RU2779652C2 - Antibodies specific to cd47 and pd-l1 - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: bispecific antibody is proposed, which specifically binds to CD47 and PD-L1. The invention also relates to nucleic acid encoding this antibody, an expression vector, a method for the production of an antibody, and to the use of antibodies and compositions including the specified antibodies in cancer therapy.

EFFECT: invention provides for specific high-affine binding of CD47 and PD-L1 and reduction in undesired effects.

23 cl, 17 dwg, 11 tbl, 23 ex

Description

Technical field

The present invention relates to the field of biotechnology, namely to antibodies or their antigen-binding fragments, as well as their use. More specifically, the present invention relates to antibodies that specifically bind to CD47 and PD-L1. The invention also relates to a nucleic acid encoding a given antibody or an antigen-binding fragment thereof, an expression vector, a method for producing an antibody, and the use of said antibodies and compositions in cancer therapy.

State of the art

Providing two separate signals to T cells is a widely accepted model of lymphocytic activation of the remaining T lymphocytes by antigen presenting cells (APC). This model fully provides recognition of one's own and others, and immune tolerance. The primary signal or antigen-specific signal is transmitted through the T cell receptor (TCR) upon recognition of a foreign antigenic peptide presented in the context of the major histocompatibility complex (MHC). The second or co-stimulatory signal is delivered to T cells by co-stimulatory molecules expressed on antigen presenting cells (APC) and induces T cells to stimulate clonal expansion, cytokine secretion and effector function. In the absence of co-stimulation, T cells can become immune to antigenic challenge, they elicit an effective immune response, and this can further lead to depletion or resistance to foreign antigens.

In the two-signal model, T cells receive both signals: both a positive and a negative secondary costimulatory signal. The regulation of such positive and negative signals is critical to maximize the host's protective immune response while maintaining immune resistance and preventing autoimmunity. Negative secondary signals appear to be required for the induction of T-cell resistance, while positive signals stimulate T-cell activation. While the simple two-signal model still provides a valid explanation for naïve lymphocytes, the immune response is a dynamic process and co-stimulatory signals to antigen-expanded T cells can also be provided. The mechanism of co-stimulation is of interest from a therapeutic point of view, since manipulation of co-stimulatory signals has been shown to provide a means of either amplifying or terminating the immune response. Recently, T cell dysfunction or anergy has been found to coexist with induced and persistent expression of the inhibitory receptor, programmed cell death polypeptide 1 (PD-1). As a result, the therapeutic targeting of PD-1 and other molecules that signal through interaction with PD-1, such as programmed death ligand 1 (PD-L1) and programmed death ligand 2 (PD-L2), is an area of intense interest.

PD-L1 is overexpressed in a variety of malignancies and is often associated with poor prognosis. Interestingly, the majority of tumor infiltrating T lymphocytes predominantly express PD-1, in contrast to T lymphocytes in normal tissues and peripheral blood T lymphocytes, which reveals upregulation of PD-1 in relation to tumor-reactive T cells and may contribute to reduced immune response. This may be due to the use of the PD-L1 signaling pathway mediated by PD-L1 expressing tumor cells and interacting with PD-1 expressing T cells, resulting in attenuation of T cell activation and evasion of immune surveillance. Thus, inhibition of PD-L1/PD-1 may enhance CD8+ T cell-mediated tumor killing.

The therapeutic focus of PD-1 and other molecules that signal through interaction with PD-1, such as PD-L1 and PD-L2, is an area of intense interest. Inhibiting PD-L1 signals has been proposed as a means to enhance T-cell immunity (eg, antitumor immunity) for the treatment of cancer and infection, including both acute and chronic infection. Inhibitors that block the interaction of PD-L1 and PD-1 are known, among other sources, from W02001014557, W02002086083, W02007005874, W02010036959, W02010077634 and WO2011066389. However, an optimal therapeutic agent targeted by this pathway has not yet been commercialized, and this is a significant unmet need in medicine.

CD47 is a cell surface glycoprotein that binds to SIRPα (otherwise referred to as SHPS-1) and SIRPγ on respective cells. This interaction leads to downregulation of immune cell function or may 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 very little data on the possible use of CD47 ligands such as SIRPα for similar therapeutic purposes. One explanation is that there is ubiquitous expression of CD47, which may preclude the use of CD47-binding polypeptides as potential drugs. Data published by Yu et al (J Invest Dermatol, 126, 2006, pp. 797-807) suggest that a fusion protein consisting of SIRPα extracellular domains fused to an immunoglobulin Fc domain may interfere with the migration of nascent dendritic cells (DCs) from the skin to the draining lymph nodes in mice and thereby attenuate (at least partially) the contact hypersensitivity response (response) in mice. The migration and function of DCs are important for immune or inflammatory responses. In a disease state, these exacerbated DC responses can lead to maintenance of the disease. Influencing the migration of pathogenic DCs from tissue to lymphoid organs may be an attractive opportunity to break the vicious cycle of promoting autoimmune or inflammatory diseases.

CD47, also known as integrin-associated protein (IAP), ovarian cancer OA3 antigen, Rh-associated antigen and MER6, is a multi-membrane transmembrane receptor belonging to the immunoglobulin superfamily. Expression and/or activity of CD47 is observed in a number of diseases and disorders. Thus, there is a need for therapies that target CD47. In addition, due to CD47 expression on platelets, there is also a need for CD47-targeting therapies (eg, antibodies) that do not cause significant levels of platelet depletion, hemagglutination, red cell depletion, and/or anemia when administered to a subject.

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

Various sources are also known describing multispecific antibodies, for example, WO/2014/087248 describes a bispecific antibody that is specific for CD47 and CD19, and WO2016023001 describes a bispecific antibody that is specific for CD47 and PD1. However, the possibility of creating and effectively using a multispecific antibody that specifically binds to CD47 and PD-L1 has not been previously described.

In connection with the foregoing, it is important to create new antibodies that effectively bind to CD47 and PD-L1.

Brief description of the invention

The present invention relates to binding molecules, eg antibodies directed to bind to CD47 and PD-L1. Such antibodies can be used to treat a disease or disorder mediated by CD47 and PD-L1.

In one aspect, the present invention provides a monoclonal antibody that specifically binds to CD47 and PD-L1 and includes one CD47 binding site and at least one PD-L1 binding site.

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

In some embodiments, an antibody of the present invention includes one or two PD-L1 binding sites.

In some embodiments, the CD47 binding site of an antibody of the present invention inhibits the interaction of the CD47 receptor and the SIRPα ligand, and/or the PD-L1 binding site inhibits the interaction of PD-L1 with the PD-1 receptor.

In some embodiments, the CD47 binding site of an antibody of the present invention comprises a heavy chain variable domain that contains the sequences CDR1, CDR2, CDR3, wherein CDR1 is at least 80% homologous to a sequence selected from the following group of SEQ ID NOs: 1-4 , that is, CDR1 is a sequence selected from the group of SEQ ID NO: 1-4 or a sequence selected from the group of SEQ ID NO: 1-4 with 1 or 2 substitutions. where CDR2 is a sequence of at least 80% homology to a sequence selected from the following group of SEQ ID NO: 6 - 15, i.e. CDR2 is a sequence selected from the group of SEQ ID NO: 6 - 15 or a sequence selected from the group of SEQ ID NO: 6 - 15 NO: 6 - 15 with 1, 2, 3, 4 or 5 replacements. where CDR3 is a sequence of at least 80% homology to a sequence selected from the following group SEQ ID NO: 17 - 20, i.e. CDR3 is a sequence selected from the group SEQ ID NO: 17 - 20 or a sequence selected from the group SEQ ID NO: 17 - 20 NO: 17 - 20 with 1, 2 or 3 replacements.

In some embodiments, the CD47 binding site of an antibody of the present invention includes a heavy chain variable domain that contains the sequences CDR1, CDR2, CDR3, where CDR1 is a sequence selected from the following group of SEQ ID NOs: 1-4, where CDR2 is a sequence, selected from the following group of SEQ ID NO: 6 - 15, where CDR3 is a sequence selected from the following group of SEQ ID NO: 17 - 20.

In some embodiments, the CD47 binding site of an antibody of the present invention includes a heavy chain variable domain according to claim 4, and a light chain variable domain that contains the sequences CDR1, CDR2, CDR3, where CDR1 is a sequence of at least 80% homologous to the sequence of the selected from the following group SEQ ID NO: 22 - 34, i.e. CDR1 is a sequence selected from the group SEQ ID NO: 22 - 34 or a sequence selected from the group SEQ ID NO: 22 - 34 with 1 or 2 substitutions, where CDR2 represents is a sequence of at least 80% homology to a sequence selected from the following group of SEQ ID NO: 36 - 48, i.e. CDR2 is a sequence selected from the group of SEQ ID NO: 36 - 48 or a sequence selected from the group of SEQ ID NO: 36 - 48 with 1, 2, or 3 substitutions, where CDR3 is at least 80% homologous to a sequence selected from the following SEQ I D NO: 50 - 64, i.e. CDR3 is the sequence of SEQ ID NO: 50 - 64 or a sequence selected from the group of SEQ ID NO: 50 - 64 with 1 or 2 substitutions..

In some embodiments, the CD47 binding site of an antibody of the present invention includes a heavy chain variable domain according to claim 4, and a light chain variable domain that contains the sequences CDR1, CDR2, CDR3, where CDR1 is a sequence selected from the following group of SEQ ID NO: 22-34, CDR2 is a sequence selected from the following group of SEQ ID NOs: 36-48, CDR3 is a sequence selected from the following group of SEQ ID NOs: 50-64.

In some embodiments, the CD47 binding site of an antibody of the present invention includes a heavy chain variable domain that contains sequences that are at least 90% homologous to sequences selected from the following group of SEQ ID NOs: 66-88, and a light chain variable domain that contains sequences at least 90% homologous to sequences selected from the following group of SEQ ID NOs: 89-106.

In some embodiments, the CD47 binding site of an antibody of the present invention includes a heavy chain variable domain that contains sequences selected from the following group of SEQ ID NOs: 66-88, and a light chain variable domain that contains sequences selected from the following group of SEQ ID NOs: : 89 - 106.

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

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

In some embodiments, the CD47 binding site of an antibody of the present invention is a Fab, scFv, scFab, or isolated VH or VHH monodomains.

In some embodiments, the PD-L1 binding site of an antibody of the present invention is a Fab, scFv, scFab, or isolated VH or VHH monodomains.

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

In some embodiments, an antibody of the present invention is characterized in that it contains an Fc fragment with at least one mutation or modification that increases antibody-dependent cell-mediated cytotoxicity (ADCC) compared to the same antibody without the mutation or modification.

In some embodiments, an antibody of the present invention is used as a drug for the treatment of cancer.

In one aspect, the present invention relates to a nucleic acid that encodes any of the antibodies described above.

In some embodiments, the nucleic acid of the present invention is DNA.

In one aspect, the present invention relates to an expression vector that contains the nucleic acid described above.

In one aspect, the present invention relates to a method for obtaining a host cell for obtaining any of the antibodies described above, which includes transforming the cell with a vector of the present invention.

In one aspect, the present invention relates to a host cell for producing any of the antibodies described above, which contains the nucleic acid described above.

In one aspect, the present invention relates to a method for producing any of the antibodies described above, which consists in culturing the host cell in a culture medium under conditions sufficient to obtain the specified antibody, if necessary, followed by isolation and purification of the resulting antibody.

In one aspect, the present invention relates to a pharmaceutical composition for the prevention or treatment of a disease or disorder mediated by PD-L1 and CD47, which contains any antibody as described above, in combination with one or more pharmaceutically acceptable excipients.

In some embodiments, the pharmaceutical composition of the present invention is for the prevention or treatment of a disease or disorder mediated by PD-L1 and CD47 selected from the group: HNSCC (head and neck squamous cell carcinoma), cervical cancer, cancer without an identified primary source, 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 high microsatellite instability), leukemia (acute or myeloid), lymphoma, multiple myeloma, breast cancer, prostate cancer, sarcoma, Hodgkin's lymphoma, T- and B-cell acute lymphoblastic leukemia, small cell lung cancer, acute myeloid leukemia, refractory B-cell non-Hodgkin's lymphoma, follicular lymphoma, B-cell marginal zone lymphoma, large B-cell diffuse lymphoma, pancreatic cancer prostate cancer, ovarian cancer, and high-risk myelodysplastic syndrome.

In one aspect, the present invention relates to a method of treating a disease or disorder mediated by PD-L1 and CD47, which includes administering to a subject any antibody of the above or a pharmaceutical composition of this invention in need of such treatment, in a therapeutically effective amount.

In some embodiments of the method of treatment according to the present invention, the disease or disorder is selected from the group: HRNC (head and neck squamous cell carcinoma), cervical cancer, cancer without an identified primary source, 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 (high microsatellite instability colorectal cancer), leukemia (acute or myeloblastic), lymphoma, multiple myeloma, breast cancer prostate cancer, bladder cancer, sarcoma, hepatocellular carcinoma, glioblastoma, Hodgkin's lymphoma, T- and B-cell acute lymphoblastic leukemia, small cell lung cancer, acute myeloid leukemia, refractory B-cell non-Hodgkin's lymphoma, follicular lymphoma, B marginal zone lymphoma, large B-cell diffuse lymphoma, pancreatic cancer, ovarian cancer chnik, and high-risk myelodysplastic syndrome.

In one aspect, the present invention relates to a method of inhibiting the biological activity of PD-L1 and/or CD47 in a subject in need of such inhibition, which includes administering to the subject an effective amount of any antibody from the above.

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

In some embodiments of the antibody of the present invention, the disease or disorder is selected from the group: HNSC (head and neck squamous cell carcinoma), cervical cancer, cancer without an identified primary source, 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 (high microsatellite instability colorectal cancer), leukemia (acute or myeloblastic), lymphoma, multiple myeloma, breast cancer prostate cancer, bladder cancer, sarcoma, hepatocellular carcinoma, glioblastoma, Hodgkin's lymphoma, T- and B-cell acute lymphoblastic leukemia, small cell lung cancer, acute myeloid leukemia, refractory B-cell non-Hodgkin's lymphoma, follicular lymphoma, B- marginal zone cell lymphoma, large B-cell diffuse lymphoma, pancreatic cancer, ovarian cancer chnik, and high-risk myelodysplastic syndrome.

Brief description of the drawings

Fig. 1. Plasmid map for transient production of human CD47-Fc in CHO-K1 mammalian cell culture.

Fig. 2. Non-reducing SDS gel electrophoresis of a human CD47-Fc preparation.

Fig. 3. SDS gel electrophoresis under reducing conditions of the preparation of the control anti-CD47 antibody B6H12

Fig. 4. SDS-gel electrophoresis under non-reducing conditions of the preparation of the control anti-CD47 antibody B6H12.

Fig. 5. ELISA plot of polyclonal phage carrying VHH antibody fragments specifically interacting with the human CD47 antigen.

Fig. 6. Schematic representation of the domain structure of anti-PD-L1/anti-CD47 bispecific antibodies, where A is based on anti-CD47 scFv fragments and B is based on anti-CD47 VHH fragments. In this case, the PD-L1 binding part is presented as a Fab fragment.

Fig. 7. Non-reducing SDS gel electrophoresis of anti-PD-L1/anti-CD47 bispecific antibodies based on anti-CD47 scFv fragments.

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

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

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

Fig. 11. Efficiency of phagocytosis of MDA-MB-231 cells by human macrophages in the presence of anti-PD-L1/anti-CD47 bispecific antibodies.

Fig. 12. Dependence of the fluorescence level on the concentration of anti-PD-L1/anti-CD47 bispecific antibodies.

Fig. 13. Dependence of the fluorescence level on the concentration of anti-PD-L1/anti-CD47 bispecific antibodies.

Fig. 14. Dependence of the fluorescence level on the concentration of anti-PD-L1/anti-CD47 bispecific antibodies.

Fig. 15. Dependence of the fluorescence level on the concentration of anti-PD-L1/anti-CD47 bispecific antibodies.

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

Fig. 17. Gel filtration profile for assessing the aggregation homogeneity of an anti-PD-L1/anti-CD47 bispecific antibody.

Description of the invention

Definitions and Common Methods

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

In addition, unless the context requires otherwise, terms in the singular include terms in the plural, and terms in the plural include terms in the singular. The generally used classification and methods of cell culture, molecular biology, immunology, microbiology, genetics, analytical chemistry, organic synthesis chemistry, medicinal and pharmaceutical chemistry, as well as hybridization and protein and nucleic acid chemistry described herein are well known to those skilled in the art and widely used in this area. Enzymatic reactions and purification methods are carried out according to the manufacturer's instructions, as is commonly done in the art, or as described herein.

Antibody Related Definitions

PD-L1 (programmed cell death receptor ligand 1), also known as cluster of differentiation 274 (CD274) or B7 homologue 1 (B7-H1), is a 40 kDa type 1 transmembrane protein. It consists of 3 domains: extracellular, represented by Ig V and C-like domains (220), transmembrane (21) and intracellular (31). It plays an important role in the suppression of the immune system during pregnancy, in transplantation of foreign tissue, and in certain diseases, such as hepatitis. Under normal conditions, in response to self antigens, a certain amount of antigen-specific CD8 + T-effector cells accumulate in the lymph nodes and spleen, in order to prevent the autoimmune process, PD-1 / PD-L1 or B7-1 / PD-L1 complexes are formed. , this leads to the transmission of an inhibitory signal that reduces the proliferation of these CD8+ T cells in the lymph nodes. Thus, the PD-1/PD-L interaction is one of the key in the development of immune tolerance.

CD47, a multiple membrane-spanning transmembrane receptor belonging to the immunoglobulin superfamily, interacts with SIRPα (signal-regulating protein α) on macrophages and thereby impairs phagocytosis. Cancer cells in which this pathway works avoid phagocytosis. Therefore, the therapeutic effect on CD47 is widely used in various cancers. Anti-CD47 antibodies may or may not be able to block the interaction between CD47 and SIRPα.

The term "binding molecule" includes antibodies and immunoglobulins.

The term "antibody" or "immunoglobulin" or "monoclonal antibody" or "bispecific antibody" or "multispecific antibody" (Ig), as used herein, includes the whole/full-length antibody or any antigen-binding fragment (i.e., "antigen-binding portion "). In addition, for example, the terms "antibody" or "immunoglobulin" or "monoclonal antibody" include any combination of antigen-binding fragments, one or more valencies and one or more specificities, and immunoglobulin constant regions, and may be similar in meaning to the terms "bispecific antibody". or "multispecific antibody". In addition, for example, the terms "antibody" or "immunoglobulin" or "monoclonal antibody" include any combination of antigen-binding fragments and immunoglobulin constant regions associated and covalently or non-covalently with any polypeptide of any nature. In addition, the term "antibody", for example, refers to a glycoprotein containing at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or an antigen-binding portion thereof. Each heavy chain contains a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. Five types of heavy chains of mammalian antibodies are known, which are denoted by Greek letters: α, δ, ε, γ and µ. The type of heavy chain present determines the class of the antibody; these chains are found in antibodies of the IgA, IgD, IgE, IgG and IgM types, respectively. Various heavy chains differ in size and composition; α and γ contain approximately 450 amino acids, while µ and ε consist of approximately 550 amino acids. Each heavy chain contains two regions, i.e. constant region and variable region. The constant region is identical in all antibodies of the same isotype, but differs in antibodies of different isotypes. Heavy chains γ, α and δ contain a constant region that consists of three constant domains CH1, CH2 and CH3 (lined up) and a hinge region that provides flexibility (Woof J., Burton D., Nat Rev Immunol 4, 2004, pp.89-99); heavy chains µ and ε contain a constant region, which consists of four constant domains CH1, CH2, CH3 and CH4. In mammals, only two types of light chains are known, which are referred to as lambda (λ) and kappa (κ). Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The approximate length of the light chain is 211-217 amino acids. Preferably the light chain is a kappa (κ) light chain and the CL constant domain is preferably C-kappa (κ).

"Antibodies" according to the invention may be antibodies 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).

The VH and VL regions can be further subdivided into regions of hypervariability, called complementarity determining regions (CDRs), scattered between regions that are more conserved, called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged from amino to carboxy in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. Antibody constant regions can mediate immunoglobulin binding to host tissues or factors, including various cells of the immune system (eg, effector cells), and the first component (Clq) of the classical complement system.

The term "antigen-binding portion" of an antibody or "antigen-binding fragment" (or simply "antibody portion" or "antibody fragment"), as used herein, refers to one or more antibody fragments 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 in the term "antigen-binding portion" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of VL, VH, CL, and CH 1 domains; (ii) an F(ab')2 fragment, a bivalent fragment, containing two Fab fragments linked by a disulfide bridge in the hinge region, (iii) an Fd fragment consisting of VH and CH 1 domains, (iv) an Fv fragment consisting of VL and VH domains in a single antibody arm, (v) dAb -fragment (Ward et al., (1989) Nature 341:544-546), which consists of a VH/VHH domain, and (vi) a distinct complementarity determining region (CDR). VHs are encoded by different genes, they can be connected by recombinant methods using a synthetic linker that allows them to be produced as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single-chain Fv (scFv); see, for example, Bird et al (1988) Science 242:423-426 ; and Huston et al. (1988) Proc. Natl. Acad. sci. USA 85:5879-5883). It is contemplated 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.

Preferably, the CDR of the antigen-binding site or the entire antigen-binding site of the antibodies of the invention is of mouse, llama, or human donor library origin, or is essentially of human origin with certain amino acid residues modified, e.g. , koff, IC50, EC50, ED50. Preferably, the framework regions of an antibody of the invention are of human origin or substantially human origin (at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% human origin).

In other embodiments, the antigen-binding site of an antibody of the invention may be from other non-human species, including, but not limited to, mouse, llama, rabbit, rat, or hamster. Alternatively, the antigen binding site may be from a human species.

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

The term "hypervariable region" as used herein refers to the amino acid residues of an antibody that are responsible for antigen binding. Typically, a hypervariable region contains amino acid residues from a "complementarity determining region" or "CDR" and/or such residues from a "hypervariable loop".

In some cases, it may also be preferable to change one or more amino acid residues of the CDR regions in order to increase the binding affinity for the target epitope. This is known as "affinity maturation" and in some cases can be performed in connection with humanization, for example, in situations where humanization of an antibody results in a decrease in binding specificity or affinity, and it is not possible to sufficiently improve the binding specificity or affinity using only back mutations. Various methods for affinity maturation are known in the art, for example, 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 proposed by Wu et al., Proc Natl Acad Sci USA 95:6037 6042 (1998).

"Framework regions" (FRs) are variable domain residues other than CDR residues. Typically, each variable domain has four FRs, defined as FR1, FR2, FR3, and FR4. If CDRs are determined according to Kabat, FR residues of the light chain are located approximately in the region of residues 1-23 (LCFR1), 35-49 (LCFR2), 57-88 (LCFR3) and 98-107 (LCFR4), and FR residues of the heavy chain are located approximately at residues 1-30 (HCFR1), 36-49 (HCFR2), 66-94 (HCFR3) and 103-113 (HCFR4) in the heavy chain. If CDR regions contain amino acid residues from hypervariable loops, light chain FR residues are located approximately at residues 1-25 (LCFR1), 33-49 (LCFR2), 53-90 (LCFR3) and 97-107 (LCFR4) in the light chain , and heavy chain FR residues are located approximately at residues 1-25 (HCFRI), 33-52 (HCFR2), 56-95 (HCFR3) and 102-113 (HCFR4) in heavy chain residues. In some examples, when the CDR contains amino acids from both the Kabat CDR and amino acids from the hypervariable loop, the FRs are adjusted accordingly. For example, when CDRH1 includes the amino acids H26-H35, heavy chain FR1 residues are at positions 1-25 and FR2 residues are at positions 36-49.

An antibody of this invention that "binds" a target antigen is an antibody that binds the antigen with sufficient affinity such that the antibody can be used as a diagnostic and/or therapeutic agent when targeted to a protein or cell or tissue expressing the antigen, and slightly cross-reacts with other proteins. According to analytical methods: fluorescence-activated cell sorting (FACS), radioimmunoprecipitation (RIA) or ELISA (ELISA), in such embodiments of the invention, the degree of binding of an antibody to a protein that is not a “target” (“non-target protein”) is less than 10 % of antibody binding to a specific target protein. In relation to the binding of an antibody to a target molecule, the term "specific binding" or the expressions "specifically binds to" or "specific to" a particular polypeptide or epitope on a particular target polypeptide means a binding that is appreciably (measurably) different from a non-specific interaction (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).

Specific binding can be quantified, for example, by determining the binding of the molecule compared to the binding of a control molecule. For example, specific binding can be determined by a competitive reaction with another molecule similar to the target, for example, with an excess of unlabeled target. In this case, specific binding is indicated if binding of the labeled target to the probe is competitively inhibited by an excess of unlabeled target. As used herein, the term "specific binding" or the expressions "specifically binds to" or "specific to" a particular polypeptide or epitope on a particular target polypeptide can be characterized by an example of a molecule having a Kd to the target of at least about 200 nM, or at least 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 higher. In one embodiment, the term "specific binding" refers to a binding in which a molecule binds to a particular polypeptide or epitope on a particular polypeptide without substantially binding to any other polypeptide or epitope on a polypeptide.

The term "Ka" as used herein refers to the rate of association of a particular antibody-antigen interaction, while the term or "Kd" refers to the rate of dissociation of a particular antibody-antigen interaction.

"Binding affinity" generally refers to the strength of the cumulative non-covalent interactions between a single binding site of a molecule (eg, an antibody) and its binding partner (eg, an antigen). Unless otherwise indicated, "binding affinity" refers to intrinsic (intrinsic, intrinsic) binding affinity, which reflects a 1:1 interaction between members of a binding pair (eg, antibody and antigen). The affinity of a molecule X for its partner Y can usually be represented by a dissociation constant (Kd). Desirably, 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 conventional methods known in the art, including the methods described herein. Low affinity antibodies usually bind antigen slowly and tend to dissociate easily, while high affinity antibodies usually bind antigen faster and tend to stay bound longer. Various methods for measuring binding affinity are known in the art, any of these methods can be used for the purposes of the present invention.

