodies are intended for therapeutic use in humans, the selection of human variable regions (both light and heavy) to be used to produce humanized antibodies is very important for reducing antigenicity and HAMA responses (human anti-mouse antibodies). According to the so-called "best fit" method, the sequence of the variable region of a rodent antibody is screened for the entire library of known human variable domain sequences. The human V domain sequence closest to the rodent V domain sequence is identified, and the human framework region (FR) within it is selected, which is suitable for humanized antibodies (Sims et al., J. Immunol. 151: 2296 (1993)). In another method, a specific framework region of a consensus sequence of certain subgroups of the light chain or heavy chain of all human antibodies is used. The same framework can be used for several different humanized antibodies (Carter et al., Proc. Natl. Acad. Sci. USA: 89: 4285 (1992)).

[0238] It is also important that humanized antibodies retain high binding affinity for antigens and other important biological properties. For this reason, according to a preferred method, humanized antibodies are prepared by analyzing parental sequences and various humanized products using conceptual three-dimensional models of parental and humanized sequences. Three-dimensional immunoglobulin models are usually available and are familiar to those skilled in the art. Computer programs are available, and they illustrate and demonstrate the possible three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these images allows analysis of the possible effects of residues in the function of candidate immunoglobulin sequences, that is, analysis of the residues that affect the ability of candidate immunoglobulin to bind antigens. In this way, FR residues can be selected and combined with receptor and input sequence to obtain desired antibody characteristics, such as an increase in affinity for target antigens. Generally speaking, hypervariable region residues directly and most substantially involve influencing antigen binding.

[0239] The humanized antibody may be an antibody fragment, such as a Fab, which is optionally conjugated to one or more cytotoxic agents to generate an immunoconjugate. Alternatively, the humanized antibody may be a full-length antibody, such as a full-length IgG1 antibody.

[0240] Human antibodies based on phage display libraries and methods

[0241] As an alternative to humanization, human antibodies can be generated. For example, it is currently possible to produce transgenic animals (e.g., mice) that are capable of producing a full range of human antibodies after immunization without endogenous immunoglobulin production. For example, homozygous deletions of antibody heavy chain joining region (JH) genes in chimeric and germline mutant mice have been described, resulting in complete suppression of endogenous antibody production. Transfer of human germline immunoglobulin gene arrays into such germline mutant mice results in the production of human antibodies after antigen attack (US 5545806, 5569825, 5591669 (all GenPharm); 5545807; and WO 97 / 17852).

[0242] Alternatively, phage display technology (McCafferty et al., Nature, 348: 552-554 (1990)) can be used to produce human antibodies and antibody fragments in vitro from immunoglobulin variable (V) region gene reservoirs from immune donors. According to this technology, antibody V region genes are cloned in frame with major or minor coat protein genes of filamentous phages (e.g., M13 or fd) and displayed as functional antibody fragments on the surface of phage particles. Because filamentous particles contain single-stranded DNA copies of the phage genome, selection based on the functional properties of antibodies also results in selection of genes encoding antibodies that exhibit said properties, and thus, phages mimic some B cell properties. Phage display can be performed in various forms. Several sources of V gene segments can be used for phage display. Clackson et al., Nature, 352: 624-628 (1991) isolated various arrays of anti-oxazolone antibodies from a small random combinatorial library of V genes derived from the spleen of immunized mice. V gene repertoires from unimmunized human donors can be constructed and antibodies directed against a diverse array of antigens, including self-antigens, can be isolated essentially following the techniques described by Marks et al., J. Mol. Biol. 222:581-597 (1991).

[0243] As described above, human antibodies can also be generated by in vitro activated B cells (see US Pat. Nos. 5,567,610 and 5,229,275).

[0244] Antibody fragments

[0245] In some cases, it may be advisable to use antibody fragments rather than whole antibodies. The small size of the fragments facilitates their rapid clearance and may facilitate better penetration into dense tumors.

[0246] Various techniques for producing antibody fragments have been developed. Routinely, these fragments are derived via proteolytic digestion of intact antibodies. However, these fragments can be directly obtained by recombinant host cells at present. Fab, Fv and ScFv antibody fragments can be expressed in Escherichia coli and secreted therefrom, thus allowing the generation of a large number of these fragments to be promoted. Antibody fragments can be separated from the antibody phage library described above. According to another embodiment, Fab'-SH fragments can 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 method, F(ab')2 fragments can be directly isolated from recombinant host cell culture. Fab and F(ab')2 with increased in vivo half-life and retained epitope binding receptor residues are described in US5869046. Other techniques for obtaining antibody fragments should be obvious to those skilled in the art. In other embodiments, the antibody of choice is a single-chain Fv fragment (scFv) (see WO 93 / 16185; US 5571894 and US 5587458). Fv and scFv are the only species with complete binding sites that lack constant regions; as a result, they are suitable for reducing nonspecific binding during in vivo use. Fusion proteins carrying scFv can be designed to produce fusions of effector proteins at the N-terminus or C-terminus of the scFv. Antibody fragments can also be "linear antibodies" as described, for example, in US5641870. Such linear antibody fragments can be monospecific or bispecific.

[0247] Multispecific Antibodies

[0248] Multispecific antibodies are antibodies that have binding specificity for at least two different epitopes. For example, a bispecific antibody can bind to two different epitopes of a protein. Other multispecific antibodies can combine binding sites for CD47 and PD-L1 with binding sites for another protein. Bispecific antibodies can be obtained as full-length antibodies or antibody fragments (e.g., F(ab')2 fragments of bispecific antibodies).

[0249] Methods for producing multispecific antibodies are known in the art. For example, the conventional production of full-length bispecific antibodies is based on the coexpression of two heavy chain immunoglobulin / light chain pairs, wherein the two chains have different specificities. Due to the random distribution of heavy chain immunoglobulin and light chain, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, wherein only one has the correct bispecific structure. Purification of the correct molecule usually completed in several steps by affinity chromatography is quite cumbersome, and the product yield is very low. A similar method is described in WO 93 / 08829.

[0250] According to different methods, the variable domain of an antibody with the desired binding specificity (the bound antigen binding site) is fused to an immunoglobulin constant domain sequence. Preferably, the fusion is performed with the Ig heavy chain constant region, which includes the hinge, C H 2 and C H Preferably, the first heavy chain constant region (C 1 ) containing the site necessary for light chain binding is present in at least one of the fusions. H 1). DNA encoding the immunoglobulin heavy chain fusion and (if necessary) the immunoglobulin light chain is inserted into various expression vectors and co-transfected into suitable host cells. When unequal ratios of the three polypeptide chains are used in the construction to provide the best yield, in an embodiment, this provides greater flexibility in selecting the mutual proportions of the three polypeptide fragments. However, when the expression of at least two polypeptide chains in equal ratios results in high yields, or when the ratio has no significant effect, it is possible to insert the coding sequence into two or all three polypeptide chains in a single expression vector.

[0251] In a preferred embodiment of this method, the bispecific antibody is a hybrid immunoglobulin heavy chain providing a first binding specificity in the first arm and a hybrid immunoglobulin heavy chain / light chain pair (providing a second binding specificity) in the second arm. It has been found that this asymmetric structure facilitates the separation of the desired bispecific molecule from the unwanted immunoglobulin chain combination because the presence of the immunoglobulin light chain in only one half of the bispecific molecule facilitates the separation. This method is disclosed in WO94 / 04690. For more details on the generation of bispecific antibodies, see, for example, Suresh et al., Methods in Enzymology 121: 210 (1986).

[0252] According to another approach described in US 5731168, the interface between a pair of antibody molecules can be constructed to maximize the percentage of heterodimers obtained from recombinant cell culture. The preferred interface comprises C HAt least a portion of region 3. According to this method, one or more small amino acids with side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). By replacing amino acids containing large side chains with amino acids containing smaller side chains (e.g., alanine or threonine), a compensatory "cavity" of equal or similar size to the large side chain is generated on the interface of the second antibody molecule. This provides a mechanism for increasing the yield of heterodimers compared to other unwanted end products.

[0253] Bispecific antibodies include cross-linked or "heteroconjugate" antibodies. For example, one antibody in the heteroconjugate can be coupled to avidin and the other to biotin. Such antibodies can be used, for example, to target immune system cells to unwanted cells (US 4676980), and to treat HIV infection (WO 91 / 00360, WO 92 / 200373 and EP 03089). Heteroconjugate antibodies can be prepared using any convenient cross-linking method. Suitable cross-linking agents are well known in the art and are disclosed in US 4676980 along with various cross-linking techniques.

[0254] Techniques for obtaining bispecific antibodies from antibody fragments have also been described in the literature. For example, bispecific antibodies can be obtained by chemical conjugation. Brennan et al., Science 229: 81 (1985) have described a procedure according to which intact antibodies are proteolytically cleaved to produce F(ab')2. These fragments are reduced in the presence of a dithiol complexing agent (such as sodium arsenite) to stabilize vicinal dithiols and prevent the formation of intermolecular disulfide bonds. The resulting Fab' fragments are then converted to thionitrobenzoate (TNB) derivatives. One Fab'-TNB derivative is then converted back to Fab'-thiol by reduction with mercaptoethylamine and mixed with an equimolar amount of another Fab'-TNB derivative to obtain a bispecific antibody. The resulting bispecific antibody can be used as a reagent for selectively immobilizing an enzyme.

[0255] Recent advances have facilitated the direct recovery of Fab'-SH fragments from E. coli, which can be chemically coupled to produce bispecific antibodies. Shalaby et al., J. Exp. Med. 175: 217-225 (1992) described the production of F(ab')2 of fully humanized bispecific antibody molecules. Each Fab' was secreted separately from E. coli and subjected to direct chemical coupling in vitro to form a bispecific antibody. The bispecific antibodies thus obtained were able to bind to cells overexpressing the ErbB2 receptor and normal human T cells, as well as trigger the lytic activity of human cytotoxic lymphocytes against human breast tumor targets.

[0256] Various techniques for obtaining and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. 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 Fab' from two different antibodies by gene fusion. Antibody homodimers were reduced at the hinge region to form monomers and then oxidized to obtain antibody heterodimers. This method can also be used to obtain homodimeric antibodies. By Hollinger et al., Proc. Natl. Acad. Sci. USA90:6444-6448 (1993) describes the "double antibody" technology as an alternative mechanism for producing bispecific antibody fragments. The fragment comprises a VH region connected to a VL region by a linker that is too short to allow pairing between two domains on the same chain. Accordingly, the VH and VL regions of one fragment should be paired with the complementary VL and VH regions of another fragment to form 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)).

[0257] The present invention also provides antibodies having more than two valencies. For example, trispecific antibodies can be generated.

[0258] Multivalent Antibodies

[0259] Multivalent antibodies can be internalized (and / or degraded) 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 (except the IgM class) (e.g., tetravalent antibodies) having three or more antigen binding sites, which can be easily obtained by recombinant expression of nucleic acids encoding antibody polypeptide chains. Multivalent antibodies may comprise a dimerization domain and three or more antigen binding sites. Preferred dimerization domains comprise (or consist of) an Fc fragment or a hinge region. In such cases, the antibody will comprise an Fc fragment and three or more antigen binding sites at the N-terminus of the Fc fragment. Preferred multivalent antibodies herein comprise (or consist of) 3 to about 8, but preferably 4 antigen binding sites. The multivalent antibody comprises at least one polypeptide chain (and preferably two polypeptide chains), wherein the polypeptide chain comprises two or more variable regions. For example, the polypeptide chain may comprise VD1-(X1)n-VD2-(X2)n-Fc, wherein VD1 refers to the first variable region, VD2 refers to the second variable region, Fc refers to a polypeptide chain of the Fc fragment, X1 and X2 refer to amino acids or polypeptides, and n is 0 or 1. For example, the polypeptide chain 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 2 (and preferably 4) light chain variable region polypeptides. The multivalent antibody herein may, for example, comprise about 2 to about 8 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.

