CD47 Antibody and Its Applications

Abstract

The present invention discloses a CD47 antibody and its applications. The CD47 monoclonal antibody 47-F provided by the present invention has a strong affinity for human CD47 protein, with a Kd value of 0.4068×10-9 M; it is of murine IgG1 subtype and the light chain is κ chain; it can compete with the commercial CD47 antibody B6H12.2 for binding to target cells; it can significantly induce murine macrophages to phagocytize B lymphoma cell Raji in vitro; it can be used to detect the expression of human CD47 protein, and can also be used alone or in combination with other methods for immunotherapy, having diagnostic and therapeutic values in tumors, autoimmune diseases, and inflammatory diseases.

Description

Technical Field

[0001] The present disclosure relates to the field of biomedicine, and in particular, to CD47 antibodies and their applications. Background Art

[0002] CD47 is a five-transmembrane glycoprotein composed of 323 amino acids with a molecular weight of approximately 50 kDa. It contains an immunoglobulin (Ig)-like extracellular domain, five transmembrane domains, and a short intracellular tail. CD47 belongs to the immunoglobulin superfamily and together with the SIRPα protein on the surface of macrophages forms the "don't eat me" signal, inhibiting the phagocytic function of macrophages. It has been reported that CD47 is overexpressed in various hematological malignancies (such as acute myeloid leukemia, myelodysplastic syndrome) and solid tumors (such as breast cancer, lung cancer, colorectal cancer). Tumor cells evade macrophage phagocytosis by highly expressing CD47. Studies have shown that the CD47 expression level inside tumors is significantly positively correlated with poor treatment prognosis. Therefore, the CD47-SIRPα pathway is considered a highly potential immunotherapy target.

[0003] Currently, a variety of antibody drugs targeting CD47 have made progress in preclinical or clinical studies: for example, Magrolimab developed by Gilead Sciences. Early clinical trials showed that Magrolimab demonstrated good efficacy in patients with high-risk myelodysplastic syndrome and acute myeloid leukemia, and it was the first CD47-targeted drug to enter phase III clinical trials. However, in February 2024, the phase III clinical trial of Magrolimab (NCT05079230) was prematurely terminated due to an increased mortality rate. Not only Magrolimab, but current antibody drugs targeting CD47 (such as B6H12.2, CC90002, etc.) generally face serious side effects such as anemia and platelet decline, and no drug has been officially applied clinically. Therefore, it is necessary to further explore and develop new antibody drugs targeting human CD47 to provide new options for subsequent clinical research and applications. Summary of the Invention

[0004] Technical Problem to be Solved:

[0005] One aspect of the present disclosure is to provide an antibody against CD47 in view of the problem in the prior art of the lack of an ideal molecule that specifically binds to human CD47.

[0006] Technical Solution:

[0007] An isolated antibody or antigen-binding portion, wherein the antibody or antigen-binding portion specifically binds to human CD47 protein, and the antibody or antigen-binding portion comprises:

[0008] The heavy chain variable region CDRH1 as shown in SEQ ID No.1, the heavy chain variable region CDRH2 as shown in SEQ ID No.2, the heavy chain variable region CDRH3 as shown in SEQ ID No.3, the light chain variable region CDRL1 as shown in SEQ ID No.4, the light chain variable region CDRL2 as shown in SEQ ID No.5, and the light chain variable region CDRL3 as shown in SEQ ID No.6.

[0009] In one embodiment, the antibody or antigen-binding portion comprises: the heavy chain variable region as shown in SEQ ID No.7 and the light chain variable region as shown in SEQ ID No.8.

[0010] In some embodiments, the antibody can be an antibody of mammalian origin, such as mouse, rabbit, sheep, horse, monkey, pig, camel, shark, chicken, etc. In other embodiments, the antibody can be a chimeric antibody, a humanized antibody or a fully human antibody.

[0011] In some embodiments, the antibody can be IgG, IgA, IgM, IgD or IgE. Preferably, in some embodiments, the type of the antibody can be IgG. Further, in some embodiments, the antibody can be one or more selected from IgG1, IgG2, IgG3 or IgG4.