In one embodiment of the invention, the "Kd" or "Kd value" of this invention is measured by surface plasmon resonance methods on a BIAcore™-2000 or BIAcore™-3000 instrument (BIAcore, Inc., Piscataway, NJ) at 25°C using chips with immobilized CM5 antigen at ~10 relative units (response units, RU). Briefly, carboxymethyldextran 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. The antigen is diluted with 10 mM sodium acetate pH 4.8 to a concentration of 5 μg/mL (~0.2 μM) and then injected at a flow rate of 5 μL/minute until about 10 relative units (RU) of bound protein is reached. After the introduction of the antigen, a 1 M solution of ethanolamine is injected to block the unreacted groups. For kinetic measurements, two-fold serial dilutions of Fab (eg, 0.78 nM to 500 nM) are injected into PBS with 0.05% Tween 20 (PBST) at 25° C. at a flow rate of about 25 μl/min. Association rate (kon) and dissociation rate (koff) values are calculated using a simple Langmuir model for one-plus-one binding (BIAcore Evaluation Software version 3.2) by simultaneously obtaining an association and dissociation sensorgram. The equilibrium dissociation constant (Kd) is calculated as the ratio koff/kon. See, for example, Chen, Y., et al., (1999) J. Mol. Biol. 293:865-881. If the association rate exceeds 10 ⁶ M ^-1 s ^-1 according to the above surface plasmon resonance method, then it can be determined by the fluorescence quenching method, which measures the increase or decrease in the intensity of fluorescent emission (excitation = 295 nm; emission (emission) = 340 nm, band 16 nm) at 25° C. of a 20 nM anti-antigen antibody (Fab form) solution in PBS, pH 7.2, in the presence of increasing concentrations of antigen measured with a spectrometer such as a stopped flow spectrophotometer (Aviv Instruments) or an SLM-spectrophotometer. Aminco (ThermoSpectronie) 8000 series with agitated cuvette.

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

The "on-rate" or "kon" of this invention can also be determined by the same surface plasmon resonance method described above on a BIAcore™-2000 or BIAcore™-3000 instrument (BIAcore, Inc., Piscataway, NJ ) at 25°C using chips with immobilized CM5 antigen at ~10 relative units (response units, RU). Briefly, carboxymethyldextran 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. The antigen is diluted with 10 mM sodium acetate pH 4.8 to a concentration of 5 μg/mL (~0.2 μM) and then injected at a flow rate of 5 μL/minute until about 10 relative units (RU) of bound protein is reached. After the introduction of the antigen, a 1 M solution of ethanolamine is injected to block the unreacted groups.

Unless specifically stated otherwise, the expressions "biologically active" and "biological activity" and "biological characteristics", in relation to the polypeptide of this invention, means having the ability to bind to a biological molecule.

The expression "biological molecule" refers to a nucleic acid, protein, carbohydrate, lipid, and combinations thereof. In one embodiment of the invention, the biological molecule exists in nature.

Antibody regions such as Fab and F(ab') 2 fragments can be prepared from whole antibodies using conventional methods such as papain or pepsin digestion of whole antibodies. Moreover, antibodies, antibody moieties, and immunoadhesion molecules can be prepared using standard recombinant DNA techniques, for example, as described herein.

The term "recombinant antibody" means an antibody that is expressed in a cell or cell line containing a nucleotide sequence(s) that encodes antibodies, said nucleotide sequence(s) not naturally associated with the cell.

The term "variant" antibody as used herein refers to an antibody having an amino acid sequence that differs from the amino acid sequence of its "parent" antibody by adding, deleting, and/or substituting one or more amino acid residues relative to the sequence of the parent antibody. In a preferred embodiment, the variant antibody contains 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; and in some embodiments implementation of the invention from one to about ten) additions, deletions and/or substitutions of amino acids relative to the parent antibody. In some embodiments, additions, deletions and/or substitutions are made to the CDR regions of the variant antibody. Sequence identity or homology to a variant antibody is defined herein as the percentage of amino acid residues in a variant antibody sequence that are identical to those of the parent antibody, after sequence alignment and gaps, if necessary, to achieve the maximum percent sequence identity. The variant antibody retains the ability to bind to the same antigen, and preferably epitope, to which the parent antibody binds, and in some embodiments of the invention, at least one property or biological activity is superior to that of the parent antibody. For example, a variant antibody may have, for example, greater binding affinity, a longer half-life, a lower IC50 value, or an increased ability to inhibit the biological activity of an antigen compared to the parent antibody. Of particular interest herein is a variant antibody exhibiting a biological activity greater than at least 2-fold (preferably at least 5-fold, 10-fold, or 20-fold) that of the parent antibody.

The term "bispecific antibody" means an antibody containing an antigen-binding domain or antigen-binding domains that are capable of specifically binding to two different epitopes on the same biological molecule, or capable of specifically binding to epitopes on two different biological molecules. A bispecific antibody is also referred to herein as having "dual specificity" or as being a "dual specific" antibody.

The term "chimeric antibody" refers broadly to an antibody that contains one or more regions from one antibody and one or more regions from one or more other antibodies, typically an antibody of part human and part non-human origin, i.e. partly from a non-human animal such as a mouse, rat or other rodent, or a camelid such as a llama or alpaca. Chimeric antibodies are preferred over non-human antibodies in order to reduce the risk of an immune response directed against antibodies in humans, for example, a response directed against mouse antibodies in humans in the case of a mouse antibody. An example of a typical chimeric antibody is one in which the variable region sequences are murine while the constant region sequences are human. In the case of a chimeric antibody, the non-human portions may be further modified to humanize the antibody.

The term "humanization" refers to the fact that when an antibody is of wholly or partly non-human origin, such as a mouse or llama antibody obtained by immunizing mice or llamas, respectively, with an antigen of interest, or is a chimeric antibody based on such a mouse or llama antibody , you can replace some amino acids, for example, in the framework regions and constant domains of the heavy and light chains, in order to avoid or minimize the immune response in humans. The specificity of the interaction of the antibody with the target antigen is mainly inherent in the amino acid residues located in the six CDR regions of the heavy and light chains. Therefore, the amino acid sequences within the CDRs are much more variable between individual antibodies than those outside the CDRs. Because CDR sequences are responsible for most antibody-antigen interactions, it is possible to express recombinant antibodies that mimic the properties of a specific natural antibody, or more generally any specific antibody with a given amino acid sequence, for example, by constructing expression vectors that express the CDR sequences. -plots from a specific antibody and framework sequences of another antibody. As a result, it is possible to "humanize" a non-human antibody and largely retain the binding specificity and affinity of the original antibody. Although it is not possible to accurately predict the immunogenicity and thus the immune response directed against an antibody in humans to a particular antibody, non-human antibodies are generally more immunogenic than human antibodies. Chimeric antibodies in which foreign (eg, rodent or camel) constant regions have been replaced with sequences of human origin have shown generally lower immunogenicity than antibodies of completely foreign origin, and there is a tendency to use humanized or fully human antibodies in therapeutic antibodies. Chimeric antibodies or other antibodies of non-human origin can thus be humanized to reduce the risk of an immune response directed against the antibody in humans.

For chimeric antibodies, humanization typically involves modification of the framework regions of the variable region sequences. Amino acid residues that are part of the complementarity determining regions (CDRs) will most often not be changed due to humanization, although in some cases it may be desirable to change individual amino acid residues of the CDR region, for example, to remove the glycosylation site, a deamidation site, an aspartate isomerization site, or an unwanted cysteine or methionine residue. N-linked glycosylation occurs by attaching an oligosaccharide chain to an asparagine residue in the Asn-X-Ser or Asn-X-Thr tripeptide sequence, where X can be any amino acid other than Pro. Removal of the N-glycosylation site can be achieved by mutating the Asn or Ser/Thr residue with another residue, preferably by conservative substitution. Deamidation of asparagine and glutamine residues can occur depending on factors such as pH and surface exposure. Asparagine residues are particularly susceptible to deamidation, especially if they are present in the Asn-Gly sequence, and to a lesser extent in other dipeptide sequences such as Asn-Ala. When there is such a deamidated site, for example Asn-Gly in the sequence of the CDR region, it may be preferable to remove this site, usually by conservative substitution to remove one of the residues involved.

Numerous methods for humanizing an antibody sequence are known in the art. One of the most commonly used methods is CDR transplantation. CDR site grafting may be based on Kabat's CDR site definitions, although a more recent publication (Magdelaine-Beuzelin et al., Crit Rev. Oncol Hematol. 64:210 225 (2007)) suggests that the IMGT® (the international ImMunoGeneTics information system®, www.imgt.org) can improve humanization outcome (see Lefranc et al., Dev. Comp Immunol. 27:55-77 (2003)). In some instances, transplantation of a CDR region may reduce the specificity and binding affinity, and therefore biological activity, of a CDR transplanted non-human antibody, compared to the parent antibody from which the CDR regions are derived. Back mutations (sometimes referred to as "frame repair") can be applied at selected CDR positions of the transplanted antibody, typically in the framework regions, in order to restore the specificity and binding affinity of the parent antibody. Determination of positions for possible backmutations can be performed using information available in the literature and in antibody databases. Amino acid residues that are candidates for backmutation are typically located on the surface of the antibody molecule, while residues that are recessed or have low surface exposure will usually not be affected. An alternative humanization method to CDR grafting and backmutation is surface alteration in which non-human residues that are not exposed on the surface are retained while surface-exposed residues are changed into human residues.

There are two technologies for obtaining fully human antibodies: using in vitro assembled phage libraries or in vivo immunization of humanized animals (mice, rats, etc.).

Phage display is the first and most widely used in vitro antibody detection technology. In 1985, Smith discovered that foreign DNA sequences could be cloned into the M13 filamentous bacteriophage in such a way that the cloned gene sequences were expressed as fusion proteins on the surface of the phage particles (Smith GP: Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface Science 1985, 228:1315-1317.). Thus, fusion proteins of interest can be selected based on their ability to bind other proteins. This discovery has been combined with PCR amplification techniques to clone the cDNA repertoire of immunoglobulin genes to create a variety of variable domain-containing phage libraries that can be used to rapidly search for target-specific monoclonal antibodies. The phage library repertoire reflects the B-lymphocyte antibody repertoire of each human or animal whose blood was used to create the library. In 1995, two papers reported the creation of genetically engineered mice that expressed fully human antibodies whose repertoire could be comparable to that obtained 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, the genes of their own endogenous heavy and k light chains of immunoglobulins were purposefully destroyed and transgenes were introduced, which are segments of the human heavy and k light chain genes. It turned out that the human gene repertoire can be used by the mouse immune system to create highly specific and high affinity antibodies to a greater variety of antigens. Although transgenic mice express B-cell receptors that are essentially a hybrid of mouse and human (human immunoglobulin, mouse Igα, Igβ, and other signaling molecules), their B cells develop and mature normally.

In some cases, it may also be preferable to change one or more amino acid residues of the CDR regions in order to increase the binding affinity for the target epitope. This is known as "affinity maturation" and in some cases can be performed in connection with humanization, for example, in situations where humanization of an antibody results in a decrease in binding specificity or affinity, and it is not possible to sufficiently improve the binding specificity or affinity using only back mutations. Various methods for affinity maturation are known in the art, for example, 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 proposed by Wu et al., Proc Natl Acad Sci USA 95:6037 6042 (1998).

The term "monoclonal antibody" or "mAb" refers to an antibody that is synthesized and isolated by a single 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, which are typically obtained by fusing individual B lymphocytes from immunized animals with individual lymphocytic tumor cells. Hybridomas are an engineered cell type and do not occur naturally.

"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 a single covalent disulfide bond, while the number of disulfide bonds between heavy chains varies between immunoglobulin isotypes. Each heavy and light chain also has evenly spaced intrachain disulfide bridges. Each heavy chain has at one end a variable domain (VH) followed by several constant domains. Each light chain has a variable domain at one end (VL) and a constant domain at the other end. The light chain constant domain is aligned with the first heavy chain constant domain, and the light chain variable domain is aligned with the heavy chain variable domain. I believe that specific amino acid residues form the interface between the variable domains of the light chain and heavy chain.

The definition of "isolated" ("isolated"), used to describe the various antibodies according to this description, means an antibody identified and isolated and/or regenerated from the cell or cell culture in which it is expressed. Impurities (contaminants) from the natural environment are materials that typically interfere with the diagnostic or therapeutic use of the polypeptide, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In preferred embodiments, the antibody is purified (1) to a degree sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence using a rotating glass cup sequencer (Edman sequencer), or (2) to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie brilliant blue or preferably silver stain. An isolated antibody includes antibodies in situ within the recombinant cells, since at least one component of the natural environment of the polypeptide is missing. Typically, an isolated polypeptide is obtained by at least one purification step.

An "isolated" nucleic acid molecule is a nucleic acid molecule that has been identified and separated from at least one contaminant nucleic acid molecule with which it is normally associated in the natural source of an antibody nucleic acid. An isolated nucleic acid molecule is different from the form or set in which it occurs naturally. Thus, an isolated nucleic acid molecule is different from a nucleic acid molecule that exists naturally in cells. However, an isolated nucleic acid molecule includes a nucleic acid molecule found in cells in which the antibody is normally expressed, for example, if the nucleic acid molecule has a different chromosomal localization from its natural cell localization.

The term "epitope" as used herein refers to the portion (determinant) of an antigen that specifically binds to a binding molecule (eg, and an antibody or related molecule, such as a bispecific binding molecule). Epitopic determinants usually consist of chemically active surface groups of molecules such as amino acids or carbohydrates or sugar side chains and typically have specific three-dimensional structural characteristics as well as specific charge characteristics. An epitope may be "linear" or "conformational". In a linear epitope, all points of interaction between a protein (such as an antigen) and an interacting molecule (such as an antibody) occur linearly along the protein's primary amino acid sequence. In a conformational epitope, points of interaction occur through amino acid residues on the protein separated from each other in the primary amino acid sequence. Once the desired antigen epitope has been determined, antibodies to that epitope can be generated using techniques well known in the art. In addition, the generation and characterization of antibodies or other binding molecules may shed light on information about desirable epitopes. Based on this information, binding molecules can then be competitively screened for binding to the same or similar epitopes, for example by conducting competition studies to find binding molecules that compete for antigen binding.

The term "peptide linker" in this document means any peptide with the ability to connect domains with a length depending on the domains that it links together, containing any amino acid sequence. Preferably, the peptide linker is greater than 5 amino acids in length and is comprised of any set of amino acids selected from G, A, S, P, E, T, D, K.

The term "in vitro" refers to a biological entity, biological process or biological response outside the body, simulated in vitro. For example, in vitro cell growth should be understood as the growth of cells in an environment outside the body, such as in a test tube, culture flask or microplate.

The term "IC ₅₀ " (50% inhibitory concentration) refers to drug concentrations at which a measurable activity or response, eg growth or proliferation of cells such as tumor cells, is inhibited by 50%. The value of IC ₅₀ can be estimated using the corresponding curves of the dependence of the response on the logarithm of the dose, using special statistical programs for curve processing.

The term GI50 (50% growth inhibition) refers to drug concentrations at which cell proliferation, such as tumor cells, is inhibited by 50%.

The term ED50 (EC50) (50% effective dose/concentration) refers to drug concentrations at which 50% of a measurable biological effect is achieved (may include cytotoxicity).

The term “effector function” of an antibody refers to the biological activities associated with the Fc region (native sequence Fc region or amino acid sequence variants of the Fc region) of an antibody, and vary depending on the isotype of the antibody. Examples of antibody effector functions are: Cl _q - binding; complement dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (eg B cell receptor, BCR) and B cell activation.

"Antibody-dependent cellular cytotoxicity" or "ADCC" refers to a cell-mediated response in which non-specific cytotoxic cells that express Fc receptors (FcR) (e.g., natural killer (NK) cells, neutrophils, and macrophages) recognize bound antibody on target cell and then cause lysis of the target cell. The primary cells for mediating ADCC, NK cells, only express FcγRIII, 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 assess the ADCC activity of a molecule of interest, in vitro ADCC assays can be performed, such as those described in US Pat. . Alternatively or additionally, the ADCC activity of a molecule of interest can be assessed in vivo, for example, in an animal model such as that described in Clynes et al. PNAS (USA) 95: 652-656 (1998).

"Human effector cells" are leukocytes that express one or more FcRs and perform effector functions. Preferably, the cells express at least FcγRIII and perform an ADCC effector function. Examples of human leukocytes that mediate ADCC include peripheral blood mononuclear cells (PBMC), natural killer (NK) cells, monocytes, cytotoxic T cells, and neutrophils; with PBMC and NK cells being preferred. Effector cells can be isolated from their natural source, such as blood or PBMC, as described in this publication.

The terms "Fc receptor" and "FcR" are 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 an IgG antibody (gamma receptor), and preferred receptors include receptors of the subclasses FcγRI, FcγRII (FcγRIIa and FcγRIIb), FcγRIII (FcγRIIIa and FcγRIIIb), including various alleles and alternatively spliced forms of these receptors. FcγRI shows high affinity for IgG, while FcγRII and FcγRIII show weaker affinity. FcγRIIa and FcγRIIIa are activating FcγRs that are expressed on monocytes/macrophages and monocytes/macrophages/natural killer cells, respectively, and can trigger human target cytotoxicity. The activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. The inhibitory receptor FcγRIIB contains in its cytoplasmic domain a tyrosine-based immunoreceptor inhibition motif (ITIM) (reviewed in Daëron, Annu. Rev. Immunol. 15: 203-234 (1997)). For an overview of FcR, see Ravetch and Kinet, Annu. Rev. Immunol 9: 457-92 (1991). Other FcRs, including FcRs that will be identified in the future, are included in the present description in the term "FcR". The term also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgG to the fetus.

"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 (eg, antibody) in complex with its antigen. To evaluate complement activation, a CDC assay can be performed, for example, as described in Gazzano-Santoro et al., J. Immunol. Methods 202: 163 (1996).

The term "identity" or "homology" should be interpreted as meaning the percentage of amino acid residues in a candidate sequence that are identical to those of the corresponding sequence to which it is being compared, after comparing the sequences and introducing "gaps", if it is necessary to achieve the maximum percent identity for the complete sequence and not considering any conservative substitutions as part of the sequence identity. Neither N- or C-terminal extensions nor insertion segments should be interpreted as reducing identity or homology. Methods and computer programs for comparison are well known. Sequence identity can be determined using sequence analysis software (eg, Sequence Analysis Software Package, Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Ave., Madison, WI 53705). This software is suitable for such sequences by determining the degree of homology for a variety of substitutions, deletions (eliminations) and other modifications.

The phrase "homologous", with respect to the polypeptide sequence of an antibody, should be interpreted as an antibody showing at least 70%, preferably 80%, more preferably 90% and most preferably 95% sequence identity relative to the polypeptide sequence. . The term in relation to a nucleic acid sequence should be interpreted as a sequence of nucleotides showing at least 85%, preferably 90%, more preferably 95% and most preferably 97% sequence identity relative to the nucleic acid sequence.

Proposed modification(s) of the amino acid sequences of the antibodies described in this publication. For example, it may be desirable to improve the binding affinity and/or other biological properties of an antibody. Amino acid sequence variants of an antibody are generated 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 in the amino acid sequences of the antibody. Any combination of deletion, insertion and substitution is carried out to obtain the final construct, provided that the final construct has the required characteristics. Amino acid changes can also change post-translational processes in the antibody, such as changing the number or position of glycosylation sites.

Modification of the amino acid sequences of antibodies using amino acid substitutions. Such a variant is the replacement of at least one amino acid residue in the antibody molecule with another residue. The sites of greatest interest for substitution mutagenesis include the hypervariable regions or CDRs, but changes in the FR or Fc region are also contemplated. Conservative substitutions are shown in Table A under the heading "preferred substitutions". If such substitutions result in a change in biological activity, then additional substantive changes may be introduced, referred to as "examples of substitutions" in Table A, or changes further described below in the description of amino acid classes, and products may be screened.

Table A original balance Replacement examples Preferred Substitutions Ala (A) Val; Leu; ile Val Arg(R) Lys; gin; Asn Lys Asn(N) gin; His; Asp, Lys; Arg gin Asp(D) Glu; Asn Glu Cys(C) Ser; Ala Ser Gln(Q) asn; Glu Asn Glu(E) asp; gin asp Gly(G) Ala Ala His(H) asn; gin; Lys; Arg Arg Ile (I) Leu; Val; met; Ala; Ph; Norleucine Leu Leu(L) Norleucine; Ile; Val; met; Ala; Phe ile Lys (K) Arg; gin; Asn Arg Met(M) Leu; Ph; ile Leu Phe(F) trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser(S) Thr Thr Thr(T) Val; Ser Ser Trp(W) Tyr; Phe Tyr Tyr(Y) trp; Ph; Thr; Ser Phe Val(V) Ile; Leu; met; Ph; Ala; Norleucine Leu

The terms "nucleic acid", "nucleic acid sequence" or "nucleic acid sequence", "polynucleotide", "oligonucleotide", "polynucleotide sequence" and "nucleotide sequence", which are used interchangeably in this specification, refer to a distinct sequence of nucleotides, whether or not modified , which defines a fragment or region of a nucleic acid, containing or not containing non-natural nucleotides and which is either double-stranded DNA or RNA, or single-stranded DNA or RNA, or transcription products of these DNA.

It should also be mentioned here that the present invention does not apply to nucleotide sequences in their natural chromosomal environment, i.e. in a natural state. The sequences of the present invention have been isolated and/or purified, i. were taken directly or indirectly, for example, by copying, while their environment was at least partially modified. Thus, isolated nucleic acids obtained by genetic recombination, for example by means of host cells, or obtained by chemical synthesis, are also to be understood here.

Reference to a nucleotide sequence embraces its complement unless otherwise indicated. Thus, reference to a nucleic acid having a particular sequence is to be understood as encompassing its complementary strand with its complementary sequence.

The expression "control sequences" refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. Control sequences suitable for prokaryotes are, for example, a promoter, optionally an operator, and a ribosome binding site. As is known, promoters, polyadenylation signals, and enhancers are present in eukaryotic cells.

A nucleic acid is "operably linked" if it is in a functional relationship with another nucleotide sequence. For example, a DNA presequence or secretory leader sequence is operably linked to the DNA of a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; a ribosome binding site is operably linked to a coding sequence if it is located such that it can facilitate translation. Typically, "operably linked" means that the linked DNA sequences are contiguous, and in the case of a secretory leader sequence, are contiguous and in read phase. However, enhancers do not need to be contiguous.

The term "vector" as used herein means a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. In some embodiments, the vector is a plasmid, i.e. a circular double-stranded piece of DNA into which additional DNA segments can be ligated. In some embodiments, the vector is a viral vector in which additional DNA segments can be ligated into the viral genome. In some embodiments, the vectors are capable of autonomous replication in the host cell into which they are introduced (eg, bacterial vectors having a bacterial origin of replication and mammalian episomal vectors). In other embodiments, the vectors (eg, non-episomal mammalian vectors) can be integrated into the genome of a host cell when introduced into the host cell, and thus replicate along with the host's genome. Moreover, some vectors are capable of directing the expression of the genes to which they are operably linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors").

The term "recombinant host cell" (or simply "host cell") as used herein means a cell into which a recombinant expression vector has been introduced. The present invention relates to host cells, which may include, for example, a vector in accordance with the present invention described above. The present invention also relates to host cells that include, for example, a nucleotide sequence encoding a heavy chain or antigen-binding portions thereof, a nucleotide sequence encoding a light chain or antigen-binding portions thereof, or both, a first binding domain and/or a second binding domain binding molecule according to this invention. It should be understood that "recombinant host cell" and "host cell" mean not only the specific claimed cell, but also the progeny of such a cell. Because modifications can take place in subsequent generations due to mutation or environmental influences, such progeny may not actually be identical to the parent cell, but such cells are still included within the scope of the term "host cell" as used herein.

The term "excipient" is used herein to describe any ingredient other than the compound(s) of this invention.

"Pharmaceutical composition" means a composition comprising an antibody according to the invention and at least one of the components selected from the group consisting of pharmaceutically acceptable and pharmacologically compatible excipients, solvents, diluents, carriers, auxiliary, distributing and receptive agents, means such as preservatives, stabilizers, fillers, grinders, humectants, emulsifiers, suspending agents, thickeners, sweeteners, flavors, flavors, antibacterial agents, fungicides, lubricants, sustained delivery regulators, the choice and ratio of which depends on the nature and method of administration and dosage . Examples of suspending agents are ethoxylated isostearyl alcohol, polyoxyethylene, sorbitol and sorbitol ether, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, as well as mixtures of these substances. Protection against the action of microorganisms can be provided with a variety of antibacterial and antifungal agents, such as parabens, chlorobutanol, sorbic acid, and the like. The composition may also include isotonic agents such as sugars, polyols, sodium chloride and the like. Prolonged action of the composition can be provided with agents that slow down the absorption of the active principle, for example, aluminum monostearate and gelatin. Examples of suitable carriers, solvents, diluents and delivery vehicles are water, ethanol, polyalcohols and mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters (such as ethyl oleate). Examples of fillers are lactose, milk sugar, sodium citrate, calcium carbonate, calcium phosphate and the like. Examples of grinders and spreaders are starch, alginic acid and its salts, silicates. Examples of lubricants are magnesium stearate, sodium lauryl sulfate, talc, and high molecular weight polyethylene glycol. Pharmaceutical composition for oral, sublingual, transdermal, intraocular, intramuscular, intravenous, subcutaneous, topical or rectal administration of the active principle, alone or in combination with another active principle, can be administered to animals and humans in a standard form of administration as a mixture with conventional pharmaceutical carriers . Suitable unit administration forms include oral forms such as tablets, gelatin capsules, pills, powders, granules, chewing gums and oral solutions or suspensions, sublingual and buccal administration forms, aerosols, implants, topical, transdermal, subcutaneous, intramuscular, intravenous, intranasal or intraocular administration forms and rectal administration forms.

"Drug (preparation)" - a substance (or a mixture of substances in the form of a pharmaceutical composition) in the form of tablets, capsules, solutions, ointments and other finished forms, intended to restore, correct or change physiological functions in humans and animals, as well as for treatment and prevention of diseases, diagnostics, anesthesia, contraception, cosmetology and other things.

The term "disease or disorder mediated by CD47 and PD-L1" means all diseases or disorders that are either directly or indirectly associated with CD47 and PD-L1, including the etiology, development, progression, persistence or pathology of the disease or disorder. "Treat", "treatment", and "therapy" refer to a method of alleviating or eliminating a biological disorder and/or at least one of its accompanying symptoms. As used herein, the term "alleviate" a disease, disease, or condition means to reduce the severity and/or frequency of occurrence of symptoms of the disease, disorder, or condition. In addition, references herein to "treatment" include references to curative, palliative, and prophylactic therapy.