[0260] Pharmaceutical composition

[0261] In another aspect, the present invention provides a pharmaceutical composition comprising a CD47 / PD-L1 specific antibody as an active ingredient (or as the only active ingredient). As described herein, the pharmaceutical composition may include at least one antibody specific for CD47 and PD-L1 and / or one or more additional binding molecules (e.g., antibodies) targeting one or more corresponding surface receptors. In some embodiments, the composition is intended to improve, prevent or treat conditions mediated by IgG.

[0262] "Pharmaceutical composition" means a composition comprising an anti-CD47 / PD-L1 antibody of the present invention and at least one component selected from pharmaceutically acceptable and pharmacologically compatible excipients, such as fillers, solvents, diluents, carriers, adjuvants, dispensing agents, delivery agents, preservatives, stabilizers, emulsifiers, suspending agents, thickeners, extended delivery control agents, the selection and proportion of which depends on the type and route of administration and the dose. The pharmaceutical composition of the present invention and the method for its preparation are undoubtedly obvious to those skilled in the art. The pharmaceutical composition should preferably be manufactured in accordance with GMP (Good Manufacturing Practice) requirements. The composition may include a buffer composition, a tonicity agent, a stabilizer and a solubilizer. The prolonged effect of the composition can be achieved by agents that slow down the absorption of the active pharmaceutical ingredient, such as aluminum monostearate and gelatin. Examples of suitable carriers, solvents, diluents and delivery agents include water, ethanol, polyols and mixtures thereof, oils and organic esters for injection.

[0263] "Medicament (drug)" is a substance in the form of tablets, capsules, powders, lyophilized preparations, injections, infusions, ointments and other ready-made forms or a mixture of substances as a pharmaceutical composition, which is intended for use in restoring, improving or changing physiological functions in humans and animals, as well as for the treatment and prevention of diseases, for diagnosis, anesthesia, contraception, beauty and others. Any method recognized in the art for administering peptides, proteins or antibodies can be appropriately used for the anti-CD47 / PD-L1 antibodies of the present invention.

[0264] The term "pharmaceutically acceptable" refers to one or more compatible liquid or solid components that are suitable for administration in a mammal, preferably a human.

[0265] The term "excipient" is used herein to describe any ingredient other than the above-mentioned ingredients of the invention. These are substances of inorganic or organic nature, which are used in the manufacture of pharmaceuticals in order to give the necessary physicochemical properties to the drug product.

[0266] As used herein, "buffer", "buffer composition", "buffer" refers to a solution that is able to resist changes in pH through the action of its acid-base conjugate components and allows the anti-CD47 / PD-L1 antibody drug to resist changes in pH. Generally, the pharmaceutical composition preferably has a pH in the range of 4.0 to 8.0. Examples of the buffer used include, but are not limited to, acetate, phosphate, citrate, histidine, succinate and other buffer solutions.

[0267] As used herein, the term "tonic agent", "osmotic agent" or "osmotic agent" refers to an excipient that can increase the osmotic pressure of a liquid antibody formulation. An "isotonic" drug is a drug that has an osmotic pressure comparable to that of human blood. Isotonic drugs typically have an osmotic pressure of about 250 to 350 mOsm / kg. Isotonic agents used include, but are not limited to, polyols, sugars and sucrose, amino acids, metal salts such as sodium chloride, and the like.

[0268] "Stabilizer" refers to an excipient or a mixture of two or more excipients that provides physical and / or chemical stability to the active agent. Stabilizers include amino acids, such as but not limited to arginine, histidine, glycine, lysine, glutamine, and proline; surfactants, such as but not limited to polysorbate 20 (trade name: Tween 20), polysorbate 80 (trade name: Tween 80), polyethylene glycol-polypropylene glycol and its copolymers (trade names: poloxamer, Pluronic), sodium dodecyl sulfate (SDS); antioxidants, such as but not limited to methionine, acetylcysteine, ascorbic acid, monothioglycerol, sulfites, etc.; chelating agents, such as but not limited to ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), sodium citrate, etc.

[0269] A pharmaceutical composition is "stable" if the active agent maintains its physical stability and / or chemical stability and / or biological activity during a specified shelf life at a storage temperature, e.g., 2-8°C. 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 testing under accelerated or natural aging conditions.

[0270] The pharmaceutical compositions of the present invention can be manufactured, packaged or widely sold in the form of a single unit dose or multiple single unit doses in the form of ready-made preparations. As used herein, the term "single unit dose" refers to a discrete number of pharmaceutical compositions containing a predetermined amount of active ingredient. The amount of active ingredient is generally equal to the dose of active ingredient to be administered to a subject, or a convenient portion of such a dose, such as half or one-third of the dose.

[0271] The pharmaceutical composition according to the present invention is generally suitable for parenteral administration as a sterile preparation, which is intended to be administered in the human body by means of injection, infusion and implantation, bypassing the gastrointestinal tract, through breaks in the skin and mucosal barriers. For example, parenteral administration includes, among others, subcutaneous, intraperitoneal, intramuscular, intrasternal, intravenous, intraarterial, intrathecal, intraventricular, intraurethral, ​​intracranial, intrasynovial, transcutaneous injection or infusion; and renal dialysis infusion techniques. Regional perfusion is also provided. Preferred embodiments include intravenous and subcutaneous routes. Any method recognized in the art for administering peptides or proteins can be appropriately used for the anti-CD47 / PD-L1 antibodies of the present invention.

[0272] Injectable formulations can be manufactured, packaged or sold in unit dosage form (without limitation), for example, in ampoules, vials, plastic containers, prefilled syringes, automatic injection devices. Formulations for parenteral administration include suspensions, solutions, emulsions in oily or aqueous bases, pastes, and the like.

[0273] In another embodiment, the present invention provides a composition for parenteral administration, comprising a pharmaceutical composition provided in dry (i.e., powder or granules) form for reconstitution with a suitable base (e.g., sterile pyrogen-free water) prior to administration. Such a formulation can be obtained, for example, by a lyophilization process, which is known in the art as freeze drying and involves freezing the product, followed by removal of the solvent from the frozen material.

[0274] The antibodies against CD47 and PD-L1 of the present invention can also be administered intranasally or by inhalation, alone or as a mixture with a suitable pharmaceutically acceptable excipient, from an inhaler (e.g., a pressurized aerosol container, pump, spray, nebulizer or atomizer) (with or without a suitable propellant), or as nasal drops or spray.

[0275] Dosage forms for parenteral administration can be formulated to be immediate release or modified release. Modified release formulations include delayed release, sustained release, pulsed release, controlled release, targeted release, and programmed release.

[0276] Therapeutic uses of the anti-CD47 / PD-L1 antibodies of the present invention

[0277] In one aspect, the anti-CD47 / PD-L1 antibodies of the invention are used to treat a condition mediated by CD47 and PD-L1, such as a disease or condition selected from the group consisting of: (HNSCC) head and neck squamous cell carcinoma, 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), kidney cancer, ovarian cancer, Hodgkin lymphoma, MSI CRC (colorectal cancer with microsatellite instability), leukemia (acute leukemia or myeloblastic leukemia), non-Hodgkin lymphoma, multiple myeloma, myelodysplastic syndrome.

[0278] In one aspect, the treatment subject or patient is a mammal, preferably a human subject. The subject can be male or female and can be of any age.

[0279] In the case of a tumor (e.g., cancer), a therapeutically effective amount of an antibody or fragment thereof (e.g., an antibody or fragment thereof that specifically binds to CD47 and PD-L1) can reduce the number of cancer cells; reduce the size of the initial tumor; inhibit (i.e., slow down to some extent and preferably stop) cancer cell infiltration into surrounding organs; inhibit (i.e., slow down to some extent and preferably stop) tumor metastasis; inhibit tumor growth to some extent; and / or alleviate one or more symptoms associated with the disorder to some extent. The antibody or fragment thereof can prevent growth and / or kill existing cancer cells to some extent, it can be cytostatic and / or cytotoxic. For cancer treatment, for example, in vivo efficacy can be measured by evaluating overall survival (OS), time to tumor progression (TTP), overall tumor response rate (ORR) to treatment, duration of response (DR), and / or quality of life.

[0280] As used herein, the terms "co-administered," "co-administered," and "in combination with" in reference to an anti-CD47 / PD-L1 antibody and one or more different therapeutic agents are intended to mean, refer to, or include:

[0281] 1) such combination of an anti-CD47 / PD-L1 antibody of the present invention and a therapeutic agent is administered simultaneously to a patient in need of treatment when such components are formulated together into a single dosage form, which releases the components to the patient at substantially the same time,

[0282] 2) when such components are separately formulated in different dosage forms, such combination of the anti-CD47 / PD-L1 antibody of the present invention and the therapeutic agent is administered to a patient in need of treatment substantially simultaneously, the introduction of said different dosage forms occurs at approximately the same time for said patient, after which these components are released to the given patient approximately simultaneously,

[0283] 3) when such components are formulated separately from each other into separate dosage forms, such combination of the anti-CD47 / PD-L1 antibody of the present invention and the therapeutic agent is administered sequentially to a patient in need of treatment, said separate dosage forms being taken by said patient at consecutive times with a significant time interval between each administration, so that said components are released to said patient at substantially different times; and

[0284] 4) Such a combination of an antibody against CD47 and PD-L1 and a therapeutic agent according to the present invention is administered sequentially to a patient in need of treatment when such components are formulated together into a single dosage form, which releases the components in a controlled manner so that they are released to the patient simultaneously, continuously or in combination at the same and / or different times, wherein each part can be administered by the same or different routes.

[0285] The anti-CD47 / PD-L1 antibodies of the invention can be administered without further therapeutic treatment, i.e. as a stand-alone therapy. In addition, treatment with the antibodies of the invention can include at least one additional therapeutic treatment (combination therapy). In some embodiments of the invention, the anti-CD47 / PD-L1 antibodies can be administered in combination with different cancer agents / drugs or formulated with different cancer agents / drugs.

[0286] As used herein, the term "cytotoxic agent" refers to a substance that inhibits or prevents the function of cells and / or causes cell destruction. It is contemplated that 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, such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and / or variants thereof.