[0012] In some embodiments, the antibody is a monoclonal antibody.

[0013] In some embodiments, the antibody or antigen-binding portion is modified, and the modifications include N-glycosylation modification, O-glycosylation modification, phosphorylation modification, methylation modification, acetylation modification or label modification.

[0014] In some embodiments, the antibody comprises an Fc portion. Preferably, in some embodiments, the Fc portion of the antibody is modified or engineered to enhance its ADCC activity, CDC activity or ADCP activity.

[0015] In some embodiments, the antigen-binding portion is Fab, Fab', F(ab') 2 , Fd, FCL, dAb or single-chain antibody scFv. Preferably, in some embodiments, the antigen-binding portion is single-chain antibody scFv.

[0016] Another aspect of the present disclosure is to provide a multivalent antibody, which comprises the above-mentioned antibody or antigen-binding portion.

[0017] The multivalent antibody can be, for example, bivalent, trivalent, tetravalent, hexavalent, nonavalent, etc. The multivalent antibody can be prepared by suitable methods in the prior art. Preferably, in some embodiments, the multivalent antibody is a bispecific antibody or a trispecific antibody.

[0018] Another aspect of the present disclosure is to provide a multispecific antibody that at least selectively binds to human CD47. The multispecific antibody comprises the above antibody or antigen-binding portion; the multispecific antibody is a monovalent antibody or a multivalent antibody.

[0019] Another aspect of the present disclosure is to provide an isolated polynucleotide that encodes the above antibody or antigen-binding portion, or encodes the above multivalent antibody. In one embodiment, the sequence of the heavy chain variable region of the polynucleotide is as shown in SEQ ID No. 9, and the sequence of the light chain variable region of the polynucleotide is as shown in SEQ ID No. 10.

[0020] Another aspect of the present disclosure is to provide a vector that contains the above polynucleotide.

[0021] Another aspect of the present disclosure is to provide a cell that includes the above antibody or antigen-binding portion, the above multivalent antibody, the above polynucleotide, or the above vector. In some embodiments, the cell can be any suitable host cell as a tool for producing target proteins. For example, SP2 / 0, YB2 / 0, IR983F, human myeloma Namalwa, PERC6 or CHO cell lines, insect cells, Escherichia coli cells.

[0022] Another aspect of the present disclosure is to provide a pharmaceutical composition that contains the above antibody or antigen-binding portion, the above multivalent antibody, the above polynucleotide, the above vector or the above cell, and a pharmaceutically acceptable carrier. To achieve better therapeutic effects, in some embodiments, the pharmaceutical composition may further contain other therapeutic drugs.

[0023] Another aspect of the present disclosure is to provide the use of the above antibody or antigen-binding portion, the above multivalent antibody, the above polynucleotide, the above vector, the above cell or the above pharmaceutical composition in the preparation of a drug for treating the following diseases: tumors, autoimmune diseases, inflammatory diseases.

[0024] Another aspect of the present disclosure is to provide the use of the above antibody or antigen-binding portion or the above multivalent antibody in the preparation of a product for detecting the presence or level of human CD47 molecules in a sample.

[0025] Advantageous effects:

[0026] The anti-human CD47 monoclonal antibody 47-F provided by the present invention has a strong affinity for human CD47 protein, with a Kd value of 0.4068×10 -9 M; it is of murine IgG1 subtype and its light chain is κ chain; it can compete with the commercial CD47 antibody B6H12.2 for binding to target cells; it can significantly induce murine macrophages to phagocytose B lymphoma cells Raji in vitro; it can be used to detect the expression of human CD47 protein and can also be used alone or in combination with other methods for immunotherapy, having diagnostic and therapeutic value in tumors, autoimmune diseases, and inflammatory diseases. Brief Description of the Drawings

[0027] Figure 1 It is a graph showing the results of flow cytometry for measuring the affinity constant of antibody 47-F in the embodiments of the present disclosure;

[0028] Figure 2 It is a graph showing the results of flow cytometry for measuring the competitive binding of antibody 47-F and commercial antibody B6H12.2 in the embodiments of the present disclosure;

[0029] Figure 3 It is a graph showing the results of high-content imaging of antibody 47-F promoting murine macrophages to phagocytose lymphoma cell line Raji in the embodiments of the present disclosure, where B6H12.2 is the commercial positive control group, the red in the figure is murine macrophages, and the green is the phagocytosed Raji cells;

[0030] Figure 4 It is a dose-dependent curve graph of antibody 47-F promoting murine macrophages to phagocytose lymphoma cell line Raji in the embodiments of the present disclosure.