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

The term "disorder" means any condition that can be improved as a result of the treatment of the present invention. The definition of this term includes chronic and acute disorders or diseases, including pathological conditions that predispose a mammal to the occurrence of this disorder. The preferred disorder to be treated according to the invention is cancer.

The terms "cancer" or "cancerous" refer to or describe a physiological condition in mammals that is typically characterized by unregulated cell growth/proliferation. This definition covers benign and malignant cancers. Examples of cancers include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More specific examples of such cancers include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, squamous cell lung carcinoma, peritoneal cancer, hepatic cell carcinoma, gastric cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, glioma, cervical cancer, cancer ovarian cancer, liver cancer, bladder cancer, breast cancer, colon cancer, colorectal cancer, endometrial and uterine carcinoma, salivary gland carcinoma, kidney cancer (renal cell carcinoma), prostate cancer (prostate cancer), cancer of the external female genital organs, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, melanoma, and various types of head and neck cancer.

The terms "immune response", "autoimmune reaction", "autoimmune inflammation" refer, for example, to the action of lymphocytes, antigen presenting cells, phagocytic cells, granulocytes, and soluble macromolecules produced by said or liver cells (including antibodies, cytokines, and complement, resulting from the selective damage, destruction, or elimination from the human body of invasive pathogens, pathogen-infected cells or tissues, cancer cells, or, in cases of autoimmunity or pathological inflammation, normal human cells or tissues).

A "therapeutically effective amount" is the amount of a therapeutic agent administered during treatment that will relieve to some extent one or more of the symptoms of the disease being treated.

The term "chronic" use refers to the continuous (long-term) use of the agent(s), as opposed to the acute (short-term) route of administration, so as to maintain the initial therapeutic effect (activity) over a long period of time.

"Intermittent" use means a treatment that is not carried out consecutively without interruption, but which is rather intermittent in nature.

In the present description and in the following claims, unless the context otherwise requires, the words "have", "include" and "comprise" or variations thereof, such as "has", "having", "includes", "including", " contains" or "comprising" should be understood to mean the inclusion of the specified integer or group of integers, but not the exclusion of any other integer or group of integers.

Detailed description of the invention

Antibody

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

In one of the embodiments of the present invention, the antibody is a full-length antibody or antigen-binding fragment

In one of the embodiments of the present invention, the antibody includes one or two binding sites for PD-L1.

In one embodiment, the CD47 binding site inhibits the interaction of the CD47 receptor and the SIRPα ligand, and/or the PD-L1 binding site inhibits the interaction of PD-L1 with the PD-1 receptor.

In one embodiment, the present invention relates to an antibody that binds CD47 and PD-L1 and whose CD47 binding site includes a heavy chain variable region comprising:

(a) a CDR1 with an amino acid sequence at least 80% or 90% homologous or identical to the sequence selected from the group SEQ ID NO: 1-4, i.e. the CDR1 is a sequence selected from the group SEQ ID NO: 1-4 or a sequence selected from the group SEQ ID NO: 1 - 4 with 1 or 2 substitutions;

(b) a CDR2 with an amino acid sequence of at least 80%, 84%, 86%, 88%, 92% or 96% homologous or identical to the sequence selected from the group SEQ ID NO: 6 to 15, i.e. CDR2 is the sequence SEQ ID NO: 6-15 or a sequence selected from the group of SEQ ID NO: 6-15 with 1, 2, 3, 4 or 5 substitutions;

(c) a CDR3 with an amino acid sequence of at least 80%, 85%, 86% 90%, 93% or 95% homologous or identical to a sequence selected from the group of SEQ ID NO: 17-20, i.e. CDR3 is a sequence, selected from the group of SEQ ID NO: 17 - 20 or a sequence selected from the group of SEQ ID NO: 17 - 20 with 1, 2 or 3 substitutions.

In one embodiment, the present invention relates to an antibody that binds CD47 and PD-L1 and whose CD47 binding site includes a heavy chain variable region comprising:

(d) CDR1 with an amino acid sequence identical to the sequence selected from the group of SEQ ID NO: 1 - 4;

(e) CDR2 with an amino acid sequence identical to the sequence selected from the group SEQ ID NO: 6 - 15;

(f) CDR3 with an amino acid sequence identical to the sequence selected from the group of SEQ ID NO: 17 - 20.

In one embodiment, the present invention provides an antibody that binds CD47 and PD-L1 and includes a CD47 binding site comprising:

(a) a heavy chain variable region comprising:

(i) a CDR1 with an amino acid sequence at least 80% or 90% homologous or identical to the sequence selected from the group of SEQ ID NO: 1-4, i.e. the CDR1 is a sequence selected from the group of SEQ ID NO: 1-4, or a sequence selected from the group SEQ ID NO: 1 - 4 with 1 or 2 substitutions;

(ii) a CDR2 with an amino acid sequence of at least 80%, 84%, 86%, 88%, 92% or 96% homologous or identical to the sequence selected from the group of SEQ ID NO: 6 to 15, i.e. CDR2 is the sequence SEQ ID NO: 6 - 15 or a sequence selected from the group of SEQ ID NO: 6 - 15 with 1, 2, 3, 4 or 5 substitutions,

(iii) a CDR3 with an amino acid sequence of at least 80%, 85%, 86%, 90%, 93% or 95% homologous or identical to the sequence selected from the group SEQ ID NO: 17-20, i.e. CDR3 is the sequence selected from the group of SEQ ID NO: 17-20 or a sequence selected from the group of SEQ ID NO: 17-20 with 1, 2 or 3 substitutions, and

(b) a light chain variable region comprising:

(i) a CDR1 with an amino acid sequence of at least 80% or 90% homology or identity to a sequence selected from the group of SEQ ID NOs: 22-34, i.e. the CDR1 is a sequence selected from the group of SEQ ID NOs: 22-34, or a sequence selected from the group SEQ ID NO: 22 - 34 with 1 or 2 substitutions,

(ii) a CDR2 with an amino acid sequence of at least 80%, 87% or 94% homologous or identical to the sequence selected from the group of SEQ ID NO: 36 - 48, i.e. CDR2 is a sequence selected from the group of SEQ ID NO: 36 - 48 or a sequence selected from the group of SEQ ID NO: 36 - 48 with 1, 2 or 3 substitutions,

(iii) a CDR3 with an amino acid sequence at least 80% or 90% homologous or identical to a sequence selected from the group of SEQ ID NO: 50-64, i.e. CDR3 is a sequence selected from the group of SEQ ID NO: 50-64 or a sequence selected from the group of SEQ ID NO: 50 - 64 with 1 or 2 substitutions.

In one embodiment, the present invention provides an antibody that binds CD47 and PD-L1 and includes a CD47 binding site comprising:

(a) a heavy chain variable region comprising:

(i) a CDR1 with an amino acid sequence selected from the group of SEQ ID NOs: 1 to 4,

(ii) a CDR2 with an amino acid sequence selected from the group of SEQ ID NO: 6-15,

(iii) a CDR3 with an amino acid sequence selected from the group of SEQ ID NOs: 17-20, and

(b) a light chain variable region comprising:

(i) a CDR1 with an amino acid sequence selected from the group of SEQ ID NOs: 22-34,

(ii) a CDR2 with an amino acid sequence selected from the group of SEQ ID NOs: 36-48,

(iii) a CDR3 with an amino acid sequence selected from the group of SEQ ID NOs: 50-64.

In one embodiment, the present invention provides an antibody that binds CD47 and PD-L1 and includes a CD47 binding site comprising:

(a) a heavy chain variable region with an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous or identical to a sequence selected from group SEQ ID NO: 66 - 88, and

(b) a light chain variable region with an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous or identical to a sequence selected from group SEQ ID NO: 89-106.

In one embodiment, the present invention provides an antibody that binds CD47 and PD-L1 and includes a CD47 binding site comprising:

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

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

In one embodiment, the present invention provides an antibody that binds CD47 and PD-L1 and includes a PD-L1 binding site comprising:

(a) a heavy chain variable region comprising:

(i) a CDR1 with an amino acid sequence at least 80% homologous or identical to the sequence of SEQ ID NO: 5, that is, the CDR1 is the sequence of SEQ ID NO: 5 or the sequence of SEQ ID NO: 5 with 1 substitution;

(ii) a CDR2 with an amino acid sequence at least 80%, 86% or 92% homologous or identical to the sequence of SEQ ID NO: 16, i.e. CDR2 is the sequence of SEQ ID NO: 16 or the sequence of SEQ ID NO: 16 with 1, 2 or 3 replacements; (iii) a CDR3 with an amino acid sequence of at least 80% or 90% homology or identity to SEQ ID NO: 21, i.e. CDR3 is SEQ ID NO: 21 or SEQ ID NO: 21 with 1 or 2 substitutions , and

(b) a light chain variable region comprising:

(i) a CDR1 with an amino acid sequence of 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 with 1, 2 or 3 replacements;

(ii) a CDR2 with an amino acid sequence at least 80% homologous or identical to SEQ ID NO: 49, i.e. CDR2 is SEQ ID NO: 49 or SEQ ID NO: 49 with 1 substitution; (iii) CDR3 with an amino acid sequence at least 80% or 90% homologous or identical to the sequence of SEQ ID NO: 65, i.e. CDR3 is the sequence of SEQ ID NO: 65 or the sequence of SEQ ID NO: 65 with 1 or 2 substitutions.

In one embodiment, the present invention provides an antibody that binds CD47 and PD-L1 and includes a PD-L1 binding site comprising:

(a) a heavy chain variable region comprising:

(i) CDR1 with the amino acid sequence of SEQ ID NO: 5,

(ii) CDR2 with the amino acid sequence of SEQ ID NO: 16,

(iii) a CDR3 with the amino acid sequence of SEQ ID NO: 21, and

(b) a light chain variable region comprising:

(i) CDR1 with the amino acid sequence of SEQ ID NO: 35,

(ii) CDR2 with the amino acid sequence of SEQ ID NO: 49,

(iii) CDR3 with the amino acid sequence of SEQ ID NO: 65.

In one embodiment, the present invention provides an antibody that binds CD47 and PD-L1, characterized in that the CD47 binding site is a Fab, scFv, scFab, or isolated VH or VHH monodomains.

In one embodiment, the present invention provides an antibody that binds CD47 and PD-L1, characterized in that the PD-L1 binding site is a Fab, scFv, scFab, or isolated VH or VHH monodomains.

In one embodiment, the present invention relates to an antibody that binds CD47 and PD-L1, is characterized in that it causes antibody-dependent cellular cytotoxicity, macrophage-mediated phagocytosis, complement-dependent cytotoxicity and/or T-cell mediated cytotoxicity against cells bearing CD47 and/or PD-L1 antigens on their surface.

In one embodiment, the present invention relates to an antibody that binds CD47 and PD-L1, is characterized in that it contains an Fc fragment with 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 mutation or modification.

Nucleic acid molecules

The present invention also relates to nucleic acid molecules, and sequences encoding the antibody to CD47 and PD-L1 according to this invention, described in this document. In some embodiments, different nucleic acid molecules encode the first domain and the second domain of the amino acid sequence of an anti-CD47 and PD-L1 antibody. Where the first domain and/or the second domain comprises a heavy chain and a light chain, in some embodiments, different nucleic acids encode the heavy chain and light chain amino acid sequences. In other embodiments, the same nucleic acid molecule encodes a heavy chain and a light chain sequence. In certain embodiments, the nucleic acid molecule may encode any combination of amino acid sequences (eg, heavy and light chain sequences) of the first and second domains. In a specific embodiment of the invention, the nucleic acid molecule may encode the amino acid sequence of the first binding domain and the amino acid sequence of the light chain of the second binding domain, optionally including any sequence of the peptide linker connecting them. Reference to a nucleotide sequence covers its complement unless otherwise noted. Thus, reference to a nucleic acid having a particular sequence is to be understood as encompassing its complementary strand with its complementary sequence. The term "polynucleotide" as used herein means a polymeric form of nucleotides, at least 10 bases in length, either ribonucleotides or deoxynucleotides, or a modified form of any type of nucleotide. The term includes single- and double-stranded forms.

In any of the above embodiments, the nucleic acid molecules may be isolated.

The nucleic acid molecule of this invention can be isolated from any source that produces an antibody to CD47 and PD-L1. In certain embodiments, the nucleic acid molecule of the invention may be synthesized rather than isolated.

In some embodiments, a nucleic acid molecule of the invention may comprise a nucleotide sequence encoding a VH domain from the first or second domain of an antibody of the invention fused at readout to a nucleotide sequence encoding a heavy chain constant domain from any source. Similarly, a nucleic acid molecule of this invention may comprise a nucleotide sequence encoding a VL domain from the first or second region of an antibody of this invention, combined in terms of reading with a nucleotide sequence encoding a light chain constant domain from any source.

In yet another aspect of the present invention, nucleic acid molecules encoding the variable domain of the heavy (VH) and/or light (VL) chains of the first or second binding domain can be "transformed" throughout the genes of the antibody. In one embodiment of the invention, nucleic acid molecules encoding VH or VL domains are converted into antibody genes over their entire length by insertion into an expression vector already encoding heavy chain (CH) or light chain (CL) constant domains, respectively, such that the VH segment is functionally connected to the CH segment(s) in the vector; and/or the VL segment is operatively connected to the CL segment in the vector. In another embodiment of the invention, nucleic acid molecules encoding VH and/or VL domains are converted into genes along the entire length of the antibody by joining, for example, ligating, a nucleic acid molecule encoding VH and/or VL domains to a nucleic acid molecule encoding CH and /or CL domains using standard molecular biology techniques. Nucleic acid molecules encoding the full length of the heavy and/or light chain can then be expressed in the cell into which they have been introduced.

Nucleic acid molecules can be used to express a large number of recombinant anti-CD47 and PD-L1 antibodies. Nucleic acid molecules can be used to generate human antibodies, humanized antibodies, chimeric antibodies, bispecific antibodies, single chain antibodies, immunoadhesins, diabodies, mutated antibodies, and antibody derivatives as described herein.

Vector

In yet another aspect, the present invention relates to a vector suitable for the expression of any of the nucleotide sequences described herein.

The present invention relates to vectors containing nucleic acid molecules that encode any of the amino acid sequences of antibodies to CD47 and PD-L1 or parts thereof (for example, the heavy chain sequences of the first and/or the heavy and/or light chain of the second binding domain) as described in this document. The present invention further relates to vectors containing nucleic acid molecules encoding fusion proteins, modified antibodies, antibody fragments.

In another embodiment of the invention, nucleic acid molecules and vectors can be used to make mutated antibodies to CD47 and PD-L1. Antibodies can be mutated in the heavy and/or light chain variable domains of the first and/or heavy and/or light chains of the second binding domain, for example, to change the binding ability of the antibodies. For example, a mutation can occur in one or more CDR regions to increase or decrease the K _D of an antibody, to increase or decrease k _off , or to change the FcRn binding specificity of an antibody. In another embodiment, one or more mutations are made to an amino acid residue known to be altered from the germline in an antibody corresponding to the first or second binding domain of an anti-CD47 and PD-L1 antibody of the invention. These mutations can be made in the CDR or framework region of the variable domain, or in the constant domain. In a preferred embodiment of the invention, the mutations are in the variable domain. In another embodiment, one or more mutations are made to an amino acid residue known to be altered from the germline in the CDR region or framework region of the variable domain of an antibody of the invention.

In some embodiments, the anti-CD47 and PD-L1 antibodies of the invention are expressed by inserting DNA encoding part or all of the first and second binding domain sequences (e.g., heavy and light chain sequences, where the binding domain contains heavy and light chain sequences) obtained as described above in expression vectors such that the genes are operably linked to the necessary expression control sequences, such as transcriptional and translational control sequences. Expression vectors include plasmids, retroviruses, adenoviruses, adeno-associated viruses (AAVs), plant viruses such as cauliflower mosaic virus, tobacco mosaic viruses, cosmids, YAC, EBV derived episomes, and the like. The DNA molecules can be ligated into the vector such that transcription and translation control sequences in the vector perform their intended function of regulating transcription and translation of the DNA. The expression vector and expression control sequences can be chosen to be compatible with the expression host cell used. DNA molecules encoding part or all of the first and second binding domain sequences (eg, heavy and light chain sequences, where the binding domain contains the heavy and light chain sequence) can be introduced into separate vectors. In one embodiment of the invention, any combination of the above DNA molecules is introduced into the same expression vector. DNA molecules can be introduced into the expression vector by standard methods (eg, ligation of complementary restriction sites on the gene fragment of the antibody and the vector, or blunt-end ligation if no restriction sites are present).

A suitable vector is one that encodes operably complete human immunoglobulin CH or CL sequences, with the appropriate restriction site designed such that any VH or VL sequence can be easily incorporated and expressed as described above. The HC- and LC-coding of the genes in such vectors may contain intron sequences that result in an overall increase in antibody protein products by stabilization consistent with the mRNA. The intron sequences are surrounded by splice-donor and splice-acceptor sites that determine where RNA splicing will occur. The location of the intron sequences can be either in the variable or constant regions of the antibody chains, or in both the variable and constant regions when multiple introns are used. Termination of polyadenylation and transcription can occur downstream of the native chromosome site of the encoded regions. The recombinant expression vector may also encode a signal peptide that facilitates production of the antibody chain by the host cell. The antibody chain gene can be cloned into a vector such that the signal peptide is linked to the amino terminus of the immunoglobulin chain. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (ie, a signal peptide of a non-immunoglobulin protein).

In addition to the antibody gene chain, the recombinant expression vector of the invention may carry control sequences that control the expression of the antibody chain genes in the host cell. Those skilled in the art will appreciate that the design of the expression vector, including the choice of regulatory sequences, may depend on such factors as the selection of the host cell for transformation, the level of expression of the desired protein, and so on. Preferred regulatory sequences for a mammalian expression host cell include viral elements that provide 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) (eg, SV40 promoter/enhancer), adenovirus (eg, adenovirus large late promoter (AdMLP)), polyoma virus, as well as strong mammalian promoters such as the native immunoglobulin promoter or the actin promoter. For further description of viral regulatory elements and their sequences, see, for example, US Pat. Methods for expressing binding molecules, such as plant antibodies, including describing promoters and vectors, as well as plant transformation, are known in the art. See, for example, US Pat. No. 6,517,529. Methods for expressing polypeptides in bacterial or fungal cells, such as yeast cells, are also well known in the art.

In addition to the antibody chain genes and regulatory sequences, the recombinant expression vectors of the invention may carry additional sequences, such as sequences that regulate the replication of the vector in host cells (eg, origins of replication) and selectable marker genes. The selectable marker gene facilitates the selection of host cells into which the vector has been introduced (see, for example, US Pat. For example, typically a selectable marker gene confers resistance to drugs such as G418, hygromycin, or methotrexate to the host cell into which the vector is introduced. For example, selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr host cells in methotrexate selection/amplification), the neo gene (for G418 selection), and the glutamate synthetase gene.

The term "expression control sequence" as used herein means polynucleotide sequences that are necessary to affect the expression and processing of the coding sequences to which they are ligated. Expression control sequences include the appropriate transcription initiation, termination, promoter, and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that increase translation efficiency (ie Kozak consensus sequence); sequences that enhance protein stability; and, optionally, sequences that enhance protein secretion. The nature of such control sequences varies depending on the host organism; in prokaryotes, such control sequences typically include a promoter, a ribosome binding site, and transcription termination sequences; in eukaryotes, such control sequences typically include promoters and transcription termination sequences. The term "control sequences" includes, at a minimum, all components whose presence is essential for expression and processing, and may also include additional components whose presence is beneficial, such as leader sequences and fused cell sequences.

Host cells

An additional aspect of the present invention relates to methods for producing antibodies to CD47 and PD-L1 according to this invention. One embodiment of the invention relates to a method for producing antibodies as defined herein, comprising introducing obtaining a recombinant host cell capable of expressing antibodies, culturing said host cell under conditions suitable for expressing antibody production, and isolating the resulting antibody. An anti-CD47 and PD-L1 antibody obtained by such expression in such recombinant host cells is referred to herein as "recombinant antibodies". The invention also relates to cell progeny of such host cells and antibodies to CD47 and PD-L1 obtained similarly.

Nucleic acid molecules encoding the anti-CD47 and PD-L1 antibodies of the invention and vectors containing these nucleic acid molecules can be used to transfect a suitable mammal or cell, plant or cell, bacterial or yeast host cell. Transformation can take place by any known method for introducing polynucleotides into a host cell. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art and include dextran-mediated transfection, transfection with a nucleic acid-positively charged polymer complex, transfection with a nucleic acid-calcium phosphate precipitate, polybren-mediated transfection, protoplast fusion, transfection with liposome-encapsulated polynucleotides, and direct microinjection of DNA into nuclei. In addition, nucleic acid molecules can be introduced into mammalian cells with viral vectors. Methods for transfecting cells are well known in the art. See, for example, US Pat. Nos. 4,399,216; 4,912,040; Methods for transforming plant cells are well known in the art, including, for example, Agrobacterium-mediated transformation, biolistic transformation, direct injection, electroporation, and viral transformation. Techniques for transforming bacterial and yeast cells are also well known in the art.

Mammalian cell lines used as hosts for transformation are well known in the art and include many immortalized cell lines available. These include, for example, Chinese Hamster Ovary (CHO) cells, NS0 cells, SP2 cells, HEK-293T cells, 293 Freestyle cells (Invitrogen), NIH-3T3 cells, HeLa cells, Hamster kidney cells (BHK), African kidney cells green monkeys (COS), human hepatocellular carcinoma cells (eg, Hep G2), A549 cells, and a number of other cell lines. Cell lines are selected by determining which cell lines have high levels of expression and provide the desired characteristics of the protein produced. Other cell lines that can be used are insect cell lines such as Sf9 or Sf21 cells. When recombinant expression vectors encoding anti-CD47 and PD-L1 antibodies are introduced into mammalian host cells, antibodies are produced by culturing the host cells for a time sufficient to express the antibodies in the host cells or, preferably, isolate the antibodies into a culture medium, in which host cells are grown. Anti-CD47 and PD-L1 antibodies can be isolated from culture media using standard protein purification methods. Plant host cells, for example, include Nicotiana, Arabidopsis, duckweed, corn, wheat, potato, and so on. Host bacterial cells include E. coli and Streptomyces species. Yeast host cells include Schizosaccharomyces pombe, Saccharomyces cerevisiae and Pichia pastoris.

In addition, the level of production of antibodies to CD47 and PD-L1 according to this invention from the producing cell line can be increased using a number of known methods. For example, the glutamine synthetase gene expression system (GS system) is common enough to enhance expression under certain conditions. The GS system is discussed in whole or in part in connection with EP 0216846, 0256055, 0323997 and 0338841.

It is likely that antibodies to CD47 and PD-L1 of different cell lines or transgenic animals will differ from each other in the glycosylation profile. However, all anti-CD47 and PD-L1 antibodies encoded by the nucleic acid molecules described herein, or containing the amino acid sequences given herein, are part of this invention, regardless of the glycosylation state of the binding molecules, and in general, regardless of the presence or absence of post-translational modifications.

Obtaining antibodies

The invention also relates to methods and processes for producing anti-CD47 and PD-L1 antibodies and antigen-binding fragments thereof.

Monoclonal antibodies

Monoclonal antibodies can be generated, for example, using the hybridoma-based method first described by Kohler et al., Nature, 256, 1975, p. 495, or using recombinant DNA techniques (US 4816567).

When using the hybridoma-based method, a mouse or other suitable host animal such as a hamster is immunized as described above to induce the production of lymphocytes that produce or can produce antibodies that have the ability to specifically bind to the protein used for immunization. In another embodiment, lymphocytes can be obtained from in vitro immunization. After immunization, the lymphocytes are isolated and then fused with a myeloma cell line using a suitable binding agent such as polyethylene glycol to obtain a hybridoma cell.

The hybridoma cells thus obtained are seeded and grown in a suitable culture medium, which preferably contains one or more substances that inhibit the growth or survival of the unfused parental myeloma cells. For example, if parental myeloma cells do not contain the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), then culture media for hybridomas would typically include hypoxanthine, aminopterine, and thymidine (HAT media), i.e. substances that inhibit the growth of HGPRT-deficient cells.

Preferred myeloma cells used as a fusion component are those that are easily fused, maintain a stable high level of antibody production by the selected antibody-producing cells, and are sensitive to a selective medium in which unbound parental cells are selected. Preferred myeloma cell lines are mouse myeloma lines, such as the mouse tumor cell lines MOPC-21 and MPC-11, which are available from the Salk Institite Cell Disrtibution Center, San Diego, CA. California, USA, and lines SP-2 or X63-Ag8-653 available from the American Type Culture Collection, Rockville, CA. Maryland, USA. The use of human myeloma and mouse-human heteromyeloma cell lines for the production of monoclonal antibodies has also been described (Kozbor, J. Immunol., 133, 1984, p. 3001).

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

The binding affinity of a monoclonal antibody can, for example, be determined using the Scatchard assay described in Munson et al., Anal. Biochem., 107, 1980, p. 220.

Once hybridoma cells have been identified that produce antibodies of the desired specificity, affinity and/or activity, clones can be subcloned using the limiting dilution method and grown by standard methods. Suitable media for this purpose include, for example, DMEM media or RPMI-1640. In addition, hydriboma cells can be grown in vivo as ascitic tumors in animals, for example, by intraperitoneal (i.p.) injection of cells into mice.

Monoclonal antibodies secreted by subclones can be separated from the culture medium, ascitic fluid or serum using conventional antibody purification methods, for example, affinity chromatography (for example, using protein A- or protein Q-sepharose), or ion exchange chromatography, chromatography on hydroxylapatites, gel electrophoresis, dialysis, etc.

DNA encoding monoclonal antibodies can be easily isolated and sequenced using conventional procedures (eg, using oligonucleotide probes that have the ability to specifically bind to the genes encoding heavy and light chains of mouse antibodies). Hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA can be incorporated into expression vectors that are then transfected into host cells, such as E. coli cells, monkey COS cells, Chinese Hamster Ovary (CHO) cells, or myeloma cells, which do not produce the antibody protein without transfection, resulting in synthesis of monoclonal antibodies in recombinant host cells. Review of articles on recombinant expression in bacteria of DNA encoding an antibody.