[0287] A "chemotherapeutic agent" is a chemical compound that can be used 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, carboquinone, meturedopa, and uredopa; ethyleneimines and methylmelamines including hexamethylmelamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolomelamine; acetogenins (e.g., bratacin and bratacinone); β-lapachone; lapachol; colchicine; betulinic acid; camptothecins (including the synthetic analog topotecan HYCAMTIN ® )、CPT-11(irinotecan, CAMPTOSAR ®), acetylcamptothecin, scopolectin, and 9-aminocamptothecin); bryostatin; callystatin; CC-1065 (including its synthetic analogs adolesin, carzelesin, and biszelesin); podophyllotoxin; podophyllic acid; teniposide; scutellarin (e.g., scutellarin 1 and scutellarin 8); dolastatin; duocarmycin (including its synthetic analogs KW-2189 and CB1-TM1); eleutherobin; water pancratistatin; sarcodictyin; spongestatin; nitrogen mustards, such as chlorambucil, naphthyl mustard, clofosamide, estramustine, ifosfamide, methylchloroethylamine, nitrogen oxide mustard hydrochloride, melphalan, new nitrogen mustard, phenylephrine, prednimustine, trofosamide, uracil mustard; nitrosoureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine and ranimustine; antibiotics, such as enediyne antibiotics (such as calicheamicin, such as calicheamicin gamma II and calicheamicin ω II (see, e.g., Agnew, Chem. Intl. Ed. Engl., 33: 183-186 (1994)); danemycins, including danemycin A; epothilones; and the neocarcinogen chromophores and related chromoprotein enediyne antibiotic chromophores), aclarubicin, actinomycin D, anthramycin, azaserine, bleomycin, actinomycin C, calabikine ( carabicin), carminomycin, carmoamicin, chromomycin, dactinomycin, daunorubicin, detopicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including ADRIAMYCIN ® Morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolido-doxorubicin, doxorubicin HCl liposome injection (DOXOL ® )、Doxorubicin Liposomal TLC D-99(MYOCETCA ® ) and pegylated doxorubicin liposomes (CAELYX ® ) and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, mexilomycin, mitomycins such as mitomycin C, mycophenolic acid, noramycin, olivetomycin, peplomycin, potfiromycin, puromycin, triferric doxorubicin, rhodorubicin, streptozocin, streptozocin, tuberculocidin, ubenimex, netastatin, daunorubicin; antimetabolites such as methotrexate, gemcitabine (GEMZAR ® ), Tegafur (UFTORAL ® ), capecitabine (XELODA ®), epothilones, and 5-fluorouracil (5-FU); folic acid analogs, such as dimethylfolate, methotrexate, pteropterin, trimetrexate; purine analogs, such as fludarabine, 6-mercaptopurine, thiopurine, thioguanine; pyrimidine analogs, such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxiflurane, enocitabine, floxuridine; antiadreners, such as aminoglutethimide, mitotane, trilostane; folic acid supplements, such as folinic acid (frolinic acid); acid); acetylglucosyl esters; aldophosphamide glycosides; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatrexate; defosfamide; demeclocycline; diacronitrile; eflornithine; elliptonium acetate; etoglucose; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansine alkaloids, such as maytansine and ansamitocin; mitoguanidine; mitoxantrone; mopidarol; nitropropanil; pentostatin; methambucil; pirarubicin; losoxantrone; 2-ethylhydrazide; procarbazine; PSK ® Polysaccharide complex (JHS Natural Products, Eugene, OR); razoxane; rhizosin; cysofol; spirogermanamine; tricholomanic acid; triazoline; 2,2',2"-trichlorotriethylamine; trichothecenes (e.g., T-2 toxin, verrucosporin A, baculosporin A, and serpentin); urethanes; dacarbazine; mannomustine; dibromomannitol; dibromodulanol; pipobroman; gacytosine; cytarabine ("Ara-C"); thiotepa; taxanes, such as paclitaxel (TAXOL ® ), albumin-modified paclitaxel nanoparticle formulation (ABRAXANE ® ) and docetaxel (TAXOTERE ® ); chlorambucil; 6-thioguanine; mercaptopurine; methotrexate; platinum agents such as cisplatin, oxaliplatin, and carboplatin; vinca alkaloids, which prevent tubulin from polymerizing to form microtubules, including VELBAN ® )、Vincristine (ONCOVIN ® )、ELDISINE ® )、FILDESIN ® ) and Navelbine ® ); etoposide (VP16); ifosfamide; mitoxantrone; leucovorin; noxolin; edatrexate; daunomycin; methotrexate; ibandronate; the topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids, such as retinoic acid, including bexarotene (TARGRETIN ® ); bisphosphonates, such as clodronate (eg, BONEFOS ®or OSTAC ® ), etidronate (DIDROCAL ® ), NE-58095, zoledronic acid / zoledronic acid salt (ZOMETA ® ), alendronate (FOSAMAJX ® ), pamidronate (AREDIA ® ), tiludronate (SKELID ® ) or risedronate (ACTONEL ® ); troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides, particularly those that inhibit the expression of genes in signaling pathways involved in abnormal cell proliferation, such as PKC-α, Raf, H-Ras, and epidermal growth factor receptor (EGF-R); vaccines, such as THERATOPE ® Vaccines and gene therapy vaccines, such as ALLOVECTIN ® Vaccines, LEUVECTIN ® Vaccines and VAXIDs ® vaccines; topoisomerase 1 inhibitors (eg, LURTOTECAN®); rmRH (eg, ABARELIX ® ); BAY439006 (sorafenib; Bayer); SU-11248 (Pfizer); perifosine, COX-2 inhibitors (such as celecoxib or etoricoxib), proteosome inhibitors (such as PS341); bortezomib (VELCADE ® ); CCI-779; tipifarnib (Rl 1577); orafenib, ABT510; Bcl-2 inhibitors, such as oblimersen sodium (GENASENSE ® ); pixenol; EGFR inhibitors (see definition below); tyrosine kinase inhibitors (see definition below); and pharmaceutically acceptable acids or derivatives of any of the foregoing; and combinations of two or more of the foregoing, such as CHOP, an abbreviation for combination therapy with cyclophosphamide, doxorubicin, vincristine, and prednisolone, and FOLFOX, an abbreviation for treatment with oxaliplatin (ELOXATIN™) in combination with 5-FU and leucovorin.

[0288] Also included in this definition are antihormonal agents that act to modulate or inhibit the effects of hormones on tumors, such as antiestrogens with a mixed agonist / antagonist profile, including tamoxifen (NOLVADEX ® ), 4-hydroxytamoxifen, toremifene (FARESTON ® ), idoxifene, droloxifene, raloxifene (EVTSTA ®), travoxifene, raloxifene (keoxifene) and selective estrogen receptor modulators (SERMs), such as SERM3; pure antiestrogens without agonist properties, such as fulvestrant (FASLODEX ® ) and EM800 (such agents can block estrogen receptor (ER) dimerization, inhibit DNA binding, increase ER turnover rate, and / or suppress ER levels); aromatase inhibitors, including steroidal aromatase inhibitors such as formestane and exemestane (AROMASIN ® ), and nonsteroidal aromatase inhibitors, such as anastrozole (AREVIIDEX ® ), letrozole (FEMARA ® ) and aminoglutethimide), and other aromatase inhibitors include vorazole (RIVISOR ® ), megestrol acetate (MEGASE ® ), fadrozole, imidazoles; luteinizing hormone-releasing hormone agonists, including leuprolide (LUPRON ® and ELIGARD ® ), goserelin, buserelin and triptorelin; sex steroids, including progestins, such as megestrol acetate and medroxyprogesterone acetate; estrogens, such as diethylstilbestrol and premarin; and androgens / retinoids, such as fluoxymesterone; all-trans retinoic acid and fenretinide; onasterone; antiprogestins; estrogen receptor downregulators (ERDs); antiandrogens, such as flutamide, nilutamide and bicalutamide; testolactone; and any pharmaceutically acceptable salts, acids or derivatives of the foregoing; and combinations of two or more of the foregoing.

[0289] Other therapeutic agents that can be used in combination with the anti-CD47 / PD-L1 antibodies of the present invention can be inhibitors of growth factor function, for example, such inhibitors include growth factor antibodies and growth factor receptor antibodies (e.g., the anti-erbB2 antibody trastuzumab [Herceptin], the anti-EGFR antibody panitumumab, the anti-erbB1 antibody cetuximab [Erbitux, C225], and any growth factor or growth factor receptor antibody disclosed by Stern et al. Critical reviews in oncology / haematology, 2005, volume 54, pages 11-29); anti-angiogenic agents, such as those that inhibit the effects of vascular endothelial growth factor [e.g., the anti-vascular endothelial cell growth factor antibody 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, such as ISIS 2503, anti-ras antisense or G3139 (Genasense), anti-bcl2 antisense; gene therapy methods, including, for example, methods to replace abnormal genes (such as abnormal p53 or abnormal BRCA1 or BRCA2), GDEPT (gene-directed enzyme prodrug therapy), methods such as methods using cytosine deaminase, thymidine kinase or bacterial nitroreductase, and methods to increase a patient's tolerance to chemotherapy or radiation therapy, such as multidrug resistance gene therapy; immunotherapy methods, including, for example, treatment with alemtuzumab (campath-1H), monoclonal antibodies against CD52, or treatment with antibodies against CD22, ex vivo and in vivo methods to increase the immunogenicity of a patient's tumor cells, treatment with cytokines (such as interleukin 2, interleukin 4 or granulocyte macrophage aggregates) methods of reducing T cell anergy, such as treatment with a monoclonal antibody that inhibits CTLA-4 function, methods using transfected immune cells, such as cytokine-transfected dendritic cells, methods using cytokine-transfected tumor cell lines, and methods using anti-idiotypic antibodies, adoptive T cell transfer using T cells that have been nonspecifically activated ex vivo or targeted to a specific antigen of interest; inhibitors of protein degradation, such as proteasome inhibitors, such as Velcade (bortezomib); biotherapeutic methods, such as those using peptides or proteins (e.g., antibodies or soluble external receptor domain constructs) that sequester receptor ligands, block ligand binding to the receptor, or reduce receptor signaling (e.g., due to enhanced receptor degradation or reduced expression levels).

[0290] Dosage and route of administration

[0291] The anti-CD47 / PD-L1 antibodies of the invention should 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. The therapeutically effective amount may vary depending on factors such as the specific condition being treated, the age, sex, and weight of the patient, and whether the anti-CD47 / PD-L1 antibody is administered as a sole therapy or in combination with one or more additional therapies.

[0292] The dosage regimen may be adjusted to provide the optimal response. For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the urgency of the therapeutic situation. It is particularly useful to manufacture parenteral compositions in standard dosage forms for ease of administration and uniformity of dosing. As used herein, a unit dosage form is intended to refer to physically discrete units suitable as unit doses for the patient / subject to be treated; each unit contains a predetermined amount of active compound calculated to produce the desired therapeutic effect and the required pharmaceutical carrier. The specifications for the standard dosage forms of the present invention are generally determined by and directly depend on the following factors: (a) the unique characteristics of the chemotherapeutic agent and the specific therapeutic or preventive effect to be achieved, and (b) the limitations inherent in the field of formulating such active compounds for treating subjects.

[0293] Therefore, those skilled in the art will recognize from the disclosure herein that dosages and dosage regimens are adjusted according to methods well known in the therapeutic field. In other words, the maximum tolerable dose can be easily established, and the effective amount that provides a detectable therapeutic effect to the patient can also be determined, and the time requirement for administering each agent to provide a detectable therapeutic effect to the patient can be determined. Therefore, although some dosages and dosage regimens are given as examples in this document, these examples are by no means limiting the dosages and dosage regimens that may be necessary for a patient when practicing the present invention.

[0294] It should be noted that the dosage value may vary with the type and severity of the condition to be alleviated, and may include a single dose or multiple doses. In addition, it should be understood that for any particular subject, the specific dosage regimen should be adjusted over time according to individual needs and the judgment of the medical professional administering the composition or supervising the administration of the composition, and the dosage ranges set forth herein are only exemplary and are not intended to limit the scope or practice of the claimed composition. In addition, the dosage regimen for the composition of the present invention can be based on various factors, including the type of disease, the age, weight, sex, medical condition of the patient, the severity of the condition, the route of administration, and the specific anti-CD47 / PD-L1 antibody used. Therefore, the dosage regimen can be widely changed, but can be routinely determined using standard methods. For example, the dosage can be adjusted based on pharmacokinetic or pharmacodynamic parameters, which can include clinical effects, such as toxic effects and / or laboratory values. Therefore, the present invention covers intra-patient dose escalation determined by a person skilled in the art. Methods for determining appropriate doses and regimens are well known in the art, and once the concepts disclosed herein are provided, the technician will understand.

[0295] Examples of suitable methods of administration are provided above.

[0296] It is believed that suitable dosages of antibodies against CD47 and PD-L1 according to the present invention will be in the range of 0.1-200 mg / kg, preferably 0.1-100 mg / kg, including about 0.5-50 mg / kg, such as about 1-20 mg / kg. Antibodies against CD47 and PD-L1 can be administered, for example, 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, such as at least 3 mg / kg, including at least 4 mg / kg, such as at least 5 mg / kg; and for example up to a maximum of 50 mg / kg, including up to a maximum of 30 mg / kg, such as up to a maximum of 20 mg / kg, including up to a maximum of 15 mg / kg. It is usually repeated at appropriate intervals, such as once a week, once every two weeks, once every three weeks, or once every four weeks, and continues as long as deemed appropriate by the responsible physician, who in some cases can increase or decrease the dosage if necessary.