According to another embodiment of the invention, monoclonal antibodies or antibody fragments can be isolated from antibody phage libraries created using the methods described in McCafferty et al., Nature, 348, 1990, cc. 552-554. In Clackson et al., Nature, 352, 1991, pp. 624-628 and Marks et al., J. Mol. Biol., 222, 1991, pp. 581-597 describe the isolation of mouse and human antibodies, respectively, using phage libraries. Subsequent publications have described the production of high affinity (nM range) human antibodies by strand shuffling (Marks et al., Bio/Technology, 10, 1992, pp. 779-783), as well as combinatorial infection and in vivo recombination as a strategy. construction of very large phage libraries (Waterhouse et al., Nucl. Acids. Res., 21, 1991, pp. 2265-2266). Thus, these methods represent a real alternative to traditional methods for isolating monoclonal antibodies based on hybridomas of monoclonal antibodies.

The DNA encoding an antibody can also be modified, for example, in such a way as to obtain chimeric or fused antibody polypeptides, for example, by replacing the sequences of the heavy and light chain constant regions (CH and CL) with homologous murine sequences (US 4816567 and Morrison et al., Proc. Natl. Acad. Sci. USA: 81, 1984, p. 6851) or by covalently linking an immunoglobulin coding sequence to all or part of the coding sequence of a non-immunoglobulin polypeptide (heterologous polypeptide). Non-immunoglobulin polypeptide sequences can be replaced with constant regions of an antibody or replaced with variable regions of an antigen-binding site of an antibody, creating a chimeric bivalent antibody that contains one antigen-binding site with specificity for an antigen and another antigen-binding site with specificity for a different antigen.

Humanized antibodies

Methods for creating "humanized" non-human antibodies are known in the art. Preferably, the humanized antibody has one or more amino acid residues inserted into it from a non-human source. These non-human derived amino acid residues are often referred to as "import" residues because they are usually derived from an "import" variable region. Humanization can generally be carried out according to the method of Winter et al. (Jones et al., Nature, 321, 1986, pp. 522-525) by replacing the hypervariable region sequences with the corresponding human antibody sequences. Thus, "humanized" antibodies are chimeric antibodies (US 4,816,567) in which a region substantially smaller than the intact human variable region has been replaced with a corresponding sequence derived from a non-human species. In practice, humanized antibodies are typically human antibodies in which some of the hypervariable region residues and possibly some of the FR residues are replaced by residues from analogous regions of rodent antibodies.

The choice of human variable regions, both light and heavy chain, intended for the production of humanized antibodies is very important to reduce antigenicity and HAMA response (human anti-mouse antibody) when the antibody is intended for human treatment. According to the so-called "best fit" method, the variable region sequence of a rodent antibody is screened against a complete library of known human variable region sequences. The human sequence of the V-region that is closest to that from the rodent organism is identified and within it a human framework region (FR) suitable for use in a humanized antibody is selected (Sims et al., J. Immunol., 151, 1993, p. 2296). Another method uses a specific framework region derived from the consensus sequence of a specific subset of the light or heavy chains of all human antibodies. The same framework region can be used for several different humanized antibodies (Carter et al., Proc. Natl. Acad. Sci. USA: 89, 1992, p. 4285).

It is also important that antibodies be humanized while retaining high antigen binding capacity and other important biological properties. To achieve this goal, in a preferred method, humanized antibodies are generated by analyzing parental sequences and various humanized products using speculative 3D models of parental and humanized sequences. Three-dimensional immunoglobulin models are publicly available and well known to those skilled in the art. Computer programs are known that illustrate and display possible three-dimensional conformational structures of selected prospective immunoglobulin sequences. The study of these images allows analysis of the possible role of the residues in the function of a promising immunoglobulin sequence, i.e. analysis of residues that affect the ability of a prospective immunoglobulin to bind to an antigen. In this way, residues in the FR can be selected and combined with recipient and import sequences to achieve desired antibody characteristics, such as increased affinity for the target antigen(s). As a rule, hypervariable region residues have a direct and most significant effect on antigen binding.

The humanized antibody may be an antibody fragment, such as a Fab fragment, optionally conjugated to one or more cytotoxic agent(s) to create an immunoconjugate. Alternatively, the humanized antibody may be a full length antibody, such as a full length IgGl antibody.

Human antibodies and a technique based on the use of a phage display library

As an alternative to humanization, human antibodies can be generated. For example, it is currently possible to obtain transgenic animals (eg, mice) that, after immunization, can produce the full range of human antibodies without endogenous immunoglobulin production. For example, it has been described that homozygous deletion of the antibody heavy chain gene junction (JH segment) in chimeric and germline mutated mice results in complete inhibition of endogenous antibody production. Transfer of the human immunoglobulin germline gene set into such germline mutant mice results in the production of human antibodies after antigen challenge (US Pat.

Alternatively, phage display technology can be used to generate human antibodies and antibody fragments in vitro from the immunoglobulin variable region (V) gene spectrum from immunized donors (McCafferty et al., Nature, 348, 1990, pp. 552-554). In this technique, antibody V region genes are cloned in-frame with either the major or minor coat protein gene of a filamentous bacteriophage, such as M13 or fd, and presented as functional antibody fragments on the surface of the phage particle. Since the filamentous particle contains a copy of the single-stranded DNA of the phage genome, selections for the functional properties of an antibody also result in the selection of a gene encoding an antibody that has these properties. Thus, the phage mimics some of the properties of the B cell. Phage presentation can be done in a variety of formats. Various sources of V gene segments can be used for phage presentation. Clackson et al., Nature, 352, 1991, pp. 624-628 isolated various sets of anti-oxazolone antibodies from a small random combinatorial library of V genes derived from the spleens of immunized mice. A spectrum of V genes derived from immunized human donors can be constructed and antibodies to a different set of antigens (including self antigens) can be isolated generally according to the methods described in Marks et al., J. Mol. Biol., 222, 1991, pp. 581-597.

As described above, human antibodies can also be produced in vitro by activated B cells (see US 5567610 and 5229275).

Antibody fragments

In certain circumstances, it is appropriate to use antibody fragments rather than complete antibodies. The smaller size of the fragments facilitates their rapid clearance and may contribute to better penetration into solid tumors.

Various methods have been developed to obtain antibody fragments. Traditionally, these fragments were obtained by proteolytic cleavage of intact antibodies. However, at present, these fragments can be obtained directly using recombinant host cells. Fab, Fv and scFv antibody fragments can be expressed and secreted from E. coli to facilitate the production of large amounts of these fragments. Antibody fragments can be isolated from the antibody phage libraries described above. Alternatively, Fab'-SH fragments can be directly isolated from E. coli and chemically linked to form F(ab') ₂ fragments (Carter et al., Bio/Technology, 10, 1992, pp. 163-167). According to another approach F(ab') ₂ - fragments can be isolated directly from the culture of recombinant host cells. Fab- and F(ab') ₂ fragments with increased in vivo half-life that retain epitope-binding receptor residues are described in US 5,869,046. Other techniques for generating antibody fragments will be apparent 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 US 5587458). Fv and scFv are the only species with intact binding sites lacking constant regions; as a result, they can be used for reduced non-specific binding when used in vivo. Fusion proteins carrying scFv can be designed to produce an effector protein fusion at either the N- or C-terminus of the scFv. An antibody fragment may also be a "linear antibody" such as that described in US 5,641,870. Such linear antibody fragments may be monospecific or bispecific.

Multispecific antibodies

Multicytic antibodies are antibodies that have binding specificity for at least two different epitopes. For example, bispecific antibodies can bind to two different epitopes of a protein. Other multispecific antibodies may carry a CD47 and PD-L1 binding site in combination with another protein binding site. Bispecific antibodies can be prepared as full length antibodies or antibody fragments (eg, F(ab') ₂ bispecific antibody fragments).

Methods for generating multispecific antibodies are known in the art. For example, the conventional production of full length bispecific antibodies is based on the co-expression of two pairs of immunoglobulin heavy and light chains, where both chains have different specificities. Due to the random selection of immunoglobulin heavy and light chains, these hybridomas (quadromas) can potentially produce a mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. Purification of the correct molecule, which is usually carried out in several steps using affinity chromatography, is quite laborious and the yield of the product is low. Similar processes are described in WO 93/08829.

In another approach, antibody variable regions with the desired binding specificity (antibody antigen binding sites) are fused to immunoglobulin constant region sequences. The fusion is preferably carried out with an Ig heavy chain constant region that includes at least a portion of the C _H 2 and C _H 3 hinge regions. Preferably, at least one of the fusions has a first heavy chain constant region ( _CH 1 ) containing the site required for light chain binding. DNA encoding immunoglobulin heavy chain fusions and, optionally, immunoglobulin light chain fusions are inserted into various expression vectors and co-transfected into an acceptable host organism. This provides more flexibility in selecting the overall proportions of the three polypeptide fragments in cases where unequal ratios of the three polypeptide chains are used in the construct in order to optimize yields. However, it is also possible to insert coding sequences into two or all three polypeptide chains in a single expression vector, when expression of at least two polypeptide chains in equal proportions provides high yields, or when the ratios are not critical.

In a preferred embodiment of this approach, the bispecific antibodies are an immunoglobulin heavy chain hybrid providing a first binding specificity in the first arm, and an immunoglobulin heavy chain-light chain pair hybrid (providing a second binding specificity) in the second arm. This asymmetric structure has been found to facilitate separation of the desired bispecific molecule from unwanted combinations of immunoglobulin chains, since the presence of an immunoglobulin light chain in only one half of the bispecific molecule facilitates separation. This approach is described in WO 94/04690. For additional detailed description of the production of bispecific antibodies, see, for example, Suresh et al., Methods in Enzymology, 121, 1986, c. 210.

According to the following approach, described in US Pat. No. 5,731,168, a contact area between a pair of antibody molecules can be designed to maximize the percentage of heterodimers that are obtained from recombinant cell culture. The preferred contact area includes at least a portion of the C- _N -3 area. According to this method, one or more small amino acids with side chains from the contact area of the first antibody molecule are replaced with molecules with larger side chains (for example, tyrosine or tryptophan). Balancing "cavities", identical or close in size to the large side chain(s), are created at the interface of the second antibody molecule by replacing amino acids with large side chains with amino acids with smaller side chains (for example, alanine or threonine ). This provides a mechanism to increase the yield of the heterodimer relative to other unwanted end products.

Bispecific antibodies include crosslinked antibodies or "heteroconjugates". For example, one of the antibodies in the heteroconjugate can be crosslinked with avidin and the other with biotin. Such antibodies can be used, for example, to target cells of the immune system to unwanted cells (US 4676980) and to treat HIV infection (WO 91/00360, WO 92/200373 and EP 03089). Heteroconjugate antibodies can be generated using any of the usual crosslinking techniques. Suitable crosslinkers are well known in the art and are described in US 4,676,980 along with various methods for introducing crosslinks.

Methods for obtaining bispecific antibodies from antibody fragments are also described in the literature. For example, bispecific antibodies can be generated by chemical linkage. Brennan et al., Science, 229, 1985, p. 81 described a procedure in which intact antibodies are subjected to proteolytic cleavage to obtain F(ab') ₂ fragments. These fragments are reduced in the presence of a dithiol complexing agent, such as sodium arsenite, to stabilize adjacent dithiols and prevent the formation of intermolecular disulfide bonds. The resulting Fab' fragments are then converted to the thionitrobenzoate (TNB) derivative. One of the Fab'-TNB derivatives is then reconverted to Fab'-thiol by reduction with mercaptoethylamine and mixed with an equimolar amount of the other Fab'-TNB derivative to form a bispecific antibody. The obtained bispecific antibodies can be used as agents for selective immobilization of enzymes.

The progress made so far makes it possible to facilitate the direct isolation from E. coli of Fab'-SH fragments, which can be chemically ligated to form bispecific antibodies. Shalaby et al., J. Exp. Med., 175, 1992, p. 217-225 describe the preparation of the F(ab') ₂ fragment of a fully humanized bispecific antibody molecule. Each Fab' fragment was individually secreted from E. coli and subjected to direct chemical linkage in vitro to obtain a bispecific antibody. The bispecific antibody thus obtained was able to bind to cells overexpressing the ErbB2 receptor and normal human T cells, and to stimulate the lytic activity of human cytotoxic lymphocytes that target human breast tumors.

Various methods for obtaining and isolating fragments of bispecific antibodies directly from recombinant cell cultures are also described. For example, bispecific antibodies have been generated using leucine zippers (Kostelny et al., J. Immunol., 148(5), 1992, pp. 1547-1553). The leucine zipper peptides from the Fos and Jun proteins were linked to the Fab' fragments of two different antibodies by gene fusion. Antibody homodimers were reduced at the hinge region to give monomers and then antibody heterodimers were obtained by re-oxidation. This method can also be used to obtain antibody homodimers. The "double antibody" technology described by Hollinger et al., Proc. Natl. Acad. sci. USA, 90, 1993, p. 6444-6448 is another mechanism for obtaining bispecific antibody fragments. The fragments contain a VH region linked to a VL region by a linker that is too short to allow pairing of two domains on the same strand to occur. Thus, the VH and VL regions of one fragment must pair with the complementary VL and VH regions of the other fragment, thereby forming two antigen-binding centers. Another strategy for obtaining bispecific antibody fragments has also been described, based on the use of single chain (Fv)-(sFv) dimers (see Gruber et al., J. Immunol, 152, 1994, p. 5368).

Antibodies having more than two valences also fall within the scope of the invention. For example, you can get trispecific antibodies.

Polyvalent antibodies

A multivalent antibody can be internalized (and/or dissimilated) by a cell expressing the antigen to which the antibody binds faster than a bivalent antibody. The antibodies of the present invention may be polyvalent antibodies (other than the IgM class) with three or more antigen-binding sites (e.g., tetravalent antibodies) that can be easily generated by recombinant expression of a nucleic acid encoding antibody polypeptide chains. A multivalent antibody may contain a dimerized domain and three or more antigen binding sites. The preferred dimerized domain contains (or consists of) an Fc fragment or a hinge region. In such a case, the antibody must contain an Fc fragment and three or more antigen binding sites located at the N-terminus relative to the Fc fragment. According to the present description, the preferred multivalent antibody contains (or consists of) from 3 to about 8, but preferably 4, antigennegative centers. A polyvalent antibody contains at least one polypeptide chain (and preferably two polypeptide chains), while the polypeptide(s) chain(s) contains(at) two or more variable regions. For example, the polypeptide(s) chain(s) may contain VDl-(X1)n-VD2-(X2)n-Fc, where VD1 denotes the first variable region, VD2 denotes the second variable region, Fc denotes one Fc polypeptide chain fragment, X1 and X2 are an amino acid or polypeptide, and n is 0 or 1. For example, the polypeptide(s) chain(s) may contain the following chain: VH-CH1 flexible linker-VH-CH1-Fc fragment; or VH-CH1-VH-CH1-Fc fragment. The multivalent antibody of the present invention preferably further comprises at least 2 (and preferably 4) light chain variable region polypeptides. A multivalent antibody of the present invention may, for example, comprise from about 2 to about 8 light chain variable region polypeptides. As used herein, light chain variable region polypeptides are intended to comprise a light chain variable region and optionally further comprise a CL region.

Pharmaceutical compositions

Another aspect of the invention is a pharmaceutical composition containing as an active ingredient (or as the only active ingredient) an antibody that is specific for CD47 and PD-L1. The pharmaceutical composition may include at least one anti-CD47 and PD-L1 antibody and/or one or more additional binding molecules (eg, antibodies) that target one or more of the appropriate surface receptors as described herein. In some embodiments, the compositions are intended to improve, prevent, or treat disorders that are mediated by IgG.

"Pharmaceutical composition" means a composition comprising an anti-CD47 and PD-L1 antibody according to the invention and at least one of the components selected from the group consisting of pharmaceutically acceptable and pharmacologically compatible excipients such as excipients, diluents, diluents, carriers, auxiliary, distributing, means of delivery, preservatives, stabilizers, emulsifiers, suspending agents, thickeners, regulators of prolonged delivery, the choice and ratio of which depends on the nature and method of administration and dosage. The pharmaceutical compositions of the present invention and methods for their manufacture will be undeniably obvious to those skilled in the art. The production of pharmaceutical compositions should preferably comply with GMP (Good Manufacturing Practice) requirements. The composition may include a buffer composition, tonic agents, stabilizers and solubilizers. Prolonged action of the composition can be provided by agents that delay the absorption of the active pharmaceutical ingredient, such as aluminum monostearate and gelatin. Examples of suitable carriers, solvents, diluents, and delivery vehicles are water, ethanol, polyalcohols, and mixtures thereof, oils, and injectable organic esters.

"Drug (preparation)" - a substance or mixture of substances in the form of a pharmaceutical composition in the form of tablets, capsules, powders, lyophilisates, injections, infusions, ointments and other ready-made forms, intended to restore, correct or change physiological functions in humans and animals , as well as for the treatment and prevention of diseases, diagnosis, anesthesia, contraception, cosmetology and other things. Any method of administering peptides, proteins, or antibodies accepted in the art can be appropriately used for the anti-CD47 and PD-L1 antibody of this invention.

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

The term "excipient" or "adjuvant" is used herein to describe any component other than those previously described in this invention. These are substances of inorganic or organic origin used in the manufacturing process, the manufacture of medicines to give them the necessary physical and chemical properties.

The term "buffer", "buffer composition", "buffer agent" means a solution capable of maintaining the pH value due to the interaction of the acidic and alkaline components that make up its composition, which allows the anti-CD47 and PD-L1 antibody preparation to be resistant to changes pH. In general, pH values of the pharmaceutical composition between 4.0 and 8.0 are preferred. As buffer agents, for example, acetate, phosphate, citrate, histidine, succinate, and the like can be used. buffer solutions, but not limited to.

The terms "tonic agent", "osmolytic" or "osmotic agent", as used herein, refer to an excipient that can supply the osmotic pressure of a liquid antibody preparation. An "isotonic" drug is a drug that has an osmotic pressure equivalent to that of human blood. Isotonic formulations typically have an osmotic pressure of about 250 to 350 mOsm/kg. As isotonic agents, polyols, mono- and disaccharides, amino acids, metal salts such as sodium chloride, and the like can be used, but not limited to.

By "stabilizer" is meant an excipient or a mixture of two or more excipients which provide physical and/or chemical stability to the active agent. As stabilizers, amino acids can be used, for example, arginine, histidine, glycine, lysine, glutamine, proline, but not limited to; surfactants, e.g. polysorbate 20 (trade name Tween 20), polysorbate 80 (trade name Tween 80), polyethylene-polypropylene glycol and its copolymers (trade names Poloxaner, Pluronic), sodium dodecyl sulfate (SDS) but not limited to; antioxidants, such as methionine, acetylcysteine, ascorbic acid, monothioglycerol, salts of sulfuric acids, and the like, but not limited to; chelating agents such as, but not limited to, ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), sodium citrate, and the like.

A pharmaceutical composition is "stable" if the active agent retains its physical stability and/or chemical stability and/or biological activity during the stated shelf life at a storage temperature, for example, 2-8°C. Preferably, the active agent retains both physical and chemical stability, as well as biological activity. The storage period is selected based on the results of a stability study with accelerated and natural storage.

The pharmaceutical composition of the present invention may be formulated, packaged, or widely sold as a single unit dose or multiple unit dose units in the form of a finished dosage form. As used herein, the term "unit dosage unit" means a discrete amount of a pharmaceutical composition containing a predetermined amount of an active ingredient. The amount of active ingredient is usually equal to the dosage of the active ingredient to be administered to the subject, or a convenient fraction of such dosage, for example, half or one third of such dosage.

Pharmaceutical compositions of the present invention are generally suitable for parenteral administration as sterile medicaments intended to be administered to the human body in violation of the integrity of the skin or mucous membranes, bypassing the gastrointestinal tract by injection, infusion or implantation. For example, parenteral administration is intended to include, but is not limited to, subcutaneous, intraperitoneal, intramuscular, intravenous, intraarterial, intrathecal, intraventricular, intraurethral, intracranial, intraarticular, transdermal injection or infusion, and renal dialysis infusion techniques. Regional perfusion is also provided. Preferred embodiments of the invention include intravenous and subcutaneous routes. Any method of administering peptides or proteins accepted in the art can be appropriately used for the anti-CD47 and PD-L1 antibody of this invention.

Injectable drugs can be manufactured, packaged or sold in unit dosage form, such as, but not limited to, ampoules, vials, plastic containers, pre-filled syringes, auto-injection devices. Drugs for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous bases, pastes, and the like.

Another embodiment of the invention is a drug for parenteral administration, where the pharmaceutical composition is provided in dry form, i.e., powder or granules for dissolution in a suitable solvent (eg, sterile, pyrogen-free water) prior to administration. Such a drug can be obtained, for example, by lyophilization, i. a process known in the art as freeze-drying, which involves freezing the preparation and then removing the solvent from the frozen contents.

The anti-CD47 and PD-L1 antibody of this invention may also be administered intranasally or by inhalation, alone, as a mixture with a suitable pharmaceutically acceptable vehicle from an inhaler such as a pressurized aerosol container, pump, spray, nebulizer) or nebulizer that uses or no suitable propellant is used, or in the form of nasal drops or spray.

The drug for parenteral administration may be immediate or modified release. Modified release drugs include delayed, sustained, pulsatile, controlled, targeted and programmed release.

Therapeutic Use of the Anti-CD47 and PD-L1 Antibody of the Invention

In one aspect, an anti-CD47 and PD-L1 antibody of the present invention is used in the treatment of disorders that are mediated by CD47 and PD-L1, e.g., the disease or disorder is selected from the group: HNSC (Head and Neck Squamous Cell Carcinoma), Cervical Cancer, Undiagnosed Cancer primary source, 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's lymphoma, MSI CRC ( colorectal cancer with high microsatellite instability), leukemia (acute or chronic), non-Hodgkin's lymphoma, multiple myeloma, myelodysplastic syndrome.

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

In the case of a tumor (eg, cancer), a therapeutically effective amount of an antibody or antibody fragment (eg, an antibody or antibody fragment that specifically binds to CD47 and PD-L1) can reduce the number of cancer cells; reduce the initial size of the tumor; inhibit (ie, slow down to some extent and preferably stop) cancer cell infiltration of peripheral organs; inhibit (ie, slow down to some extent and preferably stop) tumor metastasis; inhibit, to some extent, tumor growth; and/or alleviate, to some extent, one or more of the symptoms associated with the disorder. The antibody or antibody fragment may, to some extent, prevent the growth and/or kill existing cancer cells, it may cause a cytostatic and/or cytotoxic effect. In cancer therapy, in vivo efficacy can be determined, for example, by assessing overall life expectancy (OS), time to disease progression (TTR), overall tumor response rate (ORR), duration of response (DR) and/or quality of life.

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

1) simultaneous administration of such a combination of an anti-CD47 and PD-L1 antibody of the present invention and a therapeutic agent to a patient in need of treatment, when such components are formulated together in a single dosage form from which said components are released substantially simultaneously to said patient,

2) simultaneous administration of such a combination of an anti-CD47 and PD-L1 antibody of this invention and a therapeutic agent to a patient in need of treatment, when such components are formulated separately in different dosage forms, the administration of which occurs at almost the same time to said patient, after whereby these components are released almost simultaneously to the specified patient,

3) sequential administration of such a combination of an anti-CD47 and PD-L1 antibody of this invention and a therapeutic agent to a patient in need of treatment, when such components are formulated separately from each other in separate dosage forms that are taken sequentially in time by said patient with a significant time interval between each administration, after which said components are released at substantially different times to said patient; as well as

4) sequential administration of such a combination of an anti-CD47 and PD-L1 antibody of this invention and a therapeutic agent to a patient in need of treatment, when such components are formulated together in a single dosage form from which the release of these components occurs in a controlled manner, after which they are simultaneously, sequentially or jointly released at the same time and/or different times to the specified patient, where each part can be entered in one or different ways.

The anti-CD47 and PD-L1 antibody of this invention can be administered without additional therapeutic treatment, i. as a standalone therapy. In addition, treatment with an antibody of this invention may include at least one additional therapeutic treatment (combination therapy). In some embodiments, the anti-CD47 and PD-L1 antibody may be co-administered or formulated with another cancer medication/drug.

The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents the functioning of cells and/or causes destruction of cells. The term is intended to include radioactive isotopes (e.g., At ²¹¹ , I ¹³¹ , I ¹²⁵ , Y ⁹⁰ , Re ¹⁸⁶ , Re ¹⁸⁸ , Sm ¹⁵³ , Bi ²¹² , P ³² and Lu radioactive isotopes), chemotherapeutic agents, and toxins such as low molecular weight toxins or enzymatically active toxins originating from bacteria, fungi, plants or animals, including fragments and/or variants thereof.

A "chemotherapeutic agent" is a chemical compound useful in the treatment of cancer. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide ( ^CYTOXAN® ); alkylsulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquinone, meturedopa and uredopa; ethyleneimines and methylmelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylmelamine; acetogenins (eg bullatacin and bullatacinone); beta-lapachone; lapachol; colchicines; betulinic acid; camptothecin (including the synthetic analog of topotecan (HYCAMTIN ^® ), CPT-11 (irinotecan, CAMPTOSAR ^® ), acetylcamptothecin, scopolelectin and 9-aminocamptothecin); bryostatin; callistatin; CC-1065 (including its synthetic analogues adozelesin, carzelesin and bizelesin); podophyllotoxin; podophyllic acid; teniposide; cryptophycins (eg, cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including synthetic analogues of KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphasine, holofosfamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembihin, phenesterin, prednimustine, trofosfamide, uramustine; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine and ranimnustine; antibiotics such as enediine antibiotics (e.g., calicheamicin, e.g., calicheamicin gamma II and calicheamicin omega II (see, e.g., Agnew, Chem. Intl. Ed. Engl., 33: 183-186 (1994)); dynemycin, including dynemycin A; esperamycin; as well as the neocarcinostatin chromophore and related chromoprotein chromophores - enediine antibiotics, aclacinomysins, actinomycin, autramycin, azaserin, bleomycins, cactinomycin, carabicin, carminomycin, carsinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-oxo-diazo-5-oxo-diazo-5 -L-norleucine, doxorubicin (including ADRIAMICIN ^® , morpholinodoxorubicin, cyanomorpholinodoxorubicin, 2-pyrrolinodoxorubicin, doxorubicino-HCl in injectable liposomes (DOXOL ^® ), TLC D-99 liposomal doxorubicin (MYOCET ^® ), pegylated liposomal doxorubicin ^® (CAELYX) ), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rhodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate, gemcitabine ( ^GEMZAR® ), tegafur ( ^UFTORAL® ), capecitabine ( ^XELODA® ), epothilone, and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprin, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxyfluridine, enocytabine, floxuridine; adrenal suppressants such as aminoglutethimide, mitotane, trilostane; folic acid compensator such as folinic acid; aceglatone; aldofosfamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraksat; defofamine; demecolcin; diazichon; elfornitin; elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainin; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerin; pentostatin; phenamet; pyrarubicin; losoxantrone; 2-ethylhydrazide; procarbazine; polysaccharide complex PSK ^® (JHS Natural Products, Eugene, OR); razoxane; rhizoxin; sisofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; trichothecenes (eg, toxin T-2, verracurin A, rhoridin A, and anguidin); urethane; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; hacytosine; arabinoside ("ara-C") ; thiotepa; taxoid, such as paclitaxel (TAXOL ^® ), paclitaxel based on engineered albumin-bound nanoparticles (ABRAXANETM) and docetaxel (TAXOTERE ^® ); chlorambucil; 6-thioguanine; mercaptopurine; methotrexate; platinum-based agents such as cisplatin, oxaliplatin and carboplatin, vinca alkaloids that prevent the polymerization of tubulin from nascent microtubules, including vinblastine (VELBAN ^® ), vincristine (ONCOVIN ^® ), vindesine (ELDISINE ^® , FILDESIN ^® ) and vinorelbine (NAVELBINE ^® ); etoposide (VP-16); ifosfamide; mitoxantrone; leucovorin; novantrone; edatrexate; daunomycin; aminopterin; ibandronate; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid, including bexarotene ( ^TARGRETIN® ); bisphosphonates such as as clodronate (eg BONEFOS ^® or OSTAC ^® ), etidronate (DIDROCAL ^® ), NE-58095, zoledronic acid/zoledronate (ZOMETA ^® ), alendronate (FOSAMAX ^® ), pamidronate (AREDIA ^® ), tiludronate (SKELID ^® ) or risedronate ( ^ACTONEL® ); troxcitabine (1,3-dioxolane nucleoside analogue of cytosine); antisense oligonucleotides, for example, oligonucleotides that inhibit gene expression in signaling pathways involved in aberrant cell proliferation, such as, for example, PKC-alpha, Raf, H-Ras, and epidermal growth factor receptor (EGF-R); vaccines such as ^THERATOPE® vaccine and gene therapy vaccines such as ^ALLOVECTIN® vaccine, ^LEUVECTIN® vaccine and ^VAXID® vaccine; topoisomerase 1 inhibitor (eg LURTOTECAN ^® ); rmRH (eg ^ABARELIX® ); BAY439006 (sorafenib; Bayer); SU-11248 (Pfizer); perifosin, COX-2 inhibitor (eg celecoxib or etoricoxib), proteasome inhibitor (eg PS341); bortezomib ( ^VELCADE® ); CCI-779; tipifarnib (811577); orafenib, ABT510; a Bcl-2 inhibitor such as sodium oblimersen ( ^GENASENSE® ); pixantrone; EGFR inhibitors (see definition below); tyrosine kinase inhibitors (see definition below); and pharmaceutically acceptable salts, acids or derivatives of any of the above; as well as combinations of two or more of the above, such as CHOP, short for combination therapy with cyclophosphamide, doxorubicin, vincristine, and prednisolone, and FOLFOX, short for oxaliplatin (ELOXATINTM) in combination with 5-FU and leucovorin.

Also included in this definition are antihormonal agents that act to regulate or inhibit the action of hormones on tumors, such as antiestrogen with a mixed agonist/antagonist profile, including tamoxifen (NOLVADEX ^® ), 4-hydroxy tamoxifen, trioxifene, toremifene (FARESTON ^® ); idoxifene, droloxifene, raloxifene ( ^EVISTA® ), trioxifene, keoxifene and selective estrogen receptor modulators (SERMs) such as SERM3; pure antiestrogen without agonistic properties, such as fulvestrant (FASLODEX ^® ) and EM800 (such agents can block estrogen receptor (ER) dimerization, inhibit DNA binding, increase ER metabolism and/or reduce ER levels); aromatase inhibitors, including steroidal aromatase inhibitors such as formestane and exemestane (AROMASIN ^® ), and non-steroidal aromatase inhibitors such as anastrozole (ARIMIDEX ^® ), letrozole (FEMARA ^® ) and aminoglutethimide and other aromatase inhibitors, including vorozol (RIVISOR ^® ), megestrol acetate ( ^MEGASE® ), fadrozole, imidazole; luteinizing hormone releasing hormone agonists including leuprolide ( ^LUPRON® and ^ELIGARD® ), goserelin, buserelin and tripterelin; 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; onapriston; antiprogesterones; estrogen receptor downregulators (ERDs); antiandrogens such as flutamide, nilutamide and bicalutamide; testolactone; and pharmaceutically acceptable salts, acids or derivatives of any of the above; as well as combinations of two or more of the above means.

Other therapeutic agents that may be used in combination with the anti-CD47 and PD-L1 antibodies of the invention may be growth factor function inhibitors, 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 antibodies disclosed by Stern et al Critical reviews in oncology/haematology, 2005, Vol 54, pp11-29); anti-angiogenic agents such as inhibitory effects of vascular endothelial growth factor [eg, anti-vascular endothelial growth factor antibody bevacizumab (Avastin)], antibodies against vascular endothelial growth factor receptors such as anti-KDR antibodies and anti-flt1 antibodies; antisense nucleotides, for example directed to the above targets, such as ISIS 2503, anti-ras antisense agents or G3139 (Genasense), anti-bcl2 antisense agents; gene therapy approaches, including, for example, aberrant gene replacement approaches such as aberrant p53 or aberrant BRCA1 or BRCA2, GDEPT (Gene Targeted Enzyme Prodrug Therapy) approaches using cytosine deaminase, thymidine kinase, or a bacterial nitroreductase enzyme, and approaches aimed at increasing patient tolerance to chemotherapy or radiotherapy, such as multidrug resistance gene therapy; immunotherapeutic approaches, including, for example, treatment with Alemtuzumab (campath-1H), a monoclonal antibody directed to CD52, or treatment with antibodies directed to CD22, ex vivo and in vivo approaches to increase the immunogenicity of the patient's tumor cells, transfection with cytokines such as interleukin 2 , interleukin 4 or granulocyte-macrophage colony-stimulating factor, approaches to reduce T-cell anergy, such as treatment with monoclonal antibodies that inhibit CTLA-4 function, approaches using transfected immune cells, such as cytokine-transfected dendritic cells, approaches using cytokine-transfected tumor cell lines and approaches using anti-idiotypic antibodies, adaptive T-cell transfer using T-cells subjected to non-specific activation or targeted to a specific antigen of interest, ex vivo; protein degradation inhibitors such as a proteasome inhibitor such as Velcade (bortezomib); biotherapeutic therapeutic approaches, for example, using peptides or proteins (such as antibodies or soluble external receptor domain constructs) that sequester ligand receptors, block ligand binding to the receptor, or attenuate receptor signaling (eg, due to increased receptor degradation or reduced expression levels).

Doses and routes of administration

The anti-CD47 and PD-L1 antibody of this invention will be administered in an amount effective to treat the condition in question, i. in doses and for periods of time necessary to achieve the desired result. The therapeutically effective amount may vary depending on factors such as the particular condition being treated, the age, sex, and weight of the patient, and whether administration of the anti-CD47 and PD-L1 antibody is a stand-alone treatment or is given in combination with one or more complementary treatments.

Drug regimens can be adjusted to provide the optimal desired 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 depending on the severity of the therapeutic situation. Especially useful is the formulation of parenteral compositions in unit dosage form for ease of administration and uniformity of dosing. Unit dosage form, as used herein, refers to physically discrete units suitable as unit doses for patients/subjects to be treated; each unit contains a predetermined amount of the active compound calculated to produce the desired therapeutic effect in combination with the required pharmaceutical carrier. The specification for unit dosage forms of the present invention is generally dictated by and directly dependent on (a) the unique characteristics of the chemotherapeutic agent and the particular therapeutic or prophylactic effect to be achieved, and (b) the limitations inherent in the art of formulating such an active compound for treatment of subjects.

Thus, those skilled in the art will appreciate from the disclosure provided herein that doses and dosing regimens are adjusted in accordance with methods well known in the therapeutic field. This means that the maximum tolerated dose can be easily set and the effective amount that provides a detectable therapeutic effect for the patient can also be determined, as well as the time requirements for the administration of each agent to achieve a visible therapeutic effect for the patient. Thus, although some doses and regimens of administration are given as examples in this document, these examples in no way limit the doses and regimens of administration that may be needed for a patient in the practice of the present invention.

It should be noted that dosage values may vary depending on the type and severity of the condition to be alleviated and may include one or more doses. In addition, it should be understood that for any particular patient, specific administration schedules will need to be adjusted over time according to individual need and at the discretion of the healthcare professional who administers or supervises the administration of the compositions, and that the concentration ranges given in this specification are only by way of example and are not intended to limit the scope or practice of the claimed compositions. In addition, the dosage regimen with the compositions of this invention may be based on various factors, including the type of disease, age, weight, sex, health status of the patient, severity of the condition, route of administration, and the particular anti-CD47 and PD-L1 antibody used. Thus, the dosing regimen may vary widely, but may be determined regularly using standard methods. For example, doses may be adjusted based on pharmacokinetic and pharmacodynamic parameters, which may include clinical effects such as toxic effects or laboratory values. Thus, the present invention encompasses individual dose escalation as determined by the skilled artisan. Determination of the required dose and modes are well known in the relevant field of technology and will be clear to a person skilled in the art after reading the ideas disclosed in this document.

Examples of suitable routes of administration are provided above.

It is contemplated that a suitable dose of the anti-CD47 and PD-L1 antibody of this 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 , for example, about 1-20 mg/kg. The anti-CD47 and PD-L1 antibody can be administered, for example, at a dose of at least 0.25 mg/kg, for example, at least 0.5 mg/kg, including at least 1 mg/kg, for example, according to 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, for example up to a maximum of 20 mg/kg, including up to a maximum of 15 mg/kg. The administration will be repeated usually at suitable intervals, for example, once a week, once every two weeks, once every three weeks, or once every four weeks, and for as long as is deemed appropriate by the physician in charge, who may in some cases increase or reduce dose if necessary.

Product(s) and kits

A further embodiment of the invention is a product that contains products used in the treatment of cancer, e.g. HNCH, cervical cancer, cancer without an identified primary source, glioblastoma, esophageal cancer, bladder cancer, TNBC, CRC, hepatocellular carcinoma, melanoma, NSCLC, cancer kidney, ovarian cancer, Hodgkin's lymphoma, MSI CRC, leukemia (acute or chronic), non-Hodgkin's lymphoma, multiple myeloma, myelodysplastic syndrome. The product is a container and a label or leaflet in the package, which are placed on the container or enclosed in it. Acceptable containers are, for example, jars, vials, syringes, etc. Containers can be made from various materials such as glass or plastic. The container contains a composition effective for the treatment of a particular condition and may have a sterile inlet (for example, the container may be an intravenous solution bag or a vial fitted 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 of the invention. The label or package insert indicates that the composition is used to treat a specific condition. The label or package insert should additionally contain instructions for administering the antibody composition to the patient.

The package insert contains the usual instructions that are included in commercial packages of therapeutic products, including exemplary information about the indications, use, dose, route of administration, contraindications and/or precautions regarding the use of such therapeutic products. In one embodiment, the package insert indicates that the composition is used to treat cancer, e.g., HNCH, cervical cancer, cancer without an identified primary source, glioblastoma, esophageal cancer, bladder cancer, TNBC, CRC, hepatocellular carcinoma, melanoma, NSCLC, kidney cancer, ovarian cancer, Hodgkin's lymphoma, MSI CRC, leukemia (acute or chronic), non-Hodgkin's lymphoma, multiple myeloma, myelodysplastic syndrome.

In addition, the article may further comprise a second container containing a pharmaceutically acceptable buffer such as bacteriostatic water for injection (BAVI), phosphate buffered saline, Ringer's solution, and dextrose solution. In addition, it may include other products required from a commercial and consumer point of view, such as other buffers, diluents, filters, needles and syringes.

The invention also relates to kits that can be used for various purposes, for example, for the detection of PD-L1 in tissues, cells or body fluids of a mammal. Such a kit would be useful for screening for PD-L1 associated diseases. The kit includes a specific binding agent or antibody of the invention and means indicating the reaction of the specific binding agent or antibody with PD-L1, if present. In one embodiment, the antibody is a monoclonal antibody. In one embodiment, the PD-L1 binding antibody is labeled. In another embodiment, the antibody is an unlabeled primary antibody and the kit further includes means for detecting the primary antibody. In one embodiment, the detection means comprise a labeled second antibody that is an anti-immunoglobulin. The antibody may be labeled with a marker selected from the group consisting of a fluorochrome, an enzyme, a radionuclide, and a radioopaque material. The kit may be a kit that contains antibodies to detect and quantify PD-L1 in vitro, for example, when performing ELISA or Western blotting. Also, as in the case of the product, the kit contains a container and a label or package insert located on the surface or inside the container. The container contains a composition that includes at least one anti-PD-L1 antibody of the invention. Additional containers may contain, for example, diluents and buffers, control antibodies. The label or package insert may contain a description of the composition, as well as instructions for their use in vitro or for diagnostic purposes.

Diagnostic use and compositions

The anti-CD47 and PD-L1 antibody of the present invention are also used in diagnostic processes (eg, in vitro, ex vivo). For example, an anti-CD47 and PD-L1 antibody can be used to detect or measure the level of CD47 and/or PD-L1 in patient-derived samples (e.g., a tissue sample or body fluid sample such as inflammatory exudate, blood, serum, fluid intestines, saliva or urine). Suitable detection and measurement methods include immunological methods such as flow cytometry, enzyme-linked immunosorbent assay (ELISA), chemiluminescent assay, radioimmunoassay, and immunohistology. The invention further includes kits (eg, diagnostic kits) containing the anti-CD47 and PD-L1 antibodies described herein.

Examples

For a better understanding of the invention, the following examples are given. These examples are provided for illustrative purposes only and should not be construed as limiting the scope of the invention in any form.

All publications, patents, and patent applications referenced in this specification are incorporated herein by reference. Although the above invention has been described in some detail by way of illustration and example in order to avoid ambiguity, it will be quite clear to those skilled in the art based on the ideas disclosed in this invention that certain changes and modifications can be made without deviating from the essence and scope of the attached options. implementation of the invention.

Example 1

Production of recombinant antigens and antibodies in suspension culture of mammalian cells.

Human extracellular domain sequences CD47 (Leu19-Val134) and PD-L1 (Phe19-Arg238) (SEQ ID NO: 107-108) were cloned into a plasmid for protein production in mammalian cells with Fc tag (Fig. 1) at SalI/ restriction sites. NotI. Plasmids were generated in the required amounts in E. Coli cells and purified using the Qiagen kit.

The sequences of the variable domains of the anti-CD47 antibody (B6H12, Stanford University, US20130142786) were cloned into plasmids for protein production in IgG1 format in mammalian cells. Plasmids were generated in the required amounts in E. Coli cells and purified using the Qiagen kit.

Antibodies and antigens were produced in cells of a permanent cell line derived from Chinese hamster ovary cells (CHO-T line). Suspension culture was performed in flasks on an orbital shaker using serum-free media manufactured by Life Technologies Corporation and according to the manufacturer's instructions. For transient expression, cells at a concentration of 2*10 ⁶ /ml were transfected with linear polyethyleneimine (eg PEI MAX, Polysciences). The DNA/PEI ratio was 1:3/1:10. 5-7 days after transfection, the culture medium was centrifuged at 2000 g for 20 minutes and filtered through a filter with a pore size of 0.22 μm. Target proteins were isolated from the culture liquid using affinity chromatography.

Recombinant proteins with Fc were isolated and purified from culture broth using a protein A affinity chromatography column. four). The column was then washed with 5 column volumes of PBS to wash out the non-specific binding components. Bound antigen was eluted using 0.1 M glycine buffer pH 3. The major protein elution peak was collected and adjusted to pH neutral with 1 M Tris buffer (pH 8). All stages were carried out at a flow rate of 110 cm/h. Next, the protein was transferred to PBS (pH 7.4) using dialysis using SnakeSkin Dialysis Tubing technology, filtered (0.22 μm), transferred to test tubes and stored at -70°C.

The purity of the resulting protein solution was assessed by SDS-gel electrophoresis in 12% PAAG under non-reducing conditions.

Example 2

Obtaining full-length antibodies.

Cloning was carried out according to the standard method. PCR products containing the genes for the variable domains of the heavy and light chains of antibodies were generated with primers containing restriction sites. The heavy chain variable domain was cloned into the pEE-Hc IgG1 vector at the Sal1/Nhe1 restriction sites. The light chain variable domain was cloned into the pEE-CK vector at the Sal1/BsiW1 restriction sites. The resulting genetic constructs were transferred for transient production of proteins in the CHO-T cell line. Proteins were isolated and purified according to the standard method by affinity chromatography on bacterial protein Protein A as described in Example 1. Electrophoresis was carried out in denaturing 12% PAAG with the addition of mercaptoethanol Fig.3 and denaturing 8% PAAG without the addition of mercaptoethanol Fig.4

Example 3

Creation of a naive phage Fab-library of human antibodies MeganLibTM.

Total B-lymphocyte RNA from individual blood samples from more than a thousand human donors was isolated using the RNeasy Mini Kit according to the proposed protocol (from QIAGEN). RNA concentration was determined using a Nanovue kit (from GE Healthcare) and the quality of the isolated RNA was checked by electrophoresis on a 1.5% agarose gel.

The reverse transcription reaction was performed using the MMLV RT kit (from Evrogen) according to the recommended protocol using MMuLV reverse transcriptase and random hexamer oligonucleotides as a primer.

Reverse transcription products were used as a template in a two-step polymerase chain reaction to obtain variable domain genes flanked by restriction sites using a set of oligonucleotides according to the protocols of the authors [J Biol Chem. 1999 Jun 25; 274(26): 18218-30].

The resulting DNA preparation VL-CK-VH (treated with restriction endonucleases NheI/Eco91I and ligated into the original pH5 phagemid. The ligation products were transformed into electrocompetent cells of the E.Coli strain SS320, prepared according to the protocols [Methods Enzymol. 2000;328: 333-63.] The combinatorial phage Fab display library MeganLibTM had a transformant repertoire of 10 ^11. Phage preparations of the Fab libraries were prepared according to the procedure previously described [J Mol Biol 1991 Dec 5;222(3):581-97].

Example 4

Immunization of llama with CD47 human antigen and creation of a phage display library of llama antibody fragments.

The Lama Glama animal was sequentially immunized 5 times by subcutaneous injection of antigenic material mixed with an equal volume of complete (at the first injection) or incomplete (at the remaining injections) Freund's adjuvant. The recombinant human CD47 protein described in Example 1 was used as an antigen at 1 mg per injection. Antigen injections were performed at the following times: 0, 2, 4, 5, 8 weeks. Blood sampling (50 ml) was performed 5 days after each injection, starting from the third. As an anticoagulant, 3.8% sodium citrate was used in a ratio of 1:9. The collected llama blood was diluted 2 times with sterile saline. 30 ml of the diluted blood solution was overlaid on Lymphoprep™ medium (Axis-Shield, Norway) with a density of 1.077 g/ml in a volume of 15 ml and centrifuged for 20 min at 800g. Mononuclear cells (lymphocytes and monocytes) were collected from the plasma/Lymphoprep interphase zone and washed with sterile PBS.

The resulting titer of serum immunoglobulin against CD47, estimated according to the standard protocol, was not less than 1/100000, which is sufficient for the preparation of an antibody library.

Total llama mononuclear cell RNA was isolated using the RNeasy Mini Kit according to the proposed protocol (QIAGEN). The RNA concentration was determined using Nanovue (GE Healthcare) and the quality of the isolated RNA was checked by electrophoresis in 1.5% agarose gel.

The reverse transcription reaction was performed using the MMLV RT kit (from Evrogen) according to the recommended protocol using MMuLV reverse transcriptase and random hexamer oligonucleotides as a primer.

Reverse transcription products were used as a template in a two-step polymerase chain reaction to obtain VHH, scFv or Fab monodomain genes flanked by restriction sites using a set of oligonucleotides and the protocols of the authors [FASEB J. 2007 Nov;21(13):3490-8]. The resulting DNA preparation of the VHH genes was treated with NcoI /NotI restriction enzymes and ligated into the original pscFv phagemid, similar in all pHEN2 elements used in [FASEB J. 2007 Nov;21(13):3490-8]. The ligation products were transformed into electrocompetent cells of strain SS320 obtained according to the protocols [Methods Enzymol. 2000;328: 333-63]. The VHH-based library repertoires were 0.5-2*10E+8 independent transformants. The library repertoires based on scFv and Fab were 0.5-2*10E+9 and 1.2-2.5*10E+9, respectively. The phage library preparation was prepared according to the procedure previously described [J Mol Biol. 1991 Dec 5;222(3): 581-97].

Example 5

Selection of phage display libraries of antibody fragments

Specific CD47 phage antibodies were generated from phage Fab, VHH or scFv display libraries (Example 3, 4) by standard selection procedures previously described [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 particles and the KingFisher Flex device, since the use of this technique allows up to 96 different schemes and options to be carried out in parallel.

Human biotinylated antigen PD-L1 and CD47 (Fc, EPEA) were directed immobilized on the surface of streptavidin magnetic beads (NEB) at a concentration of 10 μg/ml for the first round, 2 μg/ml for the second, 0.4 and 0, 2 µg/ml for the third and fourth, respectively. The antigen was incubated with particles for 1 hour at room temperature on a rotator. Next, the particles were washed with PBS (pH 7.4) and the surface of the particles was blocked with a solution of 2% skimmed milk or 1% BSA in PBS (pH 7.4) for 1 hour. The MeganLibTM human phage library was diluted at 2*10 ¹³ phage particles per ml in PBS (pH 7.4) with 2% skim milk and non-target antigen with target antigen tag and preselected with magnetic particles lacking antigen on the surface to remove non-specifically binding phages. IL-5Rα-coated magnetic particles were then incubated with MeganLibTM for 1-2 hours at room temperature.

Unbound phages were removed during several washes of magnetic particles with a PBS solution (pH 7.4) with 0.1% Tween 20. -em - 12, on the 4th - 15). Phages bound to the antigen on the surface of the magnetic particles were eluted from the particles with a 100 mM Gly-HCl solution (pH 2.2) for 15 min with stirring; after elution, the 1M TRIS-HCl solution (pH 7.6) was neutralized. E. coli TG1 bacteria were infected with phages and grown in culture, after which they were isolated and used in the next round of selection. After three to four rounds, phagemid DNA was isolated from the E. coli TG1 culture according to the manufacturer's standard protocol (Qiagen). The enrichment of the library for target antigens and the assessment of the presence of non-specifically binding phage particles were performed using enzyme-linked immunosorbent assay of polyclonal phage (ELISA).

Example 6

Enzyme immunoassay of polyclonal phage for specific and nonspecific antigens.

For ELISA, the target antigen (CD47 and PD-L1) and non-target antigen (with Fc-fusion protein) were immobilized on high-sorption plates (Greiner-Bio). The protein was added at a concentration of 1 and 5 μg/ml, respectively, in 0.1 M NaHCO ₃ (pH 9.0) and titrated in steps of 2 to 7 dilutions, after which the sealed plates were incubated overnight at +4°C. All subsequent steps were performed according to a standard ELISA protocol using a high-performance automated platform based on the Tecan Freedom EVO 200 robotic system (from Tecan). Blocking buffer 2% skimmed milk or 1% BSA in PBS (pH 7.4) was added to the wells of the plates to block non-specific binding. The plates were incubated for one hour at room temperature. After washing with phosphate-buffered saline containing Tween-20 (PSBT), 50 μl per well of the tested polyclonal phage was added. After washing was added (50 μl/well) anti-M13 HRP-conjugated secondary antibody (from Pierce-ThermoScientific) at a ratio of 1:7500 in PBS. After incubation for 50 minutes at room temperature, the plates were washed with PBS three times. The colorimetric signal was developed by adding a substrate solution (H ₂ O ₂ -0.02% and TMB in CH ₃ COONa pH5.5) for 10 minutes, then the reaction was stopped by adding 1% sulfuric acid (20 μl). The color signal was measured at a wavelength of 450 nm using a suitable Tecan-Sunrise plate reader (from Tecan).

After the third and fourth rounds of selection on the target antigen, the ELISA analysis of the polyclonal phage preparation showed a significant enrichment of Fig. 5. Libraries were selected for recloning and further screening, in which a signal excess of more than 5 times was observed at the minimum dilution of phage libraries for non-homologous control antigens.

Example 7

Recloning of antibody fragment genes into an expression plasmid.

Cloning of antibody variable domain genes into an expression plasmid from a phagemid vector after successful rounds of selection was carried out according to a standard protocol using the restriction ligation method.

The resulting pool of clones enriched in VHH monodomains or scFv specific against CD47 was cloned into the expression plasmid pET-22 (Novagen) under the control of the T7 promoter, additionally containing myc-tag and His6 tag sequences at the C-terminus of VHH. Fab fragment genes for libraries containing enriched anti-CD47 antigen sequences were recloned into a pLL4 expression vector, under the control of the lac promoter, and containing additional myc-tag and His6 tag sequences at the C-terminus of the heavy chain CH1 domain.

Subsequently, expression vectors containing antibody fragments were transformed into the E. coli Bl21(DE3)Gold (Stratagene) strain to generate antibody fragments by secretion into the culture medium and to conduct a comparative analysis of the affinity of variable antibody fragments from display libraries to the antigen by ELISA using the technological platform Mabnext Flow Chart.

Example 8

Analysis of specific binding of scFv or VHH monodomain to human CD47-Fc.