[0297] Products and kits

[0298] The following embodiment of the present invention is a product containing a product for treating cancer, such as HNSCC, cervical cancer, cancer of unknown primary site, glioblastoma, esophageal cancer, bladder cancer, TNBC, CRC, hepatocellular carcinoma, melanoma, NSCLC, renal cancer, ovarian cancer, Hodgkin's lymphoma, MSICRC, leukemia (acute leukemia or myeloblastic leukemia), non-Hodgkin's lymphoma, multiple myeloma, myelodysplastic syndrome. The product is a container and a label or leaflet insert in the package, which is placed on the container or enclosed therein. Suitable containers are, for example, cans, vials, syringes, etc. The container can be made of various materials such as glass or plastic. The container contains a composition effective for treating a specific condition and can have a sterile access channel (for example, the container can be an intravenous solution bag or a vial with a stopper that can be pierced with a hypodermic needle). At least one active ingredient in the composition is an anti-PD-L1 antibody according to the present invention. The label or leaflet in the package indicates that the composition is used to treat a specific condition. The label or package leaflet included with the package should additionally contain instructions for administering the antibody composition to a patient.

[0299] The package leaflet contains the usual instructions that are included in the packaging of a therapeutic product that is about to be marketed, including some information about the indications, frequency of administration, dosage, route, contraindications and / or precautions for such therapeutic product. In one embodiment, the package insert indicates that the composition is intended for use in the treatment of cancer, such as HNSCC, cervical cancer, cancer of unknown primary site, 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.

[0300] In addition, the article of manufacture may further include a second container having a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BSVI), phosphate-buffered saline, Ringer's solution, and dextrose solution. In addition, the article of manufacture may include other products necessary from a commercial point of view and from a consumer's point of view, particularly other buffers, diluents, filters, needles, and syringes.

[0301] The present invention also relates to a kit, which can be used for various purposes, for example, for detecting PD-L1 in a mammal's tissue, cell or body fluid. Such a kit can be used for screening associated with PD-L1 disease. The kit includes a specific binding agent or antibody of the present invention, and when present, a means for indicating the reaction of a specific binding agent or anti-PD-L1 antibody. 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 first antibody, and the kit further includes a means for detecting the first antibody. In one embodiment, the detection means includes a labeled second antibody, which is an anti-immunoglobulin. The antibody can be labeled with a marker selected from a fluorescent dye, an enzyme, a radionuclide and a radiopaque material. The kit can be a kit containing antibodies to detect and quantify PD-L1 in vitro, for example by implementing ELISA or Western blot. In addition, as in the case of an in-product, the kit includes a container and a label or package insert located on or inside the container. The container holds a composition comprising at least one anti-PD-L1 antibody according to the present invention. Additional containers can include, for example, diluents and buffers, control antibodies. A label or package leaflet included with the package may contain a description of the composition and instructions for its use in vitro or for diagnostic purposes.

[0302] Diagnostic uses and compositions

[0303] The anti-CD47 / PD-L1 antibodies of the present invention are also used in diagnostic processes (e.g., in vitro, ex vivo). For example, anti-CD47 / PD-L1 antibodies can be used to detect or measure CD47 and / or PD-L1 levels in samples (e.g., tissue samples or body fluid samples, such as inflammatory exudates, blood, serum, intestinal fluid, saliva or urine) obtained from patients. Suitable methods for detection and measurement include immunoassays, such as flow cytometry, enzyme-linked immunosorbent assays (ELISA), chemiluminescence assays, radioimmunoassays, and immunohistology. The present invention further includes kits, for example, diagnostic kits comprising anti-CD47 / PD-L1 antibodies described herein. Example

[0304] The following examples are provided for a better understanding of the present invention. These examples are for illustrative purposes only and should not be construed as limiting the scope of the present invention in any way.

[0305] All publications, patents and patent applications cited in this specification are incorporated herein by reference. Although the foregoing invention has been described in some detail by way of illustration and example for the purpose of clear understanding, it is very clear to those skilled in the art that certain changes and modifications may be made based on the concepts disclosed in the present invention without departing from the essence and scope of the attached variations of the present invention.

[0306] Example 1. Production of recombinant antigens and antibodies in suspension culture of mammalian cells.

[0307] The extracellular domain sequences of human CD47 (Leu19-Val134) and PD-L1 (Phe19-Arg238) (SEQ ID NO: 107-108) were cloned into a plasmid at the SalI / NotI restriction sites for production of Fc-tagged proteins in mammalian cells ( Figure 1 ). The required amount of plasmid was produced in E. coli cells and purified using a Qiagen kit.

[0308] The sequence of the variable domain of the anti-CD47 antibody (B6H12, Stanford University, US20130142786) was cloned into a plasmid for the production of IgG1 protein in mammalian cells. The required amount of plasmid was produced in E. coli cells and purified using a Qiagen kit.

[0309] Antibodies and antigens were produced in an established cell line derived from Chinese hamster ovary cells (CHO-K1). Serum-free medium (Life Technologies Corporation) was used and suspension culture was performed in flasks on an orbital shaker according to the manufacturer's instructions. For transient expression, transfection was performed at a concentration of 2*10 6 / ml of cells. The DNA / PEI ratio was 1:3 / 1:10. Within 5-7 days after transfection, the cell culture was centrifuged at 2000 g for 20 minutes and filtered through a 0.22 µm filter. The target protein was isolated from the culture medium by affinity HPLC.

[0310] Recombinant Fc proteins were isolated and purified from cell cultures on a protein A column for affinity HPLC. The clarified culture fluid was passed through a 5 ml HiTrap rProtein A SepharoseFF 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-specifically bound components. The 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 stages were performed at a flow rate of 110 cm / hour. The protein was then dialyzed into PBS (pH 7.4) using SnakeSkin DialysisTubing technology, filtered (0.22 µm), transferred to tubes, and stored at -70°C.

[0311] The purity of the obtained protein solution was assessed by non-reducing SDS-PAGE (12% gel) ( Figure 2 ).

[0312] Example 2. Preparation of full-length antibodies.

[0313] Cloning was performed by standard techniques. PCR products containing the variable domain genes of the heavy and light chains of the antibody were generated using primers containing restriction sites. The variable domains of the heavy chain were cloned into the vector pEE-Hc IgG1 at the Sal1 / Nhe1 restriction site. The variable domains of the light chain were cloned into the vector pEE-CK at the Sal1 / BsiW1 restriction site. The resulting gene constructs were used to transiently produce proteins in the CHO-T cell line. As described in Example 1, the proteins were isolated and purified by affinity chromatography on bacterial protein A according to standard methods. The proteins were purified by 12% denaturing PAGE ( Figure 3 ) and 8% denaturing PAGE without mercaptoethanol supplementation ( Figure 4 ) to perform electrophoresis.

[0314] Example 3. Modification of the human Fab phage library MeganLibTM used for the first experiment

[0315] Total RNA from B lymphocytes of blood samples from more than one thousand individual human donors was isolated using the RNeasy Mini Kit (QIAGEN) according to the recommended protocol. RNA concentration determination was performed using the Nanovue kit (GE Healthcare); the quality of the isolated RNA was tested by means of 1.5% agarose gel electrophoresis.

[0316] Reverse transcription reactions were performed using the MMLV RT kit (Evrogen) according to the recommended protocol using MMuLV reverse transcriptase and random hexamer oligonucleotides as primers.

[0317] The reverse transcription product was used as a matrix in a two-stage polymerase chain reaction to obtain the variable domain gene flanked by restriction sites; the reaction was performed using an oligonucleotide kit according to the protocol [J Biol Chem. 1999 Jun 25;274(26):18218-30].

[0318] The obtained DNA product (VL-CK-VH) was treated with NheI / Eco91I restriction endonucleases and ligated into the original phagemid pH 5. The ligated product was transformed into SS320 E. coli electrocompetent cells prepared according to the protocol [Methods Enzymol. 2000; 328: 333-63.]. The reservoir of the combinatorial Fab phage display library MeganLibTM is 10 11 The Fab phage library products were prepared according to the procedure described earlier [J Mol Biol. 1991 Dec 5; 222(3): 581-97].

[0319] Example 4. Immunization of llamas with human CD47 antigen and generation of a phage display library of llama antibody fragments.

[0320] Animals, Lama Glama, were immunized five times in succession by subcutaneous injection of antigenic material mixed with an equal volume of complete (first injection) or incomplete (other injections) Freund's adjuvant. The recombinant human CD47 protein (1 mg / injection) of Example 1 was used as an antigen. Antigen injections were performed at the following intervals: 0, 2, 4, 5, 8 weeks. From the third injection onwards, blood samples (50 ml) were collected 5 days after each injection. 3.8% sodium citrate was used as an anticoagulant (1:9). The blood was diluted 2-fold with sterile saline solution. Then 30 ml of the diluted blood solution was layered on 15 ml Lymphoprep™ (Axis-Shield, Norway) medium (density of 1.077 g / ml) and centrifuged at 800g for 20 minutes. Mononuclear cells (lymphocytes and monocytes) were selected from the plasma / Lymphoprep medium interphase zone and washed with sterile PBS.

[0321] The titer of the obtained serum immunoglobulins against CD47 was evaluated according to standard protocols and proved to be at least 1 / 100000, which was sufficient for the preparation of an antibody library.

[0322] Total RNA from mononuclear llama cells was isolated using the RNeasy Mini Kit according to the protocol (QIAGEN). RNA concentration determination was performed using Nanovue (GE Healthcare); the quality of the isolated RNA was tested by means of 1.5% agarose gel electrophoresis.

[0323] Reverse transcription reactions were performed using the MMLV RT kit (Evrogen) with MMuLV reverse transcriptase and random hexamer primers according to the recommended protocol.

[0324] The reverse transcription product was used as a matrix in a two-stage polymerase chain reaction to obtain genes for single domain VHH, scFv or Fab flanked by restriction sites; the reaction was performed using an oligonucleotide kit and the protocol of [FASEB J. 2007 Nov; 21 (13): 3490-8]. The obtained VHH gene DNA product was treated with NcoI / NotI restriction enzymes 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 ligation product was transformed into SS320 electrocompetent cells prepared according to the protocol [Methods Enzymol. 2000; 328: 333-63.]. The reservoir of the constructed VHH-based library was 0,5-2*10E+8 independent transformants. The reservoirs of the constructed scFv / Fab based libraries were 0,5-2*10E+9 / 1,2-2,5*10E+9, respectively. Phage library products were prepared according to the procedure described earlier [J Mol Biol. 1991 Dec 5; 222(3): 581-97].

[0325] Example 5. Selection of phage display library of antibody fragments

[0326] Specific anti-CD47 phage antibodies were selected from phage Fab, VHH or scFv display libraries (Examples 3, 4) by the conventional selection procedure 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], but using magnetic beads and the KingFisherFlex device, because this technology allows up to 96 different schemes and variants to be performed simultaneously.

[0327] Human biotinylated PD-L1 / CD47 antigens (Fc, EPEA) were purposefully immobilized onto 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 antigens were incubated with the beads for 1 hour at room temperature on a rotator. The beads were then washed with PBS (pH 7.4), and the bead surface was blocked with 2% skim milk or 1% BSA in PBS (pH 7.4) for 1 hour. The human phage library MeganLibTM was incubated at 2*10 in PBS (pH 7.4) with 2% skim milk and non-target antigen containing the target antigen tag. 13The IL-5Rα-coated magnetic beads were then incubated with MeganLibTM for 1-2 hours at room temperature.

[0328] Unbound phages were removed by several cycles of washing the magnetic beads with a PBS (pH 7.4) solution containing 0.1% Tween 20. The number of washing cycles 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 the antigen on the surface of the magnetic beads were eluted from the beads with a 100 mM Gly-HCl solution (pH 2.2) during 15 minutes under stirring and then neutralized with 1M Tris-HCl (pH 7.6). E. coli TG1 bacteria were infected with phages, grown in culture medium, and subsequently used in the next selection cycle. After three or four rounds, phagemid DNA was isolated from the E. coli TG1 culture 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.

[0329] Example 6. ELISA of polyclonal phage against specific and non-specific antigens.