ELISA was used to measure the binding of the test specific antibody fragments from Example 4 to human CD47-Fc. For specific binding assay, wells of ELISA plates (from Nunc ImmunoMaxisorp) were coated with 50 μl (0.5 μg/ml in 1X carbonate coating buffer) of human CD47-Fc (Biocad), sealed and incubated overnight at 4°C. All subsequent steps were performed according to a standard ELISA protocol using a high-performance automated platform based on robotic systems GenetixQ-pix2xt (from Molecular Device) and Tecan Freedom EVO 200 (from Tecan). Blocking buffer BB (200 µl 0.5% skimmed milk in PBS) was added to block non-specific binding. The plates were incubated on a shaker for one hour at room temperature. After washes with PBS-Tween, 100 μl per well of the test cell supernatant containing the test antibody fragment was added. The plates were again incubated with shaking for one hour at room temperature, after which each well of the plates was washed five times with PBS-Tween buffer. After washing, (50 μl/well) mouse anti-MYC IgG clone 9E10 (ThermoFisher Scientific) was added at a ratio of 1:5000 in PBS-tween. The plates were shaken on a rotary shaker (50 min, room temperature) and washed five times with PBS-Tween buffer as described above. After washing was added (50 μl/well) anti-mouse IgG HRP conjugate (ThermoFisher Scientific) at a ratio of 1:10,000 in PBS-tween. The plates were shaken on a rotary shaker (50 min, room temperature) and washed five times with PBS-Tween buffer as described above. The colorimetric signal was developed by adding TMB (50 µl/well) until saturation (average 10-12 min), then further development was stopped by adding a stop solution (25 µl/well, 1% sulfuric acid). The color signal was measured at a wavelength of 450 nm using a suitable Tecan-Sunrise plate reader (from Tecan). The degree of antibody binding was proportional to the production of a color signal. Clones whose color signal exceeded the background by more than 5 times were tested in competitive ELISA analysis to identify antagonistic specific antibody fragments that block the interaction of the SIRP-Fc ligand (BIOCAD) and the human CD47-Fc receptor under conditions similar to those described [WO2016048188 A8] and with a signal reduced by 4 times relative to the control that does not contain the analyzed antibody fragments.

As a result of screening 2400 clones, 265 scFv and VHH clones were obtained with the indicated 5-fold excess in signal over background. From this panel of positive clones, 27 antagonistic clones were obtained capable of blocking the interaction of the SIRP-Fc ligand (BIOCAD) and the human CD47-Fc receptor. The nucleotide sequence of the genes of the positive clones was determined by Sanger sequencing on a 3130xl Genetic Analyzer (Applied Biosystems). As an example, six clones of VHH monodomains differing in sequence by more than 1 amino acid but having more than 90% homology of each of the CDRs with each other were obtained, indicating their origin from the same parental clone by in vivo maturation in an immunized llama ( Table 1).

Table 1. Sequences of specific CD47 binding VHH monodomains to human CD47.

Example 9

Analysis of specific binding of Fab fragments to human CD47-Fc.

Fab production was carried out according to the standard method: bacterial cells were transformed with expression vectors containing Fab genes, and the subsequent addition of an inducer that starts transcription of the lac operon to the medium during cultivation of the obtained transformants causes Fab expression.

Next, ELISA was performed to search for Fabs that bind human CD47.

The published sequence B6H12 Fab was used as a positive control (see Example 1). For specific binding assay, wells of ELISA plates (high binding from Greiner bio one) were coated with 50 μl of CD47 Fc lama (0.2 μg/ml in 1× carbonate buffer), sealed and incubated overnight at 4°C. All subsequent steps were performed according to a standard ELISA protocol using a high-performance automated platform based on robotic systems Genetix Qpix2xt (from Molecular Device) and Tecan Freedom EVO 200 (from Tecan). Blocking buffer BB (200 μl 0.5% skimmed milk in PBS) was added to block non-specific binding. The plates were incubated for one hour at room temperature. After washes with PBS-Tween, 60 μl per well of the test cell supernatant containing the test Fab was added. The plates were again incubated for one hour at room temperature, after which each well of the plates was washed three times with PBS-Tween buffer. After washing was added (50 μl/well) anti-human Fab HRP-conjugated secondary antibody (from Pierce-ThermoScientific) at a ratio of 1:7500 in PBS-tween. The plates were incubated for 1 hour at room temperature and washed three times with PBS-Tween buffer as described above. The colorimetric signal was developed by adding TMB (50 μl/well) until saturation (15 min), then further development was stopped by adding a stop solution (25 μl/well, 1% sulfuric acid). The color signal was measured at a wavelength of 450 nm using a suitable Tecan-Sunrise plate reader (from Tecan). The degree of antibody binding was proportional to the production of a color signal. Clones whose color signal exceeded the signal from the control antibody were tested in ELISA for non-specific binding.

Example 10

Analysis of non-specific binding of Fab fragments to various human antigens.

The goal of secondary screening is to select Fab producing clones that interact with the full-length CD47 antigen and do not interact with non-specific antigens, and also compete with the ligand (CD47) for binding to SIRPa.

ELISA was used to analyze the nonspecific binding of the studied Fab fragments to other antigens. The study was carried out as described above, but 3DHer3-H6E, INFα2b, PD-L1-Fc-lama (2.5 μg/ml in 1× carbonate buffer) were used as immobilization antigens. CD47 FE and CD47 Fc lama (0.2 μg/ml in 1x carbonate buffer) were used as controls for specific binding. All subsequent steps were performed according to a standard ELISA protocol using a high-performance automated platform based on robotic systems Genetix Qpix2xt (from Molecular Device) and Tecan Freedom EVO 200 (from Tecan).

Competitive ELISA was used to test previously selected anti-human CD47 specific Fabs for their ability to block interaction with the SIRPa receptor. A published sequence Fab was used as a positive antagonist control (see Example 1).

SIRPa was immobilized in the wells of ELISA plates (high binding from Greiner bio one) at 50 μl at a concentration of 0.5 μg/ml in 1× carbonate buffer and incubated overnight at 4°C. All subsequent steps were performed according to standard ELISA protocols using a high-performance automated platform based on robotic systems Genetix Qpix2xt (from Molecular Device) and Tecan Freedom EVO 200 (from Tecan). Blocking buffer BB (200 μl 0.5% skimmed milk in PBS) was added to block non-specific binding. The plates were incubated for one hour at room temperature.

In parallel, the cell supernatant containing the test Fab and CD47 Fc lama (at a final concentration of 1 μg/ml in PBS-Tween) was mixed in a 1:1 ratio in non-sorbing plates and incubated for 45 minutes at room temperature.

After washes from the BB tablet containing the SIRPa receptor, a mixture of Fab and CD47 Fc lama was transferred there, incubated for 45 minutes at room temperature. After that, each well of the plates was washed three times with PBS-Tween buffer, 50 μl/well of anti-human Fab HRP-conjugated secondary antibody (from Pierce-ThermoScientific) was added at a ratio of 1:7500 in PBS-Tween. Incubated for 45 min at room temperature, after which each well of the plates was washed three times with PBS-Tween buffer, as described above. The colorimetric signal was developed by adding TMB (50 µl/well) until saturation (average 15 min), then further development was stopped by adding a stop solution (25 µl/well, 1% sulfuric acid). The color signal was measured at a wavelength of 450 nm using a suitable Tecan-Sunrise plate reader (from Tecan). The extent of Fab binding was inversely proportional to color signal production. Clones showing blocking at the level of the control Fab antibody were marked as positive and used in further assays. The variable domain genes of the positive clones were sequenced according to standard protocols on an Applied Biosystems 3130 Genetic Analyzer (Applied Biosystems) and analyzed.

Example 11.

Comparative screening of anti-CD47 antibody fragments by kinetic dissociation constant koff (kdis).

As an example of the analysis of the kinetic parameters of the interaction of antibody fragments specifically binding to the CD47 receptor, the VHH of antibody fragments was measured. Comparative screening by dissociation constant (k _dis ) for VHH fragments was performed using an Octet Red 96 instrument and ARG2 amino-reactive biosensors (Pall-ForteBio). Affinity constants can be calculated knowing the exact concentration of the protein in the analyzed solution and, since cell growth media were used as the protein solution and protein concentrations were not measured, the candidates were compared with each other by their dissociation constant. The biosensors were preliminarily rehydrated for an hour in water. After activation of biosensors, CD47-Fc at a concentration of 10 µg/ml in acetate buffer pH4 was nonspecifically (according to NH2 groups) immobilized on the surface of the biosensor. Then the sensors were immersed in wells with a cell growth medium with specific VHH (approximately from 1 μg VHH in 1 ml of medium), where the association of the complex occurred. After that, the sensors were immersed in a buffer solution, in which the subsequent stage of complex dissociation took place. In the studied samples of the growth medium of E. Coli cells containing anti-CD47 VHH fragments, 1/10 of the volume of 10x working buffer was added and mixed. The obtained curves were analyzed using the OctetDataAnalysis software (version 7.0) according to the standard procedure using a 1:1 interaction model.

The results of koff screening of anti-CD47 VHH candidates are shown in Table 2. Specific binding of all VHH fragments to human CD47 was demonstrated, candidate BCD106-02_L.Alecto.VHHSel2_MP1_C7_49 for predominant kdis was selected for further work and recloning to obtain bispecific antibodies.

Table 2. Kinetic dissociation constants of VHH with CD47-Fc

No. clone name kdis(1/s) one 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 four 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

Example 12.

Obtaining constructs and production of asymmetric bispecific anti-CD47 and PD-L1 antibodies.

The sequences of all variable domains of the optimized scFv fragments, as well as the sequences of the genes for the synthesis of wild and mutant variants of the candidate VHH variable domain BCD106-02-VHH_C7_49 (Table 3) and the genes of the light and heavy chain variable domains of the anti-PD-L1 antibody (BCD135 - original human antibody from BIOCAD) was obtained de novo by calculation using proprietary computer algorithms and PCR synthesis from oligonucleotides obtained on ASM-2000 synthesizers (Novosibirsk) according to the standard protocol [http://www.openwetware.org/wiki/DNA_Synthesis_from_Oligos] . The long scFv genes were generated from the VH and VL genes by a two-step PCR synthesis from single-stranded DNA molecules. After PCR synthesis, the DNA fragments fractionated on agarose gel were purified on QIAquick Gel Extraction Kit (Qiagen) columns. (hole) plasmid. At the same time, pEE-Fc(knob) contains human IgG1 Fc with S354C+T366W mutations and pEE-Fc(hole) contains human IgG1 Fc with Y349C+T366S+L368A mutations, providing heterodimerization of these Fc parts with each other, with a minimum degree of homodimerization [ Nat Biotechnol. 1998 Jul;16(7):677-81.] co-expressed transiently in CHO-EBNA cells with aPD-L1 light chain variable domain similarly cloned into pEE-Clambda. After the ligation-free cloning reaction by the modified LIC method [Aslanidis C, de Jong PJ. Ligation-independent cloning of PCR products (LIC-PCR). Nucleic Acids Res. 1990;18:6069–6074]) DNA was transformed into E. coli. Sequence-correct pEE-Fc(knob)-scFv or pEE-Fc(knob)-VHH constructs were co-transfected with pEE-BCD135-VH-299P-HC-hole and pEE-BCD135-01 4LG VL hzau CL to obtain so-called asymmetric bispecific antibodies (see Fig. 6). This figure shows schematically models 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. The resulting genetic constructs were used in the production of proteins in the CHO-T cell line, according to Example 2. The antibodies after purification were highly homogeneous in composition, the production yields of bispecific antibodies varied from 60 to 260 mg/ml of the culture medium. In FIG. 7 shows examples of purified bispecific antibodies based on anti-CD47 scFv fragments. In FIG. 8 shows examples of purified bispecific antibodies based on anti-CD47 VHH fragments. The antibody variant containing the sequence indicated in Table 3 VHH47Opt3 gave an extremely low yield of the antibody and was excluded from further analyses.

Example 13

Analysis of the interaction of anti-PD-L1/anti-CD47 bispecific PD-L1 antibodies with human PD-L1 and CD47 antigens using the OctetRED96 instrument.

Analysis of the interaction of bispecific PD-L1 antibodies with human PD-L1 and CD47 antigens was performed on an OctetRed 96 instrument (Pall-ForteBio). The AR2G biosensors were previously rehydrated for one hour in mQ. After activation of biosensors, PD-L1-Fc or CD47-Fc at a concentration of 25 μg/ml in acetate buffer pH4 were nonspecifically (according to NH2 groups) immobilized on the surface of the biosensor. Then the sensors were immersed in wells with anti-PD-L1/anti-CD47 antibody solutions (10 μg/ml). In this solution, the association of the antibody-antigen complex occurred. After that, the sensors were immersed in a buffer solution for the next stage of dissociation. Binding curves with subtraction of the base signal were analyzed using Octet Data Analysis software (version 8.2) according to the standard procedure using a 1:1 interaction model.

The results of the analyzes are shown in Table 4. Thus, it can be concluded that the antibodies are high-affinity, while the affinity for the PD-L1 antigen in antibodies of this format was nM values, while the affinity for the CD47 antigen has subnM values. Such binding is considered sufficient for the interaction of the antibody with receptors on the target cells, for subsequent verification of both in vitro and in vivo therapeutic activity.

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

Example 14

Analysis of the interactions of anti-PD-L1/anti-CD47 bispecific antibodies with the CD47 and PD-L1 receptors of the cynomolgus monkey using a Forte Bio Octet RED 384 instrument.

Experiments to study the binding affinity of antibodies to the CD47 and PD-L1 antigens of animals were performed on a Forte Bio Octert RED 384 device. Antibodies at a concentration of 20 μg/ml were immobilized on the surface of AR2G sensors (Forte Bio) according to the standard protocol according to the manufacturer's instructions. The analysis was carried out at 30°C using PBS containing 0.1% Tween-20 and 0.1% BSA as working buffer. After the baseline was written in the buffer solution, the sensors were immersed in wells with a solution of antigens (CD47 of animals and PD-L1) for 300 seconds, where the association of the complex occurred. Then the dissociation of the complex in the buffer solution was detected for 600 seconds.

Binding curves with base signal subtraction were analyzed using Octet Data Analysis software (version 9.0) according to a standard procedure using a 1:1 Global interaction model. Anti-CD47 antibodies specifically bind to the macaque CD47 and PD-L1 antigens. Table 5 and Table 6.

Table 5. Kinetic values of the interaction of antibodies to the macaque antigen (CD-47).

Table 6. Kinetic values of the interaction of antibodies to the macaque antigen (PD-L1).

Example 15

Analysis of non-specific binding of anti-PD-L1/anti-CD47 bispecific antibodies to a panel of antigens on a Forte Bio Octet RED 384 instrument.

Experiments to study nonspecific binding were performed on a panel of nonspecific antigens with a GIS tag. Biosensors Anti-hIgG Fc Capture (AHC) rehydrated for 10 minutes in PBS containing 0.1% Tween-20 and 0.1% BSA as a working buffer were used for measurement.

Antibodies at a concentration of 30 μg/ml were immobilized on the surface of Anti-hIgG Fc Capture (AHC) sensors (Forte Bio). The analysis was carried out at 30°C using PBS containing 0.1% Tween-20 and 0.1% BSA as working buffer. After the baseline was written in the buffer solution, the sensors were immersed in the wells with a solution of nonspecific antigens for 300 seconds, where the association of the complex occurred. Then the dissociation of the complex in the buffer solution was detected for 600 seconds.

Binding curves with base signal subtraction were analyzed using Octet Data Analysis software (version 9.0) according to a standard procedure using a 1:1 Global interaction model. anti-PD-L1/anti-CD47 bispecific antibodies do not non-specifically bind to a panel of antigens.

Example 16

Analysis of the interaction of anti-PD-L1/anti-CD47 bispecific antibodies with the FcγRIIIa panel on the Forte Bio Octet RED 384 instrument.

Experiments to study the affinity of antibodies to a panel of Fc-binding proteins were carried out on a Forte Bio Octert RED 384 instrument using streptavidin (SAX) biosensors hydrated for 30 minutes in PBS containing 0.1% Tween-20 and 0.1% BSA .

Biotinylated Fc-binding proteins FcγRIIIa-F158 and FcγRIIIa-V158 with Avitag at a concentration of 5 μg/ml in PBS kinetic buffer containing 0.1% Tween-20 and 0.1% BSA pH 7.4 were immobilized on the surface of streptavidin ( SAX) sensors, fixed t _{RecLoad -} time to reach the signal level equal to 0.4 nm. After the baseline was written in the buffer solution, the sensors were immersed in the wells with the antibody solution for 60 seconds, where the association of the complex occurred. Then the dissociation of the complex in the buffer solution was detected for 150 seconds.

Binding curves minus the reference signal were analyzed using Octet Data Analysis software (version 9.0) according to the standard procedure using a 2:1 Global interaction model. anti-PD-L1/anti-CD47 bispecific antibodies specifically bind to the Fc-binding proteins FcγRIIIa-F158 and FcγRIIIa-V158. Table 7 (- and Table 8.

Table 7. Kinetic values of the interaction of antibodies BCD-106(02-001, 03-006, 02-013) to FcγRIIIa-F158.

Table 8. Kinetic values of the interaction of antibodies BCD-106(02-001, 03-006, 02-013) to FcγRIIIa-V158.

Example 17.

Analysis of the interaction of anti-PD-L1/anti-CD47 bispecific antibodies with FcRn on a Forte Bio Octet RED 384 instrument.

Experiments to study the affinity of antibodies to a panel of Fc-binding proteins were carried out on a Forte Bio Octert RED 384 instrument using streptavidin (SAX) biosensors hydrated for 30 minutes in PBS containing 0.1% Tween-20 and 0.1% BSA .

Biotinylated FcRn with Avitag at a concentration of 5 μg/ml in PBS kinetic buffer containing 0.1% Tween-20 pH 6 was immobilized on the surface of streptavidin (SAX) sensors, with fixation t _{RecLoad -} the time to reach a signal level of 0.4 nm . After the baseline was prescribed in the buffer solution, the sensors were immersed in wells with an antibody solution in the PBS kinetic buffer containing 0.1% Tween-20 pH 6 for 60 seconds, where the association of the complex occurred. Then the dissociation of the complex was detected in the PBS kinetic buffer containing 0.1% Tween-20 and 0.1% BSA pH 7.4 for 150 seconds.

Binding curves minus the reference signal were analyzed using Octet Data Analysis software (version 9.0) according to the standard procedure using a 2:1 Global interaction model. anti-PD-L1/anti-CD47 bispecific antibodies bind specifically to FcRn. Table 9

Table 9. Kinetic values of the interaction of antibodies BCD-106(02-001, 03-006, 02-013) to FcRn.

Example 18.

Analysis of the ability of anti-PD-L1/anti-CD47 bispecific PD-L1 antibodies to induce antibody-dependent cellular cytotoxicity on PD-L1 and CD47 positive cells.

For the analysis of ADCC (antibody-dependent cellular cytotoxicity - antibody-dependent cellular cytotoxicity), we used the MDA-MB-231 cell line expressing PD-L1 and CD47 receptors on its surface, and peripheral blood mononuclear cells (PBMC).

Production of peripheral blood mononuclear cells

PBMC was obtained by density gradient fractionation of venous blood cells from healthy donors. After isolation, cells were cultured in RPMI-1640 medium with 10% FBS at a concentration of 2-5×10 ⁶ cells/ml for 18-24 hours at 37°C 5% CO _{2 .}

Target cell preparation

MDA-MB-231 cells were cultured in DMEM with 10% FBS (fetal bovine serum) at 37°C 5% CO ₂ . Cells were removed from the plastic surface with trypsin, resuspended in DMEM with 10% FBS. Added Calcein AM to a concentration of 5 μm. After 30 minutes, the cells were washed twice from excess Calcein AM using DMEM with 10% FBS. A suspension of target cells was prepared in DMEM with 10% FBS at a concentration ^of 105 cells/ml.

Preparation of test antibody dilutions

All tested antibodies were diluted with DMEM medium with 10% FBS to a concentration of 10 μg/ml. A number of serial dilutions were prepared with a step of 5. The concentrations of the studied antibodies were (ng/ml): 10,000; 2000; 400; 80; 16; 3.2; 0.64; 0.128; 0.0256; 0.

Preparing PBMCs

Mononuclear lymphocytes were collected from vials. Centrifuged at 200×g for 5 minutes. Cell suspension was prepared in DMEM medium with 10% FBS at a concentration of 5×10 ⁶ cells/ml.

Conducting an ADCC test

In the wells of a 96-well plate, 50 μl/well of the test antibodies were added. 100 μl of the target cell suspension was added to the wells with antibodies. The plate was incubated at 37°C 5% CO ₂ for 15-20 minutes. 50 µl of PBMC suspension was added to the wells with antibodies and target cells. Three wells (maximum lysis control, KL) were filled with 50 µl of DMEM medium with 10% FBS, 100 µl of target cell suspension, and 50 µl of PBMC suspension. The tablet was incubated at 37°C 5% CO ₂ 3.5-4 hours. Lysis buffer was added to the KL wells 30 minutes before the end of the incubation.

At the end of the incubation, the plates were centrifuged at 200×g for 10 minutes. The supernatant was transferred to new 96-well plates. Fluorescence readings were measured in relative fluorescence units at an excitation/emission wavelength of 485/538 nm using a plate fluorometer.

The effectiveness of ADCC was calculated by the formula:

, где

, where

K - control of spontaneous lysis of target cells in the presence of effector cells (50 µl/well of DMEM medium with 10% FBS + 100 µl/well of target cells + 50 µl/well of PBMC)

KL - control of maximum lysis of target cells (50 µl/well of DMEM medium with 10% FBS + 100 µl/well of target cells + 50 µl/well of PBMCs + lysis buffer)

The experimental data are shown in Fig. 9 and 10.

All anti-CD47/PD-L1 antibodies were reported to have EC50 values comparable to or greater than the EC50 value of the control, replicated B6H12 anti-CD47 monospecific antibody.

Example 19.

Comparison of the activity of anti-PD-L1/anti-CD47 bispecific antibodies against CD47/PD-L1 in the test for stimulation of phagocytosis by human macrophage cells

For the analysis of ADCP (Antibody-Dependent Cellular Phagocytosis - antibody-dependent cellular phagocytosis), we used the MDA-MB-231 cell line, which has PD-L1 and CD47 receptors on its surface, and human macrophage cells.

Obtaining human macrophages

Peripheral blood mononuclear cells (PBMC) were isolated from venous blood of healthy donors by density gradient separation. Human blood monocytes were isolated using a CD14-positive human cell fraction isolation kit (Miltenyi Biotec). In the wells of a 24-well plate, 350,000 monocytes per well were cultured in 700 μl of RPMI-1640 with 10% FBS, 100 ng/ml GM-CSF (Peprotech) at 37°C 5% CO ₂ for 3 days. On the 4th day of cultivation, the medium was changed to a new medium at 700 µl RPMI-1640 with 10% FBS, 100 ng/ml GM-CSF (Peprotech), 50 ng/ml IFNγ (Peprotech) and 10 ng/ml LPS (Sigma) per well, cells were cultured at 37°C 5% CO ₂ for another 3 days.

Target cell preparation

MDA-MB-231 cells were cultured in DMEM with 10% FBS (fetal bovine serum) at 37°C 5% CO ₂ . Cells were removed from the plastic surface with trypsin and suspended in DMEM with 10% FBS. Added Calcein AM to a concentration of 5 μm. After 30 minutes, the cells were washed twice from excess Calcein AM using DMEM with 10% FBS. A suspension of target cells was prepared in DMEM with 10% FBS at a concentration ^of 105 cells/ml.

Conducting an ADCP test

The medium was taken from the wells of the plate with macrophages, 500 μl of RPMI-1640 medium with 10% FBS and 20 μg/ml of the antibodies under study were added to the wells. 500 μl of the target cell suspension was added to the wells. The plate was incubated at 37°C 5% CO ₂ for 3 hours. After the medium was taken, the cells were removed from the plastic surface using the TrypLE Express reagent, and the cells were stained with fluorescently labeled antibodies against CD14. The stained cell suspension was analyzed on a flow cytometer.

The effectiveness of ADCP was calculated by the formula:

, где

, where

Calcein ⁺ CD14 ⁺ - the number of CD14-positive cells containing calcein stain

CD14 ⁺ - the number of all CD14-positive cells

The results are shown in FIG. eleven.

According to the obtained data, a number of antibodies against CD47/PD-L1 have the efficiency of stimulation of phagocytosis of the MDA-MB-231 cell line by human macrophages comparable to the efficiency of the control reproduced B6H12 monospecific anti-CD47 antibody.

Example 20.

Comparison of the effect of candidate anti-PD-L1/anti-CD47 bispecific antibodies on human erythrocyte hemagglutination

Human erythrocytes were used to analyze the ability of antibodies to induce hemagglutination.

Preparation of a suspension of erythrocytes

Blood was taken from the vein of a healthy donor into a vacuum tube with heparin. Transfer 9 ml of blood into a 50 ml centrifuge tube. The blood was diluted to 30 ml with DPBS solution without Ca2+ and Mg2+ at room temperature. The suspension was centrifuged at 800g for 10 minutes, the supernatant was discarded. Cell washing was repeated twice with DPBS without Ca2+ and Mg2+ and centrifugation. Then 300 μl of the cell pellet was resuspended in 30 ml of DPBS, resulting in a 1% suspension of erythrocytes.

Performing a hemaglutination test

Test antibodies were diluted in DPBS to a concentration of 20 μg/ml. In a 96 well round bottom plate, 100 μl of antibody dilutions and erythrocyte suspension were mixed. The plate was incubated in a CO2 incubator for 16 hours at 37°C. The results were recorded visually on a conditional scale of 4 crosses. Reliably positive is the result of 2 crosses and above. The results are presented in Table 10.

Table 10 Hemagglutination reaction in the presence of anti-CD47/PD-L1 antibodies. The - symbol indicates the absence of agglutination.

None of the anti-CD47/PD-L1 antibodies was reported to cause hemaglutination, while the control, generic anti-CD47 monoclonal antibody B6H12 caused significant agglutination due to the bivalent nature of the antibody, and thus the ability to interact with two CD47 molecules located on different erythrocytes.

Example 21.

Complement dependent cytotoxicity (CDC) analysis of bispecific anti-CD47/PD-L1 antibodies.

For analysis of CDC (Complement-Dependent Cytotoxicity - complement-dependent cytotoxicity), the MDA-MB-231 line, which has PD-L1 and CD47 receptors on its surface, was used as target cells.

Target cell preparation

MDA-MB-231 culture was cultured in DMEM supplemented with 10% fetal bovine serum (FBS).

The cells were removed from the plastic surface with trypsin and suspended in DMEM medium with 0.1% BSA at a concentration of 1×10 ⁶ cells/ml.

Preparation of test antibody dilutions

All tested anti-CD47/PD-L1 antibodies were diluted in DMEM with 0.1% BSA to a concentration of 100 μg/ml. A number of serial dilutions were prepared with a step of 8. The concentrations of the studied antibodies were (ng/ml): 100,000; 12500; 1562.5; 195.3; 24.4; 3.05; 0.38; 0.04; 0.005.

Conducting a CDC test

Human complement was thawed and diluted 1:4 in DMEM with 0.1% BSA.

In the wells of a 96-well plate, 50 μl of each dilution of the antibodies under study, 50 μl of medium with the addition of 0.1% BSA for each antibody sample - cell control, 150 μl of DMEM medium with 0.1% BSA - control medium were added.

50 μl of MDA-MB-231 cell suspension was added to each well with test antibodies and cell control.

50 μl of diluted complement were added to all wells with test antibodies and control cells. The tablet was shaken for 2-4 min on an orbital shaker at room temperature, placed in a CO ₂ incubator for 2-3 hours at a temperature of 37°C.

Added 15 μl of Alamar Blue reagent to the wells of the analyzed plate. Shake the plates for 10-20 minutes at room temperature on an orbital shaker to mix. The plates were additionally incubated in a CO ₂ incubator for 18-24 hours.

Shake the plates for 10-20 minutes at room temperature on an orbital shaker to mix.

Fluorescence readings were evaluated in relative fluorescence units at an excitation/emission wavelength of 544/590 nm using a plate fluorimeter. The resulting fluorescence signal is proportional to the number of viable cells. The results are presented in Fig.12-15.

According to the data obtained, the studied antibodies do not cause complement-mediated lysis (CDC) of the MDA-MB-231 cell line.

Example 22.

Analysis of the antagonistic activity of anti-PD-L1/anti-CD47 bispecific antibodies in a cell culture carrying the membrane PD-L1 receptor.

To analyze the antagonistic activity of anti-PD-L1/CD47 antibodies against the PD-L1 receptor, the ability of these antibodies to reactivate the luciferase signal in the Jurkat-PD1-NFAT-Luc reporter cell line upon co-culturing with PD-L1 producing cells was evaluated.

Preparation of PD-L1 Producer Cells

MDA-MB-231 culture was cultured in DMEM supplemented with 10% fetal bovine serum (FBS).

The cells were removed from the surface of the plastic using trypsin and suspended at a concentration of 1*10 ⁵ cells/ml in DMEM with 10% FBS and 20 ng/ml interferon gamma. Then 200 µl of the cell suspension was added to the wells of a white 96-well plate and incubated for 48 hours in a CO ₂ incubator at 37°C, 5% CO ₂ .

Preparation of test antibody dilutions

All tested anti-CD47/PD-L1 antibodies were diluted with RPMI-1640 medium with 10% FBS to a concentration of 5 μg/ml. A number of serial dilutions were prepared with a step of 3. The concentrations of the studied antibodies were (ng/ml): 2500; 833.3; 277.7; 92.5; 30.8; 10.2; 3.4; 1.1.

Preparation of Jurkat-PD1-NFAT-Luc cells

On the day of the experiment, a suspension of Jurkat-PD1-NFAT-Luc cells was prepared at a concentration of 2.5*10 ⁶ cells/ml in RPMI-1640 medium with 10% FBS.

Preparation of activating antibody solution

On the day of the experiment, a 10-fold mixture of activating antibodies was prepared (4 μg/ml anti-CD3; 4 μg/ml anti-CD28; 16 μg/ml anti-mouse in RPMI-1640 medium with 10% FBS).

Conducting a test

The growth medium was removed from the MDA-MB-231 cell plate. 40 μl of antibody dilutions were added to the wells with cells. Control wells (cells without test antibodies, cells without test and activating antibodies) were filled with 40 µl of RPMI 1640 10% FBS medium. Incubated at room temperature for 30 minutes.

Next, 40 µl of Jurkat-PD1-NFAT-Luc cell suspension was added to all wells. Then, 10 μl of a 10-fold solution of activating antibodies was added to all wells, except for the control wells "cells without test and activating antibodies". Cells were incubated for 6 hours in a CO ₂ incubator at 37°C, 5% CO ₂ .

Substrate to luciferase One-Glo Luciferase Assay System "Promega" was added to all wells at the rate of 1:1 (90 μl/well). After 5-10 minutes, the level of luminescence was measured on a tablet reader.

According to the data obtained in Fig. 16, the anti-PD-L1/anti-CD47 bispecific antibodies investigated, 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 towards cells carrying the PD-L1 receptor.

Example 23.

Homogeneity analysis of anti-PD-L1/anti-CD47 bispecific antibody preparations.

Analysis of the homogeneity of preparations of bispecific antibodies was performed by size exclusion HPLC (SEC HPLC) with a UV detector. Chromatography was performed on an Agilent 1100 HPLC system on a Tosoh TSK-Gel G3000SWXL column, 7.8 mm x 30 cm, cat. No. 08541 with Tosoh TSKgel Guard SWXL, 6.0 mm x 4.0 cm, 7 µm, Cat. No. 08543. Detection was carried out at wavelengths of 220 and 280 nm. As an example, Figure 17 shows the HPLC profile of a BCD106-02-013 preparation based on VHH47Opt2 (see Example 12). According to the results of this test, it can be concluded that the BCD106-02-013 molecule gives a drug that is homogeneous 93% in terms of monomer in terms of aggregation composition and is applicable for subsequent tests in vitro and in vivo.

--->

Sequence listing

<110> CLOSED JOINT STOCK COMPANY "BIOKAD"

<120> ANTIBODIES SPECIFIC FOR CD47 and PD-L1

<140> EA201791961

<141> 2017-10-03

<160> 108

<170> BiSSAP 1.3.6

<210> 1

<211> 5

<212> PRT

<213> Artificial sequence

<400> 1

Arg Tyr Trp Met Tyr

fifteen

<210> 2

<211> 5

<212> PRT

<213> Artificial sequence

<400> 2

Ile Asn Ala Met Asn

fifteen

<210> 3

<211> 5

<212> PRT

<213> Artificial sequence

<400> 3

Lys Tyr Trp Met Tyr

fifteen

<210> 4

<211> 9

<212> PRT

<213> Artificial sequence

<400> 4

Ser Ile Phe Ser Ile Asn Ala Met Asn

fifteen

<210> 5

<211> 5

<212> PRT

<213> Artificial sequence

<400> 5

Asp Tyr Ala Met Ser

fifteen

<210> 6

<211> 23

<212> PRT

<213> Artificial sequence

<400> 6

Ile Glu Asp Ser Ser Ile Asn Thr Gly Gly Gly Thr Glu Thr Thr Tyr

1 5 10 15

Tyr Thr Asp Ser Val Lys Gly

twenty

<210> 7

<211> 24

<212> PRT

<213> Artificial sequence

<400> 7

Ser Ile Glu Asp Ser Ser Ile Asn Thr Gly Gly Gly Thr Glu Thr Thr

1 5 10 15

Tyr Tyr Thr Asp Ser Val Lys Gly

twenty

<210> 8

<211> 24

<212> PRT

<213> Artificial sequence

<400> 8

Asn Ile Lys Gln Asp Gly Ile Asn Thr Gly Gly Gly Thr Ser Glu Lys

1 5 10 15

Tyr Tyr Val Asp Ser Val Lys Gly

twenty

<210> 9

<211> 17

<212> PRT

<213> Artificial sequence

<400> 9

Ser Ile Glu Asp Ser Ser Glu Thr Thr Tyr Tyr Thr Asp Ser Val Lys

1 5 10 15

gly

<210> 10

<211> 16

<212> PRT

<213> Artificial sequence

<400> 10

Gln Ile Thr Asp Glu Gly Ile Thr Asn Tyr Val Asp Ser Val Lys Gly

1 5 10 15

<210> 11

<211> 18

<212> PRT

<213> Artificial sequence

<400> 11

Ser Ile Glu Asp Ser Ser Thr Glu Thr Thr Tyr Tyr Thr Asp Ser Val

1 5 10 15

Lys Gly

<210> 12

<211> 19

<212> PRT

<213> Artificial sequence

<400> 12

Ser Ile Glu Asp Ser Ser Ile Thr Glu Thr Thr Tyr Tyr Thr Asp Ser

1 5 10 15

Val Lys Gly

<210> 13

<211> 18

<212> PRT

<213> Artificial sequence

<400> 13

Ser Ile Asn Asp Gly Gly Gly Asp Asp Ser Thr Tyr Tyr Ala Asp Ser

1 5 10 15

Val Lys

<210> 14

<211> 16

<212> PRT

<213> Artificial sequence

<400> 14

Gly Ile Arg Thr Ser Gly Asp Thr Asp Tyr Ala Asp Ser Val Lys Gly

1 5 10 15

<210> 15

<211> 16

<212> PRT

<213> Artificial sequence

<400> 15

Gln Ile Thr Asp Glu Gly Ile Thr Asn Tyr Val Asp Ser Val Lys Gly

1 5 10 15

<210> 16

<211> 17

<212> PRT

<213> Artificial sequence

<400> 16

Asp Ile Ser Trp Ser Gly Ser Asn Thr Asn Tyr Ala Asp Ser Val Lys

1 5 10 15

gly

<210> 17

<211> 18

<212> PRT

<213> Artificial sequence

<400> 17

Gly Asp Tyr Tyr Cys Thr Thr Tyr Glu Cys Gln His Tyr Tyr Gly Met

1 5 10 15

Asp Tyr

<210> 18

<211> 9

<212> PRT

<213> Artificial sequence

<400> 18

Val Ile Thr Thr Thr Ser Glu Ile Tyr

fifteen

<210> 19

<211> 18

<212> PRT

<213> Artificial sequence

<400> 19

Gly Asp Tyr Tyr Cys Thr His Tyr Glu Cys Gln His Tyr Tyr Gly Met

1 5 10 15

Asp Tyr

<210> 20

<211> 12

<212> PRT

<213> Artificial sequence

<400> 20

Asn Ala Phe Val Ile Thr Thr Thr Ser Glu Ile Tyr

1 5 10

<210> 21

<211> 13

<212> PRT

<213> Artificial sequence

<400> 21

Ala Pro Leu Leu Leu Ala Met Thr Phe Gly Val Gly Ser

1 5 10

<210> 22

<211> 11

<212> PRT

<213> Artificial sequence

<400> 22

Arg Ala Ser Gln Ala Ile Ser Ser Tyr Leu Ala

1 5 10

<210> 23

<211> 11

<212> PRT

<213> Artificial sequence

<400> 23

Arg Ala Ser Gln Gly Ile Ser Ser Trp Leu Ala

1 5 10

<210> 24

<211> 11

<212> PRT

<213> Artificial sequence

<400> 24

Arg Ala Ser Gln Ser Ile Ser Ser Tyr Leu Asn

1 5 10

<210> 25

<211> 11

<212> PRT

<213> Artificial sequence

<400> 25

Arg Ala Ser Gln Asp Ile Gly Ser Trp Leu Ala

1 5 10

<210> 26

<211> 11

<212> PRT

<213> Artificial sequence

<400> 26

Arg Ala Ser Gln Ser Ile Ser Arg Asn Leu Asn

1 5 10

<210> 27

<211> 11

<212> PRT

<213> Artificial sequence

<400> 27

Arg Ala Ser Gln His Ile Ser Ser Trp Leu Ala

1 5 10

<210> 28

<211> 11

<212> PRT

<213> Artificial sequence

<400> 28

Arg Ala Ser Gln Ser Ile Ser His Tyr Leu Asn

1 5 10

<210> 29

<211> 11

<212> PRT

<213> Artificial sequence

<400> 29

Arg Ala Ser Gln Ser Val Ser Ser Tyr Leu Ala

1 5 10

<210> 30

<211> 12

<212> PRT

<213> Artificial sequence

<400> 30

Arg Ala Ser Gln Ser Val Ser Ser Asn Tyr Leu Ala

1 5 10

<210> 31

<211> 13

<212> PRT

<213> Artificial sequence

<400> 31

Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn Tyr Val Ser

1 5 10

<210> 32

<211> 11

<212> PRT

<213> Artificial sequence

<400> 32

Gln Gly Asp Arg Ile Glu Asn Tyr Tyr Thr Ser

1 5 10

<210> 33

<211> 13

<212> PRT

<213> Artificial sequence

<400> 33

Thr Gly Ser Ser Ser Asn Ile Gly Asp Asn Tyr Val Asn

1 5 10

<210> 34

<211> 16

<212> PRT

<213> Artificial sequence

<400> 34

Thr Ser Ser Gln Ser Leu Val Tyr Arg Asp Gly Asn Thr Tyr Leu Asn

1 5 10 15

<210> 35

<211> 14

<212> PRT

<213> Artificial sequence

<400> 35

Gly Leu Ser Ser Gly Thr Val Thr Ala Ile Asn Tyr Pro Gly

1 5 10

<210> 36

<211> 7

<212> PRT

<213> Artificial sequence

<400> 36

Ala Ala Ser Asn Leu Gln Ser

fifteen

<210> 37

<211> 7

<212> PRT

<213> Artificial sequence

<400> 37

Ala Ala Ser Ser Leu Gln Ser

fifteen

<210> 38

<211> 7

<212> PRT

<213> Artificial sequence

<400> 38

Ala Ala Ser Thr Leu Gln Arg

fifteen

<210> 39

<211> 7

<212> PRT

<213> Artificial sequence

<400> 39

Thr Ala Ser Thr Leu Gln Ser

fifteen

<210> 40

<211> 7

<212> PRT

<213> Artificial sequence

<400> 40

Ala Ala Thr Arg Leu Gln Ser

fifteen

<210> 41

<211> 7

<212> PRT

<213> Artificial sequence

<400> 41

Asp Thr Ser Asn Arg Ala Thr

fifteen

<210> 42

<211> 7

<212> PRT

<213> Artificial sequence

<400> 42

Gly Thr Ser Ser Arg Ala Thr

fifteen

<210> 43

<211> 7

<212> PRT

<213> Artificial sequence

<400> 43

Ala Ser Ser Thr Leu Gln Ser

fifteen

<210> 44

<211> 7

<212> PRT

<213> Artificial sequence

<400> 44

Asp Asn Tyr Arg Arg Pro Ser

fifteen

<210> 45

<211> 7

<212> PRT

<213> Artificial sequence

<400> 45

Gly Asn Ala Asn Arg Pro Ser

fifteen

<210> 46

<211> 7

<212> PRT

<213> Artificial sequence

<400> 46

Ser Asn Ser Asn Arg Ala Ser

fifteen

<210> 47

<211> 7

<212> PRT

<213> Artificial sequence

<400> 47

Lys Val Ser Asp Arg Asp Ser

fifteen

<210> 48

<211> 7

<212> PRT

<213> Artificial sequence

<400> 48

Gly Ala Ser Ser Arg Ala Thr

fifteen

<210> 49

<211> 7

<212> PRT

<213> Artificial sequence

<400> 49

Asn Thr Asn Thr Arg His Ser

fifteen

<210> 50

<211> 9

<212> PRT

<213> Artificial sequence

<400> 50

Gln Gln Ala Ala Ser Val Pro Leu Thr

fifteen

<210> 51

<211> 9

<212> PRT

<213> Artificial sequence

<400> 51

Gln Gln Ser Tyr Ser Thr Pro Phe Thr

fifteen

<210> 52

<211> 9

<212> PRT

<213> Artificial sequence

<400> 52

Gln Gln Ser Tyr Ser Thr Pro Trp Thr

fifteen

<210> 53

<211> 9

<212> PRT

<213> Artificial sequence

<400> 53

Gln Gln Ser His Ser Leu Pro Leu Thr

fifteen

<210> 54

<211> 9

<212> PRT

<213> Artificial sequence

<400> 54

Gln Gln Ser Tyr Arg Thr Pro Phe Thr

fifteen

<210> 55

<211> 9

<212> PRT

<213> Artificial sequence

<400> 55

Gln Gln Ala Thr Ser Phe Pro Leu Thr

fifteen

<210> 56

<211> 9

<212> PRT

<213> Artificial sequence

<400> 56

Gln Gln Ser Tyr Ser Ala Leu Phe Thr

fifteen

<210> 57

<211> 9

<212> PRT

<213> Artificial sequence

<400> 57

Gln Gln Arg Ser Asn Trp Pro Pro Thr

fifteen

<210> 58

<211> 9

<212> PRT

<213> Artificial sequence

<400> 58

Gln Gln Ser Asp Thr Ser Pro Arg Val

fifteen

<210> 59

<211> 10

<212> PRT

<213> Artificial sequence

<400> 59

Gln Gln Ser Leu Ser Ile Pro Gln Val Thr

1 5 10

<210> 60

<211> 11

<212> PRT

<213> Artificial sequence

<400> 60

Gly Thr Trp Asp Ser Ser Leu Ser Ala Val Val

1 5 10

<210> 61

<211> 9

<212> PRT

<213> Artificial sequence

<400> 61

Gln Ser Gly Ser Ser Thr Glu Asn Ile

fifteen

<210> 62

<211> 10

<212> PRT

<213> Artificial sequence

<400> 62

Gln Ser Tyr Glu Ile Ser Gly Tyr Pro Val

1 5 10

<210> 63

<211> 9

<212> PRT

<213> Artificial sequence

<400> 63

Met Gln Gly Thr His Trp Pro Ser Thr

fifteen

<210> 64

<211> 10

<212> PRT

<213> Artificial sequence

<400> 64

Gln Gln Tyr Gly Ser Ser Pro Gln Leu Thr

1 5 10

<210> 65

<211> 10

<212> PRT

<213> Artificial sequence

<400> 65

Ala Leu Tyr Met Gly Asn Gly Gly His Met

1 5 10

<210> 66

<211> 134

<212> PRT

<213> Artificial sequence

<400> 66

Glu Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Thr Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Arg Tyr

20 25 30

Trp Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Glu Asp Ser Ser Ile Asn Thr Gly Gly Gly Thr Glu Thr

50 55 60

Thr Tyr Tyr Thr Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp

65 70 75 80

Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Lys Ser Glu

85 90 95

Asp Thr Ala Val Tyr Tyr Cys Ala Lys Gly Asp Tyr Tyr Cys Thr Thr

100 105 110

Tyr Glu Cys Gln His Tyr Tyr Gly Met Asp Tyr Trp Gly Lys Gly Thr

115 120 125

Met Val Thr Val Ser Ser

130

<210> 67

<211> 134

<212> PRT

<213> Artificial sequence

<400> 67

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Tyr

20 25 30

Trp Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Glu Asp Ser Ser Ile Asn Thr Gly Gly Gly Thr Glu Thr

50 55 60

Thr Tyr Tyr Thr Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp

65 70 75 80

Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu

85 90 95

Asp Thr Ala Val Tyr Tyr Cys Ala Lys Gly Asp Tyr Tyr Cys Thr Thr

100 105 110

Tyr Glu Cys Gln His Tyr Tyr Gly Met Asp Tyr Trp Gly Gln Gly Thr

115 120 125

Thr Val Thr Val Ser Ser

130

<210> 68

<211> 134

<212> PRT

<213> Artificial sequence

<400> 68

Glu Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Thr Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Arg Tyr

20 25 30

Trp Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Glu Asp Ser Ser Ile Asn Thr Gly Gly Gly Thr Glu Thr

50 55 60

Thr Tyr Tyr Thr Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp

65 70 75 80

Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Lys Ser Glu

85 90 95

Asp Thr Ala Val Tyr Tyr Cys Ala Lys Gly Asp Tyr Tyr Cys Thr Thr

100 105 110

Tyr Glu Cys Gln His Tyr Tyr Gly Met Asp Tyr Trp Gly Lys Gly Thr

115 120 125

Thr Val Thr Val Ser Ser

130

<210> 69

<211> 134

<212> PRT

<213> Artificial sequence

<400> 69

Glu Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Thr Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Arg Tyr

20 25 30

Trp Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Glu Asp Ser Ser Ile Asn Thr Gly Gly Gly Thr Glu Thr

50 55 60

Thr Tyr Tyr Thr Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp

65 70 75 80

Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Lys Ser Glu

85 90 95

Asp Thr Ala Val Tyr Tyr Cys Ala Lys Gly Asp Tyr Tyr Cys Thr Thr

100 105 110

Tyr Glu Cys Gln His Tyr Tyr Gly Met Asp Tyr Trp Gly Lys Gly Thr

115 120 125

Thr Val Thr Val Ser Ser

130

<210> 70

<211> 134

<212> PRT

<213> Artificial sequence

<400> 70

Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Thr Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Arg Tyr

20 25 30

Trp Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Glu Asp Ser Ser Ile Asn Thr Gly Gly Gly Thr Glu Thr

50 55 60

Thr Tyr Tyr Thr Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp

65 70 75 80

Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Lys Ser Glu

85 90 95

Asp Thr Ala Val Tyr Tyr Cys Ala Lys Gly Asp Tyr Tyr Cys Thr Thr

100 105 110

Tyr Glu Cys Gln His Tyr Tyr Gly Met Asp Tyr Trp Gly Lys Gly Thr

115 120 125

Leu Val Thr Val Ser Ser

130

<210> 71

<211> 134

<212> PRT

<213> Artificial sequence

<400> 71

Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Thr Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Arg Tyr

20 25 30

Trp Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Glu Asp Ser Ser Ile Asn Thr Gly Gly Gly Thr Glu Thr

50 55 60

Thr Tyr Tyr Thr Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp

65 70 75 80

Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Lys Ser Glu

85 90 95

Asp Thr Ala Val Tyr Tyr Cys Ala Lys Gly Asp Tyr Tyr Cys Thr Thr

100 105 110

Tyr Glu Cys Gln His Tyr Tyr Gly Met Asp Tyr Trp Gly Lys Gly Thr

115 120 125

Leu Val Thr Val Ser Ser

130

<210> 72

<211> 134

<212> PRT

<213> Artificial sequence

<400> 72

Glu Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Thr Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Arg Tyr

20 25 30

Trp Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Glu Asp Ser Ser Ile Asn Thr Gly Gly Gly Thr Glu Thr

50 55 60

Thr Tyr Tyr Thr Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp

65 70 75 80

Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Lys Ser Glu

85 90 95

Asp Thr Ala Val Tyr Tyr Cys Ala Lys Gly Asp Tyr Tyr Cys Thr Thr

100 105 110

Tyr Glu Cys Gln His Tyr Tyr Gly Met Asp Tyr Trp Gly Lys Gly Thr

115 120 125

Leu Val Thr Val Ser Ser

130

<210> 73

<211> 134

<212> PRT

<213> Artificial sequence

<400> 73

Glu Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Thr Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Arg Tyr

20 25 30

Trp Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Glu Asp Ser Ser Ile Asn Thr Gly Gly Gly Thr Glu Thr

50 55 60

Thr Tyr Tyr Thr Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp

65 70 75 80

Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Lys Ser Glu

85 90 95

Asp Thr Ala Val Tyr Tyr Cys Ala Lys Gly Asp Tyr Tyr Cys Thr Thr

100 105 110

Tyr Glu Cys Gln His Tyr Tyr Gly Met Asp Tyr Trp Gly Lys Gly Thr

115 120 125

Leu Val Thr Val Ser Ser

130

<210> 74

<211> 134

<212> PRT

<213> Artificial sequence

<400> 74

Glu Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Thr Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Arg Tyr

20 25 30

Trp Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Glu Asp Ser Ser Ile Asn Thr Gly Gly Gly Thr Glu Thr

50 55 60

Thr Tyr Tyr Thr Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp

65 70 75 80

Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Lys Ser Glu

85 90 95

Asp Thr Ala Val Tyr Tyr Cys Ala Lys Gly Asp Tyr Tyr Cys Thr Thr

100 105 110

Tyr Glu Cys Gln His Tyr Tyr Gly Met Asp Tyr Trp Gly Lys Gly Thr

115 120 125

Leu Val Thr Val Ser Ser

130

<210> 75

<211> 134

<212> PRT

<213> Artificial sequence

<400> 75

Gln Val Gln Leu Gln Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Thr Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Arg Tyr

20 25 30

Trp Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Glu Asp Ser Ser Ile Asn Thr Gly Gly Gly Thr Glu Thr

50 55 60

Thr Tyr Tyr Thr Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp

65 70 75 80

Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Lys Ser Glu

85 90 95

Asp Thr Ala Val Tyr Tyr Cys Ala Lys Gly Asp Tyr Tyr Cys Thr Thr

100 105 110

Tyr Glu Cys Gln His Tyr Tyr Gly Met Asp Tyr Trp Gly Lys Gly Thr

115 120 125

Met Val Thr Val Ser Ser

130

<210> 76

<211> 134

<212> PRT

<213> Artificial sequence

<400> 76

Gln Val Gln Leu Gln Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Thr Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Arg Tyr

20 25 30

Trp Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Glu Asp Ser Ser Ile Asn Thr Gly Gly Gly Thr Glu Thr

50 55 60

Thr Tyr Tyr Thr Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp

65 70 75 80

Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Lys Ser Glu

85 90 95

Asp Thr Ala Val Tyr Tyr Cys Ala Lys Gly Asp Tyr Tyr Cys Thr Thr

100 105 110

Tyr Glu Cys Gln His Tyr Tyr Gly Met Asp Tyr Trp Gly Lys Gly Thr

115 120 125

Met Val Thr Val Ser Ser

130

<210> 77

<211> 134

<212> PRT

<213> Artificial sequence

<400> 77

Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Thr Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Arg Tyr

20 25 30

Trp Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Glu Asp Ser Ser Ile Asn Thr Gly Gly Gly Thr Glu Thr

50 55 60

Thr Tyr Tyr Thr Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp

65 70 75 80

Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Lys Ser Glu

85 90 95

Asp Thr Ala Val Tyr Tyr Cys Ala Lys Gly Asp Tyr Tyr Cys Thr Thr

100 105 110

Tyr Glu Cys Gln His Tyr Tyr Gly Met Asp Tyr Trp Gly Lys Gly Thr

115 120 125

Leu Val Thr Val Ser Ser

130

<210> 78

<211> 134

<212> PRT

<213> Artificial sequence

<400> 78

Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Thr Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Arg Tyr

20 25 30

Trp Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Glu Asp Ser Ser Ile Asn Thr Gly Gly Gly Thr Glu Thr

50 55 60

Thr Tyr Tyr Thr Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp

65 70 75 80

Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Lys Ser Glu

85 90 95

Asp Thr Ala Val Tyr Tyr Cys Ala Lys Gly Asp Tyr Tyr Cys Thr Thr

100 105 110

Tyr Glu Cys Gln His Tyr Tyr Gly Met Asp Tyr Trp Gly Lys Gly Thr

115 120 125

Met Val Thr Val Ser Ser

130

<210> 79

<211> 134

<212> PRT

<213> Artificial sequence

<400> 79

Glu Val Gln Leu Gln Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Thr Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Arg Tyr

20 25 30

Trp Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Glu Asp Ser Ser Ile Asn Thr Gly Gly Gly Thr Glu Thr