[0330] Target antigens (CD47 / PD-L1) and non-target antigens (with Fc fusion proteins) were immobilized on high-absorbency plates (Greiner-Bio) to perform ELISA. Proteins were added at concentrations of 1 μg / ml and 5 μg / ml in 0.1 M NaHCO3 (pH 9.0), respectively, and titrated in increments of 2 to 7 dilutions, and the sealed plates were then incubated overnight at 4°C. All subsequent steps were performed according to the standard ELISA protocol using a high-performance automated Tecan Freedom EVO 200-based robotic platform (Tecan). To block nonspecific binding, a blocking buffer containing 2% skim milk or 1% BSA in PBS (pH 7.4) was added to the plate wells. The plates were incubated for 1 hour at room temperature. After several washing cycles with phosphate-saline buffer containing Tween 20 (PBST), 50 μl / well of the 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 incubation for 50 min at room temperature, the plate was washed three times with PBST. The colorimetric signal was obtained by adding substrate solution (H2O2-0.02% and TMB in CH3COONa pH 5.5) for 10 min; the color development was then blocked by adding 1% sulfuric acid (20 μl). The color signal was measured at 450 nm using an appropriate Tecan-Sunrise plate reader (Tecan).

[0331] After the third and fourth rounds of selection for the target antigen, ELISA of the polyclonal phage products showed significant enrichment. The library was selected for recloning and further screening, where at the minimum dilution of the phage library, a signal exceeding 5-fold over the non-cognate control antigen was observed.

[0332] Example 7

[0333] Reclone the gene of the antibody fragment into the expression plasmid

[0334] After successful rounds of selection, recloning of the antibody variable domain genes from the phagemid vector into an expression plasmid was performed using restriction ligation techniques according to standard protocols.

[0335] The resulting pool of clones enriched for VHH single domains or scFvs specific for CD47 was recloned into the expression plasmid pET-22 (Novagen) carrying myc- and His6-tag sequences at the C-terminus of the VHH under the control of the T7 promoter. The Fab genes of the library containing enriched sequences for the CD47 antigen were recloned into the expression vector pLL4, which further contained myc- and His6-tag sequences at the C-terminus of the heavy chain CH1 domain, under the control of the lac promoter.

[0336] Subsequently, the expression vectors containing the antibody fragments were transformed into E. coli Bl21 (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 displayed library to the antigen was performed by ELISA using the Mabnext Flow Chart platform.

[0337] Example 8. Analysis of specific binding of scFv or VHH single domains to human CD47-Fc.

[0338] ELISA was used to measure the binding of the specific test antibody fragment of Example 4 to human CD47-Fc. ELISA well plates (Nunc ImmunoMaxisorp) were covered with 50 µl / well of human CD47-Fc (Biocad) (0.5 µg / ml in 1X coating carbonate buffer), sealed and incubated overnight at 4°C. All further stages were performed according to standard ELISA protocols using high-performance automation platforms based on robotic systems such as GenetixQ-pix2xt (Molecular Devices) and TecanFreedom EVO 200 (Tecan). Nonspecific binding was blocked by adding blocking buffer BB (200 µl of 0.5% skim milk in PBS). The plates were incubated on a shaker at room temperature for 1 hour. After washing with PBS-Tween, each cell was coated with 100 μl of cell supernatant containing the test antibody fragment. The plates were incubated on a shaker at room temperature for 1 hour; further, each plate well was washed 5 times with PBS-Tween buffer. After washing, mouse anti-MYC IgG clone 9E10 (ThermoFisher Scientific) (50 µl / well) was added in PBS-Tween (1:5000). The plate was shaken on a rotary shaker (50 min at room temperature) and then washed 5 times with PBS-Tween buffer as described above. After washing, anti-mouse IgG HRP conjugate (ThermoFisherScientific) (50 µl / well) was added in PBS-Tween (1:10000). The plate was shaken on a rotary shaker (50 min at room temperature) and then washed 5 times with PBS-Tween buffer as described above. The colorimetric signal was obtained by adding ТМВ (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 an appropriate Tecan-Sunrise plate reader (Tecan). Antibody binding is proportional to the signal generated. Clones in which the color signal exceeded the background signal by more than 5-fold were tested in a competitive ELISA assay to identify antagonistic specific antibody fragments that blocked the interaction between SIRP-Fc ligand (BIOCAD) and human CD47-Fc receptor under conditions similar to those described in [WO2016048188 A8] and at a 4-fold reduction in signal compared to a control not containing the tested antibody fragment.

[0339] Screening of 2400 clones resulted in 265 scFv and VHH clones that demonstrated a 5-fold increase over background in signal. The positive clone group produced 27 antagonistic clones capable of blocking the interaction between SIRP-Fc ligand (BIOCAD) and human CD47-Fc receptor. The nucleotide sequence of the positive clone genes was determined by Sanger sequencing on a 3130xl Genetic Analyzer (Applied Biosystems). Six exemplary VHH single domain clones were obtained, whose sequences differed by at least 1 amino acid, but had CDR regions that were at least 90% homologous to each other, thus indicating that they were derived from a parental clone by in vivo maturation in immune llamas (Table 1).

[0340] Table 1. Sequences of CD47-specific VHH single domains targeting human CD47

[0341]

[0342]

[0343] Example 9. Analysis of specific binding of Fab to human CD47-Fc.

[0344] Fab production is carried out according to standard techniques: bacterial cells are transformed with an expression vector containing the Fab gene, and during the culture of the resulting transformants, an inducer for the transcription of the lac operon is added to the culture medium, resulting in the expression of Fab.

[0345] ELISA was then performed to search for Fabs that bind to human CD47.

[0346] B6H12 Fab with a public sequence (see Example 1) was used as a positive control. To test specific binding, ELISA wells (medium binding, Greiner bio one) were covered with 50 μl / well of CD47 Fc llama (0.2 μg / ml in 1X carbonate buffer), sealed and incubated overnight at 4°C. All further stages were performed according to standard ELISA protocols using high-performance automation platforms based on robotic systems such as GenetixQpix2xt (Molecular Devices) and Tecan Freedom EVO 200 (Tecan). Nonspecific 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. After washing with PBS-Tween, each cell was coated with 60 μl / well of cell supernatant containing the test Fab. The plates were incubated for 1 hour at room temperature; each plate well was then washed 3 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 plates were incubated for 1 hour at room temperature and washed 3 times with PBS-Tween buffer as described above. The colorimetric signal was obtained by adding TMV (50 µl / well) until saturation (15 minutes); 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). Antibody binding is proportional to the signal generated. Clones in which the color signal exceeded the signal from the control antibody were tested for nonspecific binding by ELISA.

[0347] Example 10. Analysis of non-specific binding of Fab to various human antigens.

[0348] The secondary screening was aimed at selecting Fab-producing clones that interacted with the full-length CD47 antigen and did not interact with nonspecific antigens and also competed with the ligand (CD47) for binding to SIRPa.

[0349] ELISA was used to analyze the nonspecific binding of the test Fab to other antigens. The analysis was performed as described above, but using 3DHer3-H6E, INFα2b, PD-L1-Fc-llama (2.5 μg / ml in 1× carbonate buffer) as antigens for immobilization. CD47 FE and CD47 Fc llama (0.2 μg / ml in 1× carbonate buffer) were used as specific binding controls. All further stages were performed according to standard ELISA protocols using high-performance automated platforms based on robotic systems such as Genetix Qpix2xt (Molecular Device) and Tecan Freedom EVO200 (Tecan).

[0350] Competitive ELISA was used to test the ability of preselected anti-human CD47 specific Fabs to block the interaction with SIRPα receptor. Fab with a published sequence (see Example 1) was used as a positive control for antagonists.

[0351] ELISA plates (high binding, Greiner bio one) were covered with 50 µl / well of SIRPa (0.5 µg / ml in 1X carbonate buffer) and incubated overnight at 4°C. All further stages were performed according to the standard ELISA protocol using a high-performance automation platform based on, for example, the robotic systems Genetix Qpix2xt (Molecular Devices) and TecanFreedom EVO 200 (Tecan). Nonspecific binding was blocked by adding blocking buffer BB (200 µl 0.5% skim milk in PBS). The plates were incubated for 1 hour at room temperature.

[0352] In parallel, cell supernatants containing test Fab and CD47 Fc llama (at a final concentration of 1 μg / ml in PBS-Tween) were mixed at a 1:1 ratio in a non-absorbent plate and incubated for 45 minutes at room temperature.

[0353] After washing the plates containing the SIRPα receptors to remove the BB, the mixture of Fab and CD47 Fc llama was transferred to the plates 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 plates were incubated for 45 min at room temperature and washed three times with PBS-Tween as described above. The colorimetric signal was obtained by adding TMV (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 generated. Clones showing blocking at the level of the control Fab were noted as positive and used in further assays. The genes of the variable domains of positive clones were sequenced and analyzed on an Applied Biosystems 3130 GeneticAnalyzer (Applied Biosystems) according to standard protocols.

[0354] Example 11. Screening of anti-CD47 antibody fragments by comparison of the kinetic dissociation constant koff (kdis).

[0355] VHH antibody fragments were measured to provide an example of analyzing the kinetic parameters of the interaction of antibody fragments that specifically bind to the CD47 receptor. Using Octet Red 96 and ARG2 amino-reactive biosensors (Pall-ForteBio), the dissociation constant (k)-based analysis of the VHH fragments was performed. dis). Affinity constants can be calculated based on the exact protein concentration in the test solution, and since cell growth medium was used as protein solution and protein concentration was not measured, candidates were compared against each other by using their dissociation constants. The biosensor was prehydrated in water for 1 hour. After activation of the biosensor, CD47-Fc at a concentration of 10 µg / ml in acetate buffer pH 4 was non-specifically immobilized (via NH2 groups) to the biosensor. The sensor was then immersed into a well containing cell growth medium with specific VHH (approximately 1 μg VHH from 1 ml of medium), where the complex associates. The sensor was then immersed into a buffer solution, where the subsequent dissociation phase of the complex occurs. 1 / 10 volume of 10x working buffer was added to the test sample in E. coli growth medium containing the anti-CD47 VHH fragment. The obtained curves were analyzed with a 1:1 interaction model using Octet Data Analysis (version 7.0) according to standard procedures.

[0356] The results of the koff screening of anti-CD47 VHH candidates are shown in Table 2. All VHH fragments were confirmed to bind specifically to human CD47; based on the dominant kdis, candidate BCD106-02_L.Alecto.VHHSel2_MP1_C7_49 was selected for further studies and recloning to obtain bispecific antibodies.

[0357] Table 2. Kinetic dissociation constants of VHH and CD47-Fc

[0358] serial number Clone Name kdis (1 / s) 1 BCD106-02_L.Alecto.VHHSel2_MP1_B9_64 8.00E-03 2 BCD106-02_L.Alecto.VHHSel2_MP1_C7_49 5.00E-03 3 BCD106-02_L.Alecto.VHHSel2_MP1_F10_76 1.00E-02 4 BCD106-02_L.Alecto.VHHSel2_MP1_G5_37 6.00E-03 5 BCD106-02_L.Alecto.VHHSel2_MP1_G7_53 8.00E-03 6 BCD106-02_L.Alecto.VHHSel2_MP1_H10_78 6.00E-03 7 BCD106-02_L.Alecto.VHHSel2_MP1_B9_64 8.00E-03

[0359] Example 12. Obtaining constructs and producing asymmetric bispecific anti-CD47 / PD-L1 antibodies.