50 55 60

Thr Tyr Tyr Thr Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp

65 70 75 80

Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Lys Ser Glu

85 90 95

Asp Thr Ala Val Tyr Tyr Cys Ala Lys Gly Asp Tyr Tyr Cys Thr Thr

100 105 110

Tyr Glu Cys Gln His Tyr Tyr Gly Met Asp Tyr Trp Gly Lys Gly Thr

115 120 125

Pro Val Thr Val Ser Ser

130

<210> 80

<211> 134

<212> PRT

<213> Artificial sequence

<400> 80

Gln Val Thr Leu Arg Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Thr Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Arg Tyr

20 25 30

Trp Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Glu Asp Ser Ser Ile Asn Thr Gly Gly Gly Thr Glu Thr

50 55 60

Thr Tyr Tyr Thr Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp

65 70 75 80

Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Lys Ser Glu

85 90 95

Asp Thr Ala Val Tyr Tyr Cys Ala Lys Gly Asp Tyr Tyr Cys Thr Thr

100 105 110

Tyr Glu Cys Gln His Tyr Tyr Gly Met Asp Tyr Trp Gly Lys Gly Thr

115 120 125

Met Val Thr Val Ser Ser

130

<210> 81

<211> 134

<212> PRT

<213> Artificial sequence

<400> 81

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Tyr

20 25 30

Trp Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Asn Ile Lys Gln Asp Gly Ile Asn Thr Gly Gly Gly Thr Ser Glu

50 55 60

Lys Tyr Tyr Val Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp

65 70 75 80

Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu

85 90 95

Asp Thr Ala Val Tyr Tyr Cys Ala Lys Gly Asp Tyr Tyr Cys Thr Thr

100 105 110

Tyr Glu Cys Gln His Tyr Tyr Gly Met Asp Tyr Trp Gly Gln Gly Thr

115 120 125

Thr Val Thr Val Ser Ser

130

<210> 82

<211> 127

<212> PRT

<213> Artificial sequence

<400> 82

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Tyr

20 25 30

Trp Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Glu Asp Ser Ser Glu Thr Thr Tyr Tyr Thr Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Lys Gly Asp Tyr Tyr Cys Thr Thr Tyr Glu Cys Gln His Tyr Tyr

100 105 110

Gly Met Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120 125

<210> 83

<211> 118

<212> PRT

<213> Artificial sequence

<400> 83

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Arg Ser Ile Phe Ser Ile Asn

20 25 30

Ala Met Asn Trp Tyr Arg Gln Ala Pro Gly Lys Gly Thr Glu Trp Val

35 40 45

Ala Gln Ile Thr Asp Glu Gly Ile Thr Asn Tyr Val Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Asn

85 90 95

Ala Phe Val Ile Thr Thr Thr Ser Glu Ile Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser

115

<210> 84

<211> 128

<212> PRT

<213> Artificial sequence

<400> 84

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Tyr

20 25 30

Trp Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Glu Asp Ser Ser Thr Glu Thr Thr Tyr Tyr Thr Asp Ser

50 55 60

Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu

65 70 75 80

Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ala Lys Gly Asp Tyr Tyr Cys Thr Thr Tyr Glu Cys Gln His Tyr

100 105 110

Tyr Gly Met Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120 125

<210> 85

<211> 129

<212> PRT

<213> Artificial sequence

<400> 85

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Tyr

20 25 30

Trp Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Glu Asp Ser Ser Ile Thr Glu Thr Thr Tyr Tyr Thr Asp

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Ala Lys Gly Asp Tyr Tyr Cys Thr Thr Tyr Glu Cys Gln His

100 105 110

Tyr Tyr Gly Met Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser

115 120 125

Ser

<210> 86

<211> 129

<212> PRT

<213> Artificial sequence

<400> 86

Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Gln Ala Ser Gly Phe Thr Phe Arg Arg Tyr

20 25 30

Trp Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Asn Asp Gly Gly Gly Asp Asp Ser Thr Tyr Tyr Ala Asp

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Val Lys Ser Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Ala Lys Gly Asp Tyr Tyr Cys Thr His Tyr Glu Cys Gln His

100 105 110

Tyr Tyr Gly Met Asp Tyr Trp Gly Lys Gly Thr Leu Val Thr Val Ser

115 120 125

Ser

<210> 87

<211> 126

<212> PRT

<213> Artificial sequence

<400> 87

Glu Val Gln Leu Val Gln Ser Gly Gly Asp Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Lys Tyr

20 25 30

Trp Met Tyr Trp Val Arg Gln Thr Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Gly Ile Arg Thr Ser Gly Asp Thr Asp Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Lys Gly Asp Tyr Tyr Cys Thr Thr Tyr Glu Cys Gln His Tyr Tyr Gly

100 105 110

Met Asp Tyr Trp Gly Lys Gly Thr Thr Val Thr Val Ser Ser

115 120 125

<210> 88

<211> 118

<212> PRT

<213> Artificial sequence

<400> 88

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Arg Ser Ile Phe Ser Ile Asn

20 25 30

Ala Met Asn Trp Tyr Arg Gln Ala Pro Gly Lys Gly Thr Glu Trp Val

35 40 45

Ala Gln Ile Thr Asp Glu Gly Ile Thr Asn Tyr Val Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Asn

85 90 95

Ala Phe Val Ile Thr Thr Thr Ser Glu Ile Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Val Thr Val Ser Ser

115

<210> 89

<211> 107

<212> PRT

<213> Artificial sequence

<400> 89

Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ala Ile Ser Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Asn Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Ala Ser Val Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 90

<211> 107

<212> PRT

<213> Artificial sequence

<400> 90

Val Ile Trp Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Phe

85 90 95

Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys

100 105

<210> 91

<211> 107

<212> PRT

<213> Artificial sequence

<400> 91

Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 92

<211> 107

<212> PRT

<213> Artificial sequence

<400> 92

Asn Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Gly Ser Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly His Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Thr Leu Gln Arg Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Gly Gly Tyr Gly Thr Glu Phe Thr Leu Thr Ile Asp Ser Leu Gln Ser

65 70 75 80

Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Ser His Ser Leu Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Gln Gly Gly Asn Gln

100 105

<210> 93

<211> 107

<212> PRT

<213> Artificial sequence

<400> 93

Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Thr Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Arg Asn

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Asn Leu Leu Ile

35 40 45

Tyr Thr Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Thr Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Ile Tyr His Cys Gln Gln Ser Tyr Arg Thr Pro Phe

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 94

<211> 107

<212> PRT

<213> Artificial sequence

<400> 94

Asp Ile Gln Leu Thr Gln Ser Pro Ser Ala Val Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln His Ile Ser Ser Trp

20 25 30

Leu Ala Trp Phe Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Pro Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Thr Ser Phe Pro Leu

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 95

<211> 107

<212> PRT

<213> Artificial sequence

<400> 95

Ala Ile Arg Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser His Tyr

20 25 30

Leu Asn Trp Tyr Gln Trp Lys Pro Gly Lys Val Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ala Thr Arg Leu Gln Ser Gly Val Pro Pro Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Ile Tyr Phe Cys Gln Gln Ser Tyr Ser Ala Leu Phe

85 90 95

Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys

100 105

<210> 96

<211> 107

<212> PRT

<213> Artificial sequence

<400> 96

Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Arg Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Thr Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 97

<211> 108

<212> PRT

<213> Artificial sequence

<400> 97

Glu Thr Thr Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Asn

20 25 30

Tyr Leu Ala Trp Tyr Gln His Lys Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

Ile Tyr Gly Thr Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asp Arg Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Asp Thr Ser Pro

85 90 95

Arg Val Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 98

<211> 108

<212> PRT

<213> Artificial sequence

<400> 98

Asn Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ala Ser Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Ser Leu Ser Ile Pro Gln

85 90 95

Val Thr Phe Gly Gly Gly Thr Lys Val Asp Ile Lys

100 105

<210> 99

<211> 110

<212> PRT

<213> Artificial sequence

<400> 99

Gln Ser Val Leu Thr Gln Pro Ser Val Ser Ala Ala Pro Ala Gln

1 5 10 15

Lys Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn

20 25 30

Tyr Val Ser Trp Tyr Gln Gln Val Pro Gly Thr Ala Pro Glu Leu Leu

35 40 45

Ile Tyr Asp Asn Tyr Arg Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Thr Leu Ala Ile Thr Gly Leu Gln

65 70 75 80

Thr Gly Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Trp Asp Ser Ser Leu

85 90 95

Ser Ala Val Val Phe Gly Gly Gly Thr Gln Leu Thr Val Leu

100 105 110

<210> 100

<211> 104

<212> PRT

<213> Artificial sequence

<400> 100

Glu Leu Thr Gln Pro Ser Ala Val Ser Val Ser Leu Gly Gln Thr Val

1 5 10 15

Arg Ile Thr Cys Gln Gly Asp Arg Ile Glu Asn Tyr Tyr Thr Ser Trp

20 25 30

Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr Gly Asn

35 40 45

Ala Asn Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser Ser Ser

50 55 60

Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu Asp Glu

65 70 75 80

Ala Asp Tyr Tyr Cys Gln Ser Gly Ser Ser Thr Glu Asn Ile Phe Gly

85 90 95

Gly Gly Thr Lys Leu Thr Val Leu

100

<210> 101

<211> 107

<212> PRT

<213> Artificial sequence

<400> 101

Leu Pro Val Leu Thr Gln Pro Ser Ala Val Ser Val Ser Leu Gly Gln

1 5 10 15

Thr Ala Arg Ile Thr Cys Gln Gly Asp Arg Ile Glu Asn Tyr Tyr Thr

20 25 30

Ser Trp Phe Arg Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr

35 40 45

Gly Asn Ala Asn Arg Pro Ser Gly Val Pro Glu Arg Phe Ser Gly Ser

50 55 60

Ser Ser Gly Val Thr Ala Thr Leu Thr Ile Ser Gly Ala Gln Ala Glu

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Gly Ser Ser Thr Glu Asn Ile

85 90 95

Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gln

100 105

<210> 102

<211> 107

<212> PRT

<213> Artificial sequence

<400> 102

Leu Pro Val Leu Thr Gln Pro Ser Ala Val Ser Val Ser Leu Gly Gln

1 5 10 15

Thr Ala Arg Ile Thr Cys Gln Gly Asp Arg Ile Glu Asn Tyr Tyr Thr

20 25 30

Ser Trp Phe Arg Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr

35 40 45

Gly Asn Ala Asn Arg Pro Ser Gly Val Pro Glu Arg Phe Ser Gly Ser

50 55 60

Ser Ser Gly Val Thr Ala Thr Leu Thr Ile Ser Gly Ala Gln Ala Glu

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Gly Ser Ser Thr Glu Asn Ile

85 90 95

Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gln

100 105

<210> 103

<211> 107

<212> PRT

<213> Artificial sequence

<400> 103

Leu Pro Val Leu Thr Gln Pro Ser Ala Val Ser Val Ser Leu Gly Gln

1 5 10 15

Thr Ala Arg Ile Thr Cys Gln Gly Asp Arg Ile Glu Asn Tyr Tyr Thr

20 25 30

Ser Trp Phe Arg Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr

35 40 45

Gly Asn Ala Asn Arg Pro Ser Gly Val Pro Glu Arg Phe Ser Gly Ser

50 55 60

Ser Ser Gly Val Thr Ala Thr Leu Thr Ile Ser Gly Ala Gln Ala Glu

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Gly Ser Ser Thr Glu Asn Ile

85 90 95

Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gln

100 105

<210> 104

<211> 110

<212> PRT

<213> Artificial sequence

<400> 104

Asn Phe Met Leu Thr Gln Pro Ser Val Ser Gly Ser Pro Gly Gln

1 5 10 15

Lys Phe Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Asp Asn

20 25 30

Tyr Val Asn Trp Tyr Gln His Leu Pro Gly Thr Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Ser Asn Ser Asn Arg Ala Ser Gly Val Pro Asp Arg Phe Ser

50 55 60

Gly Ser Ser Ser Gly Gly Thr Ala Thr Leu Thr Ile Ser Gly Ala Gln

65 70 75 80

Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Tyr Glu Ile Ser Gly

85 90 95

Tyr Pro Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gln

100 105 110

<210> 105

<211> 112

<212> PRT

<213> Artificial sequence

<400> 105

Glu Ile Val Leu Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Thr Ser Ser Gln Ser Leu Val Tyr Arg

20 25 30

Asp Gly Asn Thr Tyr Leu Asn Trp Phe Gln Gln Arg Pro Gly Gln Ser

35 40 45

Pro Arg Arg Leu Ile Tyr Lys Val Ser Asp Arg Asp Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Gly

85 90 95

Thr His Trp Pro Ser Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 106

<211> 108

<212> PRT

<213> Artificial sequence

<400> 106

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro Gln

85 90 95

Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 107

<211> 116

<212> PRT

<213> Artificial sequence

<400> 107

Gln Leu Leu Phe Asn Lys Thr Lys Ser Val Glu Phe Thr Phe Cys Asn

1 5 10 15

Asp Thr Val Val Ile Pro Cys Phe Val Thr Asn Met Glu Ala Gln Asn

20 25 30

Thr Thr Glu Val Tyr Val Lys Trp Lys Phe Lys Gly Arg Asp Ile Tyr

35 40 45

Thr Phe Asp Gly Ala Leu Asn Lys Ser Thr Val Pro Thr Asp Phe Ser

50 55 60

Ser Ala Lys Ile Glu Val Ser Gln Leu Leu Lys Gly Asp Ala Ser Leu

65 70 75 80

Lys Met Asp Lys Ser Asp Ala Val Ser His Thr Gly Asn Tyr Thr Cys

85 90 95

Glu Val Thr Glu Leu Thr Arg Glu Gly Glu Thr Ile Ile Glu Leu Lys

100 105 110

Tyr Arg Val Val

115

<210> 108

<211> 220

<212> PRT

<213> Artificial sequence

<400> 108

Phe Thr Val Thr Val Pro Lys Asp Leu Tyr Val Val Glu Tyr Gly Ser

1 5 10 15

Asn Met Thr Ile Glu Cys Lys Phe Pro Val Glu Lys Gln Leu Asp Leu

20 25 30

Ala Ala Leu Ile Val Tyr Trp Glu Met Glu Asp Lys Asn Ile Ile Gln

35 40 45

Phe Val His Gly Glu Glu Asp Leu Lys Val Gln His Ser Ser Tyr Arg

50 55 60

Gln Arg Ala Arg Leu Leu Lys Asp Gln Leu Ser Leu Gly Asn Ala Ala

65 70 75 80

Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr Arg Cys

85 90 95

Met Ile Ser Tyr Gly Gly Ala Asp Tyr Lys Arg Ile Thr Val Lys Val

100 105 110

Asn Ala Pro Tyr Asn Lys Ile Asn Gln Arg Ile Leu Val Val Asp Pro

115 120 125

Val Thr Ser Glu His Glu Leu Thr Cys Gln Ala Glu Gly Tyr Pro Lys

130 135 140

Ala Glu Val Ile Trp Thr Ser Ser Asp His Gln Val Leu Ser Gly Lys

145 150 155 160

Thr Thr Thr Thr Asn Ser Lys Arg Glu Glu Lys Leu Phe Asn Val Thr

165 170 175

Ser Thr Leu Arg Ile Asn Thr Thr Thr Asn Glu Ile Phe Tyr Cys Thr

180 185 190

Phe Arg Arg Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu Val Ile

195 200 205

Pro Glu Leu Pro Leu Ala His Pro Asn Glu Arg

210 215 220

<---

Claims (111)

1. A bispecific antibody that specifically binds to CD47 and PD-L1 and includes: i) a first antigen-binding fragment that specifically binds to CD47 and includes: a) heavy chain variable domain, which contains the sequences CDR1, CDR2, CDR3, where CDR1 is the sequence of SEQ ID NO: 1, where CDR2 is a sequence selected from the following group: SEQ ID NO: 6-9, 11, 12, where CDR3 is the sequence of SEQ ID NO: 17, and b) a light chain variable domain that contains the sequences CDR1, CDR2, CDR3, where CDR1 is the sequence of SEQ ID NO: 32, CDR2 is the sequence of SEQ ID NO: 45, CDR3 is the sequence of SEQ ID NO: 61; ii) a second antigen-binding fragment that specifically binds to PD-L1 and includes: a) a heavy chain variable domain which contains the following sequences: SEQ ID NO: 5, SEQ ID NO: 16 and SEQ ID NO: 21, and b) a light chain variable domain that contains the following sequences: SEQ ID NO: 35, SEQ ID NO: 49 and SEQ ID NO: 65; and iii) Fc fragment. 2. The bispecific antibody of claim 1, wherein the first antigen-binding fragment that specifically binds to CD47 comprises: (i) (a) a heavy chain variable domain comprising: CDR1 with amino acid sequence SEQ ID NO: 1, CDR2 with the amino acid sequence of SEQ ID NO: 6 and CDR3 with the amino acid sequence of SEQ ID NO: 17; and (b) a light chain variable domain comprising: CDR1 with amino acid sequence SEQ ID NO: 32, CDR2 with the amino acid sequence of SEQ ID NO: 45 and CDR3 with the amino acid sequence of SEQ ID NO: 61; (ii) (a) a heavy chain variable domain comprising: CDR1 with amino acid sequence SEQ ID NO: 1, CDR2 with the amino acid sequence of SEQ ID NO: 7 and CDR3 with the amino acid sequence of SEQ ID NO: 17; and (b) a light chain variable domain comprising: CDR1 with amino acid sequence SEQ ID NO: 32, CDR2 with the amino acid sequence of SEQ ID NO: 45 and CDR3 with the amino acid sequence of SEQ ID NO: 61; (iii) (a) a heavy chain variable domain comprising: CDR1 with amino acid sequence SEQ ID NO: 1, CDR2 with the amino acid sequence of SEQ ID NO: 8 and CDR3 with the amino acid sequence of SEQ ID NO: 17; and (b) a light chain variable domain comprising: CDR1 with amino acid sequence SEQ ID NO: 32, CDR2 with the amino acid sequence of SEQ ID NO: 45 and CDR3 with the amino acid sequence of SEQ ID NO: 61; (iv) (a) a heavy chain variable domain comprising: CDR1 with amino acid sequence SEQ ID NO: 1, CDR2 with the amino acid sequence of SEQ ID NO: 9 and CDR3 with the amino acid sequence of SEQ ID NO: 17; and (b) a light chain variable domain comprising: CDR1 with amino acid sequence SEQ ID NO: 32, CDR2 with the amino acid sequence of SEQ ID NO: 45 and CDR3 with the amino acid sequence of SEQ ID NO: 61; (v) (a) a heavy chain variable domain comprising: CDR1 with amino acid sequence SEQ ID NO: 1, CDR2 with the amino acid sequence of SEQ ID NO: 11 and CDR3 with the amino acid sequence of SEQ ID NO: 17; and (b) a light chain variable domain comprising: CDR1 with amino acid sequence SEQ ID NO: 32, CDR2 with the amino acid sequence of SEQ ID NO: 45 and CDR3 with the amino acid sequence of SEQ ID NO: 61; or (vi) (a) a heavy chain variable domain comprising: CDR1 with amino acid sequence SEQ ID NO: 1, CDR2 with the amino acid sequence of SEQ ID NO:12 and CDR3 with the amino acid sequence of SEQ ID NO: 17; and (b) a light chain variable domain comprising: CDR1 with amino acid sequence SEQ ID NO: 32, CDR2 with the amino acid sequence of SEQ ID NO: 45 and CDR3 with the amino acid sequence of SEQ ID NO: 61. 3. A bispecific antibody according to claim 1, characterized in that the first antigen-binding fragment that specifically binds to CD47 is a Fab, scFv, scFab. 4. A bispecific antibody according to claim 1, characterized in that the second antigen-binding fragment that specifically binds to PD-L1 is a Fab, scFv, scFab. 5. A bispecific antibody according to claim 1, characterized in that the antibody is a full length antibody. 6. A bispecific antibody according to claim 1, characterized in that the first antigen-binding fragment that specifically binds to CD47 inhibits the interaction of the CD47 receptor and the SIRP ligand, and/or the second antigen-binding fragment that specifically binds to PD-L1 inhibits the interaction of PD- L1 with PD-1 receptor. 7. A bispecific antibody according to claim 1, characterized in that the first antigen-binding fragment that specifically binds to CD47 includes a heavy chain variable domain that contains a sequence selected from the following group: SEQ ID NO: 67, 75, 76, 81, 82, 84, 85. 8. The bispecific antibody of claim 1, characterized in that the first antigen-binding fragment that specifically binds to CD47 includes a light chain variable domain that contains a sequence selected from the following group: SEQ ID NOs: 100-103. 9. The bispecific antibody of claim 1, characterized in that the first antigen-binding fragment that specifically binds to CD47 comprises: a) a heavy chain variable domain which contains sequences selected from the following group: SEQ ID NOs: 67, 75, 76, 81, 82, 84, 85, and b) a light chain variable domain that contains sequences selected from the following group: SEQ ID NO: 100-103. 10. A bispecific antibody according to claim 1, characterized in that the first antigen-binding fragment that specifically binds to CD47 includes: i) a) a heavy chain variable domain which contains the amino acid sequence of SEQ ID NO: 67 and b) a light chain variable domain that contains the amino acid sequence of SEQ ID NO: 100; (ii) a) a heavy chain variable domain which contains the amino acid sequence of SEQ ID NO: 85, and b) a light chain variable domain that contains the amino acid sequence of SEQ ID NO: 100; (iii) a) a heavy chain variable domain which contains the amino acid sequence of SEQ ID NO: 84, and b) a light chain variable domain that contains the amino acid sequence of SEQ ID NO: 100; (iv) a) a heavy chain variable domain which contains the amino acid sequence of SEQ ID NO: 82, and b) a light chain variable domain that contains the amino acid sequence of SEQ ID NO: 100; (v) a) a heavy chain variable domain which contains the amino acid sequence of SEQ ID NO: 81, and b) a light chain variable domain that contains the amino acid sequence of SEQ ID NO: 100; (vi) a) a heavy chain variable domain which contains the amino acid sequence of SEQ ID NO: 75, and b) a light chain variable domain that contains the amino acid sequence of SEQ ID NO: 101; (vii) a) a heavy chain variable domain which contains the amino acid sequence of SEQ ID NO: 76, and b) a light chain variable domain that contains the amino acid sequence of SEQ ID NO: 101; (viii) a) a heavy chain variable domain which contains the amino acid sequence of SEQ ID NO: 75, and b) a light chain variable domain that contains the amino acid sequence of SEQ ID NO: 102; (ix) a) a heavy chain variable domain which contains the amino acid sequence of SEQ ID NO: 76, and b) a light chain variable domain that contains the amino acid sequence of SEQ ID NO: 102; (x) a) a heavy chain variable domain which contains the amino acid sequence of SEQ ID NO: 75, and b) a light chain variable domain that contains the amino acid sequence of SEQ ID NO: 103; or (xi) a) a heavy chain variable domain which contains the amino acid sequence of SEQ ID NO: 76, and b) a light chain variable domain that contains the amino acid sequence of SEQ ID NO: 103. 11. A bispecific antibody according to claim 1, characterized in that it causes antibody-dependent cellular cytotoxicity, macrophage-mediated phagocytosis and/or T-cell mediated cytotoxicity against cells bearing CD47 and/or PD-L1 antigens on the surface. 12. A bispecific antibody according to claim 1, characterized in that it contains an Fc fragment with 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 mutation or modification. 13. A nucleic acid that encodes a bispecific antibody according to any one of paragraphs. 1-12. 14. The nucleic acid of claim 13, wherein the nucleic acid is DNA. 15. An expression vector containing a nucleic acid according to any one of paragraphs. 13, 14. 16. The method of obtaining a host cell to obtain bispecific antibodies according to any one of paragraphs. 1-12, which includes transforming the cell with the vector of claim 15. 17. A host cell to obtain a bispecific antibody according to any one of paragraphs. 1-12, containing a nucleic acid according to any one of paragraphs. 13, 14. 18. A method for producing a bispecific antibody according to any one of paragraphs. 1-12, which consists in culturing the host cell according to claim 17 in a culture medium under conditions sufficient to obtain the specified antibody, if necessary, followed by isolation and purification of the resulting antibody. 19. A pharmaceutical composition for the prevention or treatment of a disease or disorder mediated by PD-L1 and CD47, containing a bispecific antibody according to any one of paragraphs. 1-12 in combination with one or more pharmaceutically acceptable excipients. 20. Pharmaceutical composition according to claim 19, intended for the prevention or treatment of a disease or disorder mediated by PD-L1 and CD47, selected from the group: HNSCC (squamous cell carcinoma of the head and neck), cervical cancer, cancer without an identified primary source, glioblastoma, cancer esophagus, 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 high microsatellite instability), leukemia (acute or myeloid), lymphoma, multiple myeloma, breast cancer, prostate cancer, sarcoma, hepatocellular carcinoma, Hodgkin's lymphoma, T- and B-cell acute lymphoblastic leukemia, small cell lung cancer, acute myeloid leukemia, refractory B-cell non-Hodgkin's lymphoma, follicular lymphoma, marginal zone B-cell lymphoma, large B-cell diffuse lymphoma, squamous cell head and neck cancer, pancreatic cancer, ovarian cancer, acute myeloid leukemia, and high-risk myelodysplastic syndrome. 21. A method of inhibiting the biological activity of PD-L1 and/or CD47 in a subject in need of such inhibition, comprising administering to the subject an effective amount of a bispecific antibody according to any one of paragraphs. 1-12. 22. The use of a bispecific antibody according to any one of paragraphs. 1-12 for treatment in a subject in need of such treatment for a disease or disorder mediated by PD-L1 and CD47. 23. The use of an antibody according to claim 22, where the disease or disorder is selected from the group: HNSCC (squamous cell carcinoma of the head and neck), cervical cancer, cancer without an identified primary source, glioblastoma, esophageal cancer, bladder cancer, TNBC (three times negative breast cancer gland), CRC (colorectal cancer), hepatocellular carcinoma, melanoma, NSCLC (non-small cell lung cancer), kidney cancer, ovarian cancer, MSI CRC (high microsatellite instability colorectal cancer), leukemia (acute or myeloblastic), lymphoma, multiple myeloma, breast cancer, colorectal cancer, prostate cancer, bladder cancer, sarcoma, hepatocellular carcinoma, glioblastoma, Hodgkin's lymphoma, T- and B-cell acute lymphoblastic leukemia, small cell lung cancer, acute myeloid leukemia, refractory B-cell non-Hodgkin's lymphoma, follicular lymphoma, marginal zone B-cell lymphoma, diffuse large B-cell lymphoma, head and neck squamous cell carcinoma, pancreatic cancer, ovarian cancer, acute myeloid leukemia and high-risk myelodysplastic syndrome.

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