[0360] The sequences of all variable domains of the optimized scFv fragments and the gene sequences of wild-type and mutant variants of the variable domain VHH of the candidate BCD106-02-VHH_C7_49 (Table 3) and the genes of the variable domains of the light and heavy chains 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 gene was derived 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 agarose gel were purified on a QIAquick Gel Extraction Kit (Qiagen) column. The scFv and VHH genes were individually linked into the plasmid pEE-Fc (knob), while the variable heavy chain domains of the aPD-L1-specific antibody were individually linked into the plasmid pEE-Fc (hole). pEE-Fc (knob) contains human IgG1 Fc with mutations S354C+T366W, while pEE-Fc (hole) contains human IgG1 Fc with mutations Y349C+T366S+L368A, providing heterodimerization of these Fc parts with each other at a minimal degree of homodimerization [Nat Biotechnol. 1998 Jul; 16(7): 677-81.], accompanied by co-transient expression in CHO-EBNA cells with the aPD-L1 light chain variable domain similarly cloned into pEE-Cλ. 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. The constructs pEE-Fc(knot)-scFv or pEE-Fc(knot)-VHH with the correct sequence were co-transfected with pEE-BCD135-VH-299P-HC-hole and pEE-BCD135-01 4LG VL hzau CL to obtain the so-called asymmetric bispecific antibodies (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 fragments, B-based on anti-CD47 VHH and anti-PD-L1 Fab binding fragments. According to Example 2, the resulting genetic constructs were used to produce proteins in a CHO-T cell line. After purification, the antibodies were highly homogeneous in composition, and the production yield of the bispecific antibodies ranged from 60 to 260 mg / ml culture medium. Figure 7 An example of a purified bispecific antibody based on an anti-CD47 scFv fragment is shown. Figure 8 Examples of purified bispecific antibodies based on anti-CD47 VHH fragments are shown. The antibody variant containing the sequence VHH47Opt3 shown in Table 3 produced very low antibody yields and was excluded from further testing.

[0361] Table 3. Amino acid sequences of wild-type / mutant variants of the variable domains of anti-CD47 VHH and anti-PD-L1 BCD135 from the BCD106-02-VHH_C7_49 clone. Grey indicates anti-CD47 VHH positions with mutations that differ from those of the wild-type.

[0362]

[0363] Example 13. Analysis of the interaction of anti-PD-L1 / anti-CD47 PD-L1-bispecific antibody with human PD-L1 / CD47 antigen on OctetRED96.

[0364] The interaction analysis of PD-L1-bispecific antibodies with human PD-L1 / CD47 antigens was performed on OctetRed 96 (Pall-ForteBio). AR2G biosensors were prehydrated in mQ for 1 h. After activation of the biosensor, PD-L1-Fc or CD47-Fc at a concentration of 25 µg / ml in acetate buffer pH 4 was nonspecifically immobilized to the biosensor (via NH2 groups). The sensor was then immersed in a well containing an anti-PD-L1 / anti-CD47 antibody solution (10 μg / ml), in which the antibody-antigen complex associates. The sensor was then immersed in a buffer solution for the subsequent dissociation step. Binding curves were analyzed using Octet Data Analysis software (version 8.2) according to standard procedures and using a 1:1 interaction model after subtracting the reference signal.

[0365] The results of the analysis are shown in Table 4. Therefore, it can be concluded that the antibody has high affinity, with this form of the antibody having nM values ​​for the PD-L1 antigen and sub-nM values ​​for the CD47 antigen. Such binding is considered sufficient to enable the antibody to interact with receptors on target cells for subsequent testing of therapeutic activity in vitro and in vivo.

[0366] Table 4. Kinetic dissociation constants of anti-CD47 / anti-PD-L1 bispecific antibodies.

[0367]

[0368] Example 14. Analysis of the interaction of anti-PD-L1 / anti-CD47 bispecific antibodies with cynomolgus monkey CD47 and PD-L1 receptors on the Forte Bio Octet RED 384.

[0369] The experimental study of antibody affinity for animal CD47 / PD-L1 antigens was performed on a Forte Bio Octert RED 384. Antibodies were immobilized to AR2G sensors (Forte Bio) at a concentration of 20 μg / ml according to the standard protocol and manufacturer's instructions. The analysis was performed at 30°C using PBS containing 0.1% Tween 20 and 0.1% BSA as working buffer. After baseline recording, the sensor was immersed in a well containing the antigen solution (animal CD47 and PD-L1) for 300 seconds, where the complexes associated. The dissociation of the complexes in the buffer solution was then detected for 600 seconds.

[0370] Binding curves were analyzed using Octet Data Analysis (version 9.0) software according to standard procedures and using a 1:1 global interaction model after subtracting the reference signal. Anti-CD47 antibodies specifically bind to the cynomolgus antigens CD47 and PD-L1. Tables 5 and 6.

[0371] Table 5. Kinetic values ​​for the interaction of antibodies against cynomolgus monkey antigen (CD-47).

[0372] name KD(M) KD Error kon(1 / Ms) kon error kdis(1 / s) kdis error BCD106-02-001 6.32E-09 3.69E-11 1.88E+05 9.64E+02 1.18E-03 3.28E-06 BCD106-02-006 6.99E-10 3.99E-12 8.64E+05 3.61E+03 6.04E-04 2.34E-06 BCD106-02-013 3.06E-10 3.56E-11 5.31E+04 1.60E+02 1.63E-05 1.89E-06

[0373] Table 6. Kinetic values ​​of the interaction of antibodies with cynomolgus monkey antigen (PD-L1).

[0374] name KD(M) KD Error kon(1 / Ms) kon error kdis(1 / s) kdis error BCD106-02-001 8.75E-10 6.01E-12 1.67E+06 1.07E+04 1.46E-03 3.55E-06 BCD106-02-006 7.27E-10 6.40E-12 1.73E+06 1.34E+04 1.26E-03 5.25E-06 BCD106-02-013 1.34E-09 1.02E-11 1.65E+06 1.19E+04 2.21E-03 5.66E-06

[0375] Example 15. Analysis of non-specific binding of anti-PD-L1 / anti-CD47 bispecific antibodies to a panel of antigens on the Forte Bio Octet RED 384.

[0376] For the nonspecific histidine-tagged antigen group, nonspecific binding experiments were performed. Anti-hIgG FcCapture (AHC) biosensors prehydrated for 10 min in PBS containing 0.1% Tween-20 and 0.1% BSA as working buffer were used for measurements.

[0377] The antibody was immobilized to the Anti-hIgG Fc Capture (AHC) sensor (ForteBio) at a concentration of 30 μg / ml. PBS containing 0.1% Tween 20 and 0.1% BSA was used as the working buffer and the analysis was performed at 30°C. After the baseline was established in the buffer solution, the sensor was immersed in the well with a solution of nonspecific antigen for 300 seconds, in which the complex was associated. The dissociation of the complex in the buffer solution was then detected for 600 seconds.

[0378] Binding curves were analyzed using Octet Data Analysis (version 9.0) according to standard procedures using a 1:1 global interaction model (after subtracting the reference signal). The anti-PD-L1 / anti-CD47 bispecific antibody did not bind nonspecifically to the antigen panel.

[0379] Example 16. Analysis of the interaction between anti-PD-L1 / anti-CD47 bispecific antibody and FcγRIIIa group on Forte Bio Octet RED 384.

[0380] Experimental studies of antibody affinity to a panel of Fc binding proteins were performed on a Forte Bio OctertRED 384 using streptavidin (SAX) biosensors pre-hydrated for 30 min in PBS containing 0.1% Tween-20 and 0.1% BSA.

[0381] Biotinylated Fc-binding Avi-tagged proteins FcγRIIIa-F158 and FcγRIIIa-V158 were immobilized to streptavidin (SAX) sensors at a concentration of 5 μg / ml in FSB kinetic buffer containing 0.1% Tween-20 and 0.1% BSA pH 7.4, with the required signal level to reach 0.4 nM. t RecLoad Time fixation. After baseline recording, the sensor was immersed in a well containing antibody solution for 60 seconds, where the complex associates. The dissociation of the complex was then monitored in buffer solution for 150 seconds.

[0382] Binding curves (after subtraction of reference signals) were analyzed using Octet Data Analysis (version 9.0) according to standard procedures using a 2:1 global interaction model. The anti-PD-L1 / anti-CD47 bispecific antibody specifically binds to the Fc binding proteins FcγRIIIa-F158 and FcγRIIIa-V158. Tables 7 and 8.

[0383] Table 7. Kinetic values ​​of the interaction of antibody BCD-106 (02-001, 03-006, 02-013) against FcγRIIIa-F158.

[0384] name KD(M) KD Error kdis(1 / s) kdis error kon(1 / Ms) kon error BCD106-02-001 1.61E-06 9.86E-07 6.27E-08 1.04E-07 1.73E+05 1.19E+04 BCD106-02-006 1.47E-06 1.73E-07 6.03E-08 8.40E-08 1.35E+05 1.67E+04 BCD106-02-013 1.30E-06 2.00E-06 3.79E-08 1.88E-07 2.22E+05 1.55E+04

[0385] Table 8: Kinetic values ​​of the interaction of antibody BCD-106 (02-001, 03-006, 02-013) against FcγRIIIa-V158.

[0386] name KD(M) KD Error kdis(1 / s) kdis error kon(1 / Ms) kon error BCD106-02-001 9.32E-07 1.62E-07 2.86E-08 1.86E-08 2.33E+05 5.72E+04 BCD106-02-006 4.63E-06 <1.0E-12 1.07E-06 5.25E-07 4.75E+04 8.58E+03 BCD106-02-013 3.73E-07 1.45E-07 1.83E-09 1.07E-08 6.10E+05 5.77E+04

[0387] Example 17. Analysis of the interaction between anti-PD-L1 / anti-CD47 bispecific antibody and FcRn on Forte Bio Octet RED 384.

[0388] Experimental studies of antibody affinity to a panel of Fc binding proteins were performed on a Forte Bio OctertRED 384 using streptavidin (SAX) biosensors prehydrated for 30 min in PBS containing 0.1% Tween-20 and 0.1% BSA.

[0389] Biotinylated Avi-tagged FcRn was immobilized to a streptavidin (SAX) sensor at a concentration of 5 μg / ml in PBS kinetic buffer containing 0.1% Tween-20 pH 6, with the required 5 μg / ml to achieve a signal level of 0.4 nm. t RecLoad After baseline recording in buffer solution, the sensor was immersed in a well containing antibody solution in PBS kinetic buffer containing 0.1% Tween-20 pH 6 for 60 seconds, in which the complex associates. The dissociation of the complex was then monitored in PBS kinetic buffer containing 0.1% Tween-20 and 0.1% BSA pH 7.4 for 150 seconds.

[0390] Binding curves were analyzed using Octet Data Analysis (version 9.0) according to standard procedures using a 2:1 global interaction model (after subtracting the reference signal). Anti-PD-L1 / anti-CD47 bispecific antibodies specifically bind to FcRn. Table 9.

[0391] Table 9. Kinetic values ​​of the interaction of antibody BCD-106 (02-001, 03-006, 02-013) against FcRn.

[0392] name KD(M) KD Error kdis(1 / s) kdis error kon(1 / Ms) kon error BCD106-02-001 1.61E-06 9.86E-07 6.27E-08 1.04E-07 1.73E+05 1.19E+04 BCD106-02-006 1.47E-06 1.73E-07 6.03E-08 8.40E-08 1.35E+05 1.67E+04 BCD106-02-013 1.30E-06 2.00E-06 3.79E-08 1.88E-07 2.22E+05 1.55E+04

[0393] Example 18. Analysis of the ability of the anti-PD-L1 / anti-CD47 bispecific antibody PD-L1 to induce antibody-dependent cellular cytotoxicity on PD-L1 / CD47 positive cells.

[0394] To perform ADCC (antibody-dependent cellular cytotoxicity), the MDA-MB-231 cell line expressing PD-L1 / CD47 receptors on its surface and peripheral blood mononuclear cells (PBMCs) were used.

[0395] Obtain peripheral blood mononuclear cells

[0396] PBMCs were obtained by density gradient fractionation of venous blood cells from healthy donors. After isolation, cells were cultured at 2–5 × 10 6 The cells were cultured at a concentration of 10 cells / ml at 37°C and 5% CO2 for 18-24 hours.

[0397] Target cell preparation

[0398] MDA-MB-231 cells were cultured in DMEM medium containing 10% FBS (fetal bovine serum) at 37°C and 5% CO2. Cells were removed from the plastic surface using trypsin and resuspended in DMEM containing 10% FBS. Calcein AM was added to a concentration of 5 μM. After 30 minutes, the cells were washed twice with DMEM containing 10% FBS to remove excess Calcein AM. A concentration of 10 μM was prepared in DMEM containing 10% FBS. 5 cells / ml of target cell suspension.

[0399] Preparation of test antibody dilutions

[0400] All test antibodies were diluted to a concentration of 10 μg / ml in DMEM medium containing 10% FBS. A series of serial dilutions were prepared in 5-fold increments. The concentrations of the test antibodies were (ng / ml): 10000; 2000; 400; 80; 16; 3,2; 0,64; 0,128; 0,0256; 0.

[0401] Preparation of PBMCs

[0402] Mononuclear lymphocytes were collected from vials and centrifuged at 200 × g for 5 min. A concentration of 5 × 10 6 cells / ml of cell suspension.

[0403] Perform ADCC assay

[0404] Add 50 μl / well of test antibody to the wells of a 96-well plate. Add 100 μl / well of target cell suspension to the wells containing antibody. Incubate the plate at 37°C and 5% CO2 for 15-20 minutes. Add 50 μl / well of PBMC suspension to the wells containing antibody and target cells. Add 50 μl / well of DMEM medium containing 10% FBS, 100 μl of target cell suspension, and 50 μl of PBMC suspension to three wells (control for maximum lysis, "KL"). Incubate the plate at 37°C and 5% CO2 for 3.5-4 hours. 30 minutes before the end of the incubation, add lysis buffer to the KL wells.

[0405] After incubation, the plate was centrifuged at 200 xg for 10 minutes. The supernatant fluid was transferred to a new 96-well plate. Fluorescence was measured in relative fluorescence units at excitation / emission wavelengths of 485 / 538 nm using a plate fluorimeter.

[0406] ADCC efficacy was calculated by the following formula:

[0407]

[0408] ,in

[0409] 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 PBMC)

[0410] KL- Control for maximum lysis of target cells (50 μl / well of DMEM medium containing 10% FBS + 100 μl / well of target cells + 50 μl / well of PBMC + lysis buffer).

[0411] Results are shown in Fig. 9 and 10 middle.

[0412] Based on the data obtained, all anti-CD47 / PD-L1 antibodies showed comparable or higher EC50 values ​​than the control monospecific anti-CD47 antibody (clone B6H12).

[0413] Example 19. Comparison of the activity of anti-PD-L1 / anti-CD47 bispecific antibodies against CD47 / PD-L1 in a test on stimulation of phagocytosis by human macrophages

[0414] To perform ADCP (antibody-dependent cellular phagocytosis), the MDA-MB-231 cell line and human macrophages that have PD-L1 / CD47 receptors on their surface were used.

[0415] Obtaining human macrophages

[0416] Peripheral blood mononuclear cells (PBMCs) were isolated from venous blood of healthy donors by density gradient separation. Human blood mononuclear cells were isolated using a kit for isolation of human CD14-positive human cell fraction (Miltenyi Biotec). Wells of a 24-well plate were used to culture 350,000 mononuclear cells / well in 700 μl of RPMI-1640 containing 10% FBS, 100 ng / ml GM-CSF (Peprotech) at 37°C and 5% CO2 for 3 days. On the 4th day of culture, the culture medium was replaced with new culture medium containing 700 μl of RPMI-1640 per well containing 10% FBS, 100 ng / ml GM-CSF (Peprotech), 50 ng / ml IFNγ (Peprotech) and 10 ng / ml LPS (Sigma), and the cells were cultured for another 3 days at 37°C and 5% CO2.

[0417] Target cell preparation

[0418] MDA-MB-231 cells were cultured in DMEM medium containing 10% FBS (fetal bovine serum) at 37°C and 5% CO2. Cells were removed from the plastic surface by trypsinization and resuspended in DMEM containing 10% FBS. Calcein AM was added to a concentration of 5 μM. After 30 minutes, the cells were washed twice with DMEM containing 10% FBS to remove excess Calcein AM. A concentration of 10 μM was prepared in DMEM containing 10% FBS. 5 cells / ml of target cell suspension.

[0419] Perform ADCP assay

[0420] Select medium from the wells containing macrophages, and add 500 μl of RPMI-1640 medium containing 10% FBS and 20 μg / ml test antibody to the wells. Add 500 μl / well of target cell suspension to the wells. Incubate the plate at 37°C and 5% CO2 for 3 hours. Then select medium, remove cells from the plastic surface by TrypLE Express reagent, and stain with fluorescently labeled anti-CD14 antibody. Analyze the suspension of stained cells on a flow cytometer.

[0421] ADCP efficacy is calculated by the following formula:

[0422] ,in

[0423] Calcein + CD14 + is the number of CD14-positive cells containing calcein dye.

[0424] CD14 + is the number of all CD14 positive cells

[0425] Results are shown in Fig.11 middle.

[0426] 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 that was comparable to the efficacy of a control monospecific anti-CD47 antibody (clone B6H12).

[0427] Example 20. Comparison of the effects of anti-PD-L1 / anti-CD47 bispecific antibody candidates on human red blood cell hemagglutination

[0428] Human red blood cells are used to analyze the ability of antibodies to cause hemagglutination.

[0429] Preparation of red blood cell suspension

[0430] Blood samples were drawn from the vein of healthy donors into vacuum heparin tubes. 9 ml of blood was transferred into a 50 ml centrifuge tube. The blood was diluted to 30 ml with DPBS without Ca2+ and Mg2+ at room temperature. The suspension was centrifuged at 800g for 10 minutes and the supernatant was decanted. The cell washing procedure was repeated twice with DPBS without Ca2+ and Mg2+ and centrifugation. Then 300 μl of the cell pellet was resuspended in 30 ml DPBS to obtain a 1% red blood cell suspension.

[0431] Perform hemagglutination assay

[0432] The test antibody was diluted to a concentration of 20 μg / ml in DPBS. 100 μl of the antibody dilution and the red blood cell suspension were mixed in a 96-well round bottom plate. The plate was incubated in a CO2 incubator at 37°C for 16 hours. The results were recorded visually using an arbitrary 4 plus sign scale. Significantly positive results were 2 plus signs and more. The results are shown in Table 10.

[0433] Table 10. Hemagglutination reaction in the presence of anti-CD47 / PD-L1 antibodies. “–” indicates the absence of agglutination.

[0434]

[0435] According to the data obtained, none of the anti-CD47 / PD-L1 antibodies caused hemagglutination, whereas the reference anti-CD47 monoclonal antibody (clone B6H12) caused significant agglutination, due to the bivalent nature of this antibody and thus the ability to interact with two CD47 molecules located on different erythrocytes.

[0436] Example 21. Analysis of complement-dependent cytotoxicity (CDC) of bispecific anti-CD47 / PD-L1 antibodies.

[0437] 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.

[0438] Target cell preparation

[0439] MDA-MB-231 cultures were grown in DMEM medium supplemented with 10% fetal bovine serum (FBS).

[0440] The cells were removed from the plastic surface by trypsin and cultured at 1 × 10 6 The cells were suspended at a concentration of 1 cell / ml in DMEM medium containing 0.1% BSA.

[0441] Preparation of test antibody dilutions

[0442] All tested anti-CD47 / PD-L1 antibodies were diluted to a concentration of 100 μg / ml in DMEM medium containing 0.1% BSA. A series of serial dilutions were prepared in 8-fold increments. The concentrations of the tested antibodies were (ng / ml): 100000; 12500; 1562,5; 195,3; 24,4; 3,05; 0,38; 0,04; 0,005.

[0443] Get a CDC test

[0444] Human complement was thawed and dissolved 1:4 in DMEM medium containing 0.1% BSA.

[0445] 50 μl / well of each dilution of the test antibody was added to the wells of a 96-well plate, and for each antibody sample, 50 μl / well of medium supplemented with 0.1% BSA was added - cell control, and 150 μl / well of DMEM medium containing 0.1% BSA was added - medium control.

[0446] 50 μl / well of the suspension of MDA-MB-231 cells was added to each well containing the test antibody and cell control.

[0447] Pour 50 μl / well of diluted complement into all wells containing test antibodies and cell controls. Shake the plate on an orbital shaker at room temperature for 2-4 minutes and place in a CO2 incubator at 37ºC for 2-3 hours.

[0448] Add 15 µl of Alamar Blue reagent to the wells of the assay plate. Shake the plate on an orbital shaker at room temperature for 10–20 minutes. Incubate the plate further in a CO2 incubator for 18-24 hours.

[0449] The plate was shaken on an orbital shaker at room temperature for 10-20 minutes.

[0450] Fluorescence was measured using relative fluorescence units at excitation / emission wavelengths of 544 / 590 nm using a plate fluorimeter. The fluorescence signal obtained is proportional to the number of viable cells. The results are displayed in Figure 12-15 middle.

[0451] According to the data obtained, the tested antibodies did not induce complement-dependent cytotoxicity (CDC) on the MDA-MB-231 cell line.

[0452] Example 22. Analysis of the antagonistic activity of anti-PD-L1 / anti-CD47 bispecific antibodies against cell cultures bearing PD-L1 membrane receptors.

[0453] To analyze the antagonistic activity of anti-PD-L1 / CD47 antibodies against the PD-L1 receptor, the ability of the antibodies to reactivate luciferase signaling in the Jurkat-PD1-NFAT-Luc reporter cell line during co-culture with PD-L1 producing cells was assessed.

[0454] Preparation of PD-L1 producing cells

[0455] MDA-MB-231 cultures were grown in DMEM medium supplemented with 10% fetal bovine serum (FBS).

[0456] The cells were removed from the plastic surface by trypsin and plated at 1 × 10 5 The cells were suspended in DMEM medium containing 10% FBS and 20 ng / ml interferon gamma at a concentration of 10 cells / ml. 200 μl / well of the cell suspension was then added to the wells of a white 96-well plate and incubated in a CO2 incubator at 37°C and 5% CO2 for 48 hours.

[0457] Preparation of test antibody dilutions

[0458] All tested anti-CD47 / PD-L1 antibodies were diluted to a concentration of 5 μg / ml in RPMI-1640 medium containing 10% FBS. A series of serial dilutions in 3-fold increments were prepared. The concentrations of the tested antibodies were (ng / ml): 2500; 833,3; 277,7; 92,5; 30,8; 10,2; 3,4; 1,1.

[0459] ● Preparation of Jurkat-PD1-NFAT-Luc cells

[0460] ● On the day of the experiment, prepare a 2.5*10 6 Suspension of Jurkat-PD1-NFAT-Luc cells at 1 cell / ml.

[0461] ● Preparation of activation antibody solution

[0462] ● On the day of the experiment, a 10x mix of activating antibodies was prepared (anti-CD3 at 4 μg / ml; anti-CD28 at 4 μg / ml; anti-mouse at 16 μg / ml in RPMI-1640 medium containing 10% FBS).

[0463] ● Conducting the Test

[0464] ● Remove growth medium from the plate containing MDA-MB-231 cells. Add 40 μl / well of antibody dilutions to the wells containing cells. Add 40 μl / well of RPMI 1640 medium containing 10% FBS to control wells (cells without test antibody, cells without test antibody and activating antibody) and incubate at room temperature for 30 minutes.

[0465] ● Then 40 μl / well of Jurkat-PD1-NFAT-Luc cell suspension was added to all wells. Then, 10 μl / well of 10-fold solution of activation antibody was added to all wells except the control well "cells without test antibody and activation antibody". The cells were incubated in a CO2 incubator at 37°C and 5% CO2 for 6 hours.

[0466] ● Introduce the luciferase substrate One-Glo Luciferase Assay System "Promega" into all wells at a 1:1 ratio (90 μl / well). After 5-10 minutes, measure the luminescence level using a plate reader.

[0467] ● Based on the data 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, therefore, can stimulate T cell-dependent cytotoxicity against cells carrying the PD-L1 receptor.

[0468] Example 23. Homogeneity analysis of anti-PD-L1 / anti-CD47 bispecific antibody products.

[0469] The homogeneity of the bispecific antibodies was analyzed by size exclusion HPLC (SEC HPLC) with UV detection. Chromatography was performed on a HPLC system (Agilent) on a column Tosoh TSK-Gel G3000SWXL (7.8 mm x 30 cm, order number 08541) and a precolumn Tosoh TSKgel Guard SWXL (6.0 mm x 4.0 cm, with a particle size of 7 μm, order number 08543). Detection was performed at wavelengths of 220 and 280 nm. Fig.17 An exemplary HPLC profile of the product BCD106-02-013 based on VHH47Opt2 (see Example 12) is shown. Based on the test results, it can be concluded that the product generated by the molecule BCD106-02-013 is 93% homogeneous in the aggregate composition of monomers and can be applied to subsequent in vitro and in vivo tests.

Claims

1. A bispecific antibody that specifically binds to CD47 and PD-L1, comprising one binding site for CD47 and at least one binding site for PD-L1, characterized in that The binding sites for CD47 include (i) a heavy chain variable domain comprising a CDR1 of the sequence shown in SEQ ID NO: 1, a CDR2 of the sequence shown in SEQ ID NO: 6 or SEQ ID NO: 7; and a CDR3 of the sequence shown in SEQ ID NO: 17; and A light chain variable domain comprising CDR1 of the sequence shown in SEQ ID NO:22, CDR2 of the sequence shown in SEQ ID NO:36, and CDR3 of the sequence shown in SEQ ID NO:50; or CDR1 of the sequence shown in SEQ ID NO:25, CDR2 of the sequence shown in SEQ ID NO:38, and CDR3 of the sequence shown in SEQ ID NO:53; or CDR1 of the sequence shown in SEQ ID NO:29, CDR2 of the sequence shown in SEQ ID NO:48, and CDR3 of the sequence shown in SEQ ID NO:64; or CDR1 of the sequence shown in SEQ ID NO:28, CDR2 of the sequence shown in SEQ ID NO:40, and CDR3 of the sequence shown in SEQ ID NO:56; or CDR1 of the sequence shown in SEQ ID NO:23, CDR2 of the sequence shown in SEQ ID NO:37, and CDR3 of the sequence shown in SEQ ID NO:51; or CDR1 of the sequence shown in SEQ ID NO:30, CDR2 of the sequence shown in SEQ ID NO:42, and CDR3 of the sequence shown in SEQ ID NO:58; or CDR1 of the sequence shown in SEQ ID NO:24, CDR2 of the sequence shown in SEQ ID NO:43, and CDR3 of the sequence shown in SEQ ID NO:59; or CDR1 of the sequence shown in SEQ ID NO:32, CDR2 of the sequence shown in SEQ ID NO:45, and CDR3 of the sequence shown in SEQ ID NO:61; or (ii) a light chain variable domain comprising CDR1 of the sequence shown in SEQ ID NO:32, CDR2 of the sequence shown in SEQ ID NO:45, and CDR3 of the sequence shown in SEQ ID NO:61, and A heavy chain variable domain comprising CDR1 of the sequence shown in SEQ ID NO: 1, CDR2 of the sequence shown in SEQ ID NO: 8, and CDR3 of the sequence shown in SEQ ID NO: 17; or CDR1 of the sequence shown in SEQ ID NO: 1, CDR2 of the sequence shown in SEQ ID NO: 9, and CDR3 of the sequence shown in SEQ ID NO: 17; or CDR1 of the sequence shown in SEQ ID NO: 1, CDR2 of the sequence shown in SEQ ID NO: 11, and CDR3 of the sequence shown in SEQ ID NO: 17; or CDR1 of the sequence shown in SEQ ID NO: 1, CDR2 of the sequence shown in SEQ ID NO: 12, and CDR3 of the sequence shown in SEQ ID NO: 17, or (iii) a heavy chain variable domain comprising a CDR1 of the sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4, a CDR2 of the sequence shown in SEQ ID NO: 10; and a CDR3 of the sequence shown in SEQ ID NO: 18 or SEQ ID NO: 20; and The binding site for PD-L1 comprises a heavy chain variable domain and a light chain variable domain, the heavy chain variable domain comprises the following sequences: SEQ ID NO: 5, SEQ ID NO: 16 and SEQ ID NO: 21, and the light chain variable domain comprises the following sequences: SEQ ID NO: 35, SEQ ID NO: 49 and SEQ ID NO:

65.

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

3. The antibody according to claim 1, characterized in that The antibodies include one or two binding sites for PD-L1.

4. The antibody according to claim 1, characterized in that The binding site for CD47 inhibits the interaction between the CD47 receptor and the SIRPα ligand, and / or the binding site for PD-L1 inhibits the interaction between PD-L1 and the PD-1 receptor.

5. The antibody according to claim 1, characterized in that The binding site for CD47 comprises (i) a heavy chain variable domain comprising the sequence of SEQ ID NO: 66, and a light chain variable domain comprising the sequence of SEQ ID NO: 92; or (ii) a heavy chain variable domain comprising the sequence of SEQ ID NO:68 or SEQ ID NO:69, and a light chain variable domain comprising the sequence of SEQ ID NO:95; or (iii) a heavy chain variable domain comprising the sequence of SEQ ID NO:70 or SEQ ID NO:71, and a light chain variable domain comprising the sequence of SEQ ID NO:89; or (iv) a heavy chain variable domain comprising the sequence of SEQ ID NO: 78, and a light chain variable domain comprising the sequence of SEQ ID NO: 106; or (v) a heavy chain variable domain comprising the sequence of SEQ ID NO:72, SEQ ID NO:73 or SEQ ID NO:74, and a light chain variable domain comprising the sequence of SEQ ID NO:90 or SEQ ID NO:97; or (vi) a heavy chain variable domain comprising the sequence of SEQ ID NO: 80 and a light chain variable domain comprising the sequence of SEQ ID NO: 98; or (vii) a heavy chain variable domain comprising the sequence of SEQ ID NO:75 or SEQ ID NO:76, and a light chain variable domain comprising the sequence of SEQ ID NO:101, SEQ ID NO:102 or SEQ ID NO:103; or (viii) a heavy chain variable domain comprising the sequence of SEQ ID NO:67, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:84 or SEQ ID NO:85, and a light chain variable domain comprising the sequence of SEQ ID NO:100; or (ix) a heavy chain variable domain VHH comprising the sequence SEQ ID NO:88, SEQ ID NO:83, or AVQLVDSGGGLVQPGGSLRLSCAASRSIFSINAMNWYRQAPGKGTEWVAQITDEGITNYVD SVKGRFTITRDNAKNTMYLQMNSLKPEDTAVYYCNAFVITTTSEIYWGQGTTVTVSS; AVQLVDSGGGLVQPGGSLRLSCAASRSIFSINAMNWYRQPPGNRREWVAQITDEGITNYVD SVKGRFTITRDNAKNTMYLQMNSLKPEDTAVYYCNAFVITTTSEIYWGQGTTVTVSS; QVQLVESDGGLVQPGGSLRLSCAASRFTFSINAMNWVRQAPGKGLEWVSQITDEGITNYV DSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCAAFVITTTSEIYWGQGTLVTVSS; QVKLEESGGGLVQPGGSLRLSCAASRSISSINAMNWYRQAPGKRREWVAQITGEGITNYRD SVKGRFTITSDNAKNTMYLQMNSLKPEDTAVYYCNAFVIHTTSEVYWGQGTLVTVSS; AVQLVDSGGGLVQPGGSLRLSCAASRSIFSINAMNWYRQAPGNRREWVAQITDEGITNYVD SVKGRFTITRDNAKNTMYLQMNSLKPEDTAVYYCNAFVITTTSEIYWGQGTTVTVSS; QVKLEESGGGLVQPGGSLTLSCAASGIISSINAMNWYRQAPGKRREWVAQITGEGITNCRDS WKGRFSITSDSANNTMYLQMNNLKPDDTDVYYCNAFVIHTTSEIYWGLGTTVTVSS; DVQLVESGGGLVQPGGSLRLSCAASRNIFSINAMNWYRQAPGKRREWVAQITSEGITNYVD SVKGRFTITRDNAKNTMYLQMNSLKPEDTAVYYCNAFVITASSEVYWGQGTTVTVSS; DVQLVESGGGLVQPGGSLTLSCAASRNIFRINAMNWYRQAPGKRREWVAPITSEGITNYVD SVKGRFTITRDNAKNTMYLQMNSLKPEDTAVYYCNACLITASSEVYWGQGTLVTVSS.

6. The antibody according to claim 1, characterized in that The binding site for PD-L1 includes the heavy chain variable domain containing the following EVQLVESGGGVVRPGGSLRLSCAASGFTFDDYAMSWVRQAPGKGLEWVSDISWSGSNTNYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYHCARAPLLLAMTFGVGSWGQGTLVTVSS, and a light chain variable domain comprising TVVTQEPSLSVSPGGTVTLTCGLSSGTVTAINYPGWYQQTPGQAPRTLIYNTNTRHSGVPDR FSGSISGNKAALTITGAQAEDEADYYCALYMGNGGHMFGGGTKLTVL.

7. The antibody according to claim 1, characterized in that It causes antibody-dependent cellular cytotoxicity, macrophage-mediated phagocytosis and / or T-cell-mediated cytotoxicity, ratios against cells bearing CD47 and / or PD-L1 antigens on their surface.

8. The antibody according to claim 1, characterized in that It includes 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.

9. The antibody according to any one of claims 1 to 7 for use as a medicament for cancer treatment.

10. A nucleic acid encoding the antibody according to any one of claims 1 to 7.

11. The nucleic acid according to claim 10, wherein the nucleic acid is DNA.

12. An expression vector comprising the nucleic acid according to any one of claims 10-11.

13. A method for obtaining a host cell to produce an antibody according to any one of claims 1 to 7, comprising transforming the cell with an expression vector according to claim 12.

14. A host cell for obtaining an antibody according to any one of claims 1 to 7, comprising a nucleic acid according to any one of claims 10 to 11.

15. A method for obtaining an antibody according to any one of claims 1 to 7, comprising: The host cell according to claim 14 is cultured in a culture medium under conditions sufficient to obtain the specified antibody, and the obtained antibody is subsequently isolated and purified, if necessary.

16. A pharmaceutical composition comprising the antibody according to any one of claims 1 to 7 in combination with one or several pharmaceutically acceptable excipients.

17. Use of an antibody according to any one of claims 1 to 7 or a pharmaceutical composition according to claim 16 for the preparation of a medicament for treating a disease or condition mediated by PD-L1 and CD47 in a subject in need thereof, The disease or condition is selected from (HNSCC) head and neck squamous cell carcinoma, cervical cancer, carcinoma of unknown primary, glioblastoma, esophageal cancer, bladder cancer, CRC (colorectal cancer), hepatocellular carcinoma, melanoma, NSCLC (non-small cell lung cancer), renal cancer, ovarian cancer, leukemia, lymphoma, multiple myeloma, breast cancer, prostate cancer, sarcoma, small cell lung cancer, pancreatic cancer and high-risk myelodysplastic syndrome.

18. Use according to claim 17, wherein the disease or disorder is selected from TNBC (triple negative breast cancer), acute leukemia or myeloblastic leukemia, refractory non-Hodgkin's B-cell lymphoma, follicular lymphoma, marginal zone B-cell lymphoma and diffuse large B-cell lymphoma.

19. Use according to claim 17 or 18, wherein the CRC (colorectal cancer) is MSI CRC (colorectal cancer with microsatellite instability) and the acute leukemia is T-cell and B-cell acute lymphoblastic leukemia.

20. The use according to claim 17, wherein the lymphoma is Hodgkin's lymphoma.

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