[0031] Sequence Description

[0032] Detailed Embodiments

[0033] The present invention discloses an anti-human CD47 antibody and its application. Those skilled in the art can draw on the content of this article and appropriately modify the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. And relevant personnel can obviously make modifications or appropriate changes and combinations to the content described herein without departing from the content, spirit, and scope of the present invention to implement and apply the technology of the present invention.

[0034] In this disclosure, unless otherwise specified, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Unless otherwise clearly indicated, throughout the specification and claims, the term "comprising" or its variations such as "including" or "having" etc. will be understood to include the stated elements or components, without excluding other elements or other components. The term "a" (or "an") includes plural referents. The term "plural" means two or more. Terms such as "such as" and "for example" are intended to indicate exemplary embodiments and are not intended to limit the scope of this disclosure.

[0035] In this disclosure, when a range of values is provided, it should be understood that unless the context clearly indicates otherwise, the endpoints are included in the range and each intermediate value between the upper and lower limits of the range and any other specified value or intermediate value within the specified range, as well as any value within the smaller range between the specified values, are all encompassed.

[0036] In this disclosure, the term "about" generally means varying within a range of 0.5% - 10% above or below the specified value, for example, varying within a range of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below the specified value.

[0037] In the present disclosure, unless otherwise specified, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Definitions of common terms in molecular biology can be found in Lewin’s GENES, Twelfth Edition, Jocelyn E. Krebs, Elliott S. Goldstein, Stephen T. Kilpatrick, Publisher: Jones & Bartlett Learning. Definitions of common terms in biochemistry can be found in Lehninger Principles of Biochemistry, Eighth Edition, David L. Nelson, Michael M. Cox, Publisher: W. H. Freeman. Definitions of common terms in cell biology can be found in Molecular Biology of the Cell, Sixth Edition, Bruce Alberts, Alexander Johnson, Julian Lewis, David Morgan, Martin Raff, Keith Roberts, Peter Walter, Publisher: Garland Science. Definitions of common terms in genetics can be found in Genetics: Analysis of Genes and Genomes, Eighth Edition, Daniel L. Hartl, Maryellen Ruvolo, Publisher: Jones & Bartlett Learning.

[0038] Unless otherwise specified, experimental techniques herein employ conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA, which can be found in standard books such as: 《Molecular Cloning: A Laboratory Manual》; 《Cell Biology: A Laboratory Handbook》, etc.

[0039] Definitions:

[0040] As used herein, the term "isolated" refers to a substance or entity that has been removed from its natural environment or the environment in which it existed prior to isolation and is separated from other components. For example, an isolated protein is substantially free of cellular material or other proteins from the cell or tissue source from which it is derived. The proportion of separation can be, for example, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%. Isolated substances may have different purity levels compared to the substances prior to their isolation.

[0041] As used herein, the term "antibody" refers to an immunoglobulin molecule typically composed of two pairs of polypeptide chains (each pair having one "light" (L) chain and one "heavy" (H) chain). Antibody light chains can be classified as κ and λ light chains. Heavy chains can be classified as μ, δ, γ, α, or ε, and the isotypes of antibodies are defined as IgM, IgD, IgG, IgA, and IgE, respectively. Within the light and heavy chains, the variable and constant regions are joined by a "J" region of approximately 12 or more amino acids, and the heavy chain also contains a "D" region of approximately 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain, CL. The constant region of an antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The VH and VL regions can also be further divided into regions of high variability (called complementarity-determining regions (CDRs)), interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminus to the carboxyl terminus. The variable regions (VH and VL) of each heavy chain / light chain pair form the antibody binding site. The term "antibody" is not limited by any particular method of antibody production. For example, it includes, in particular, recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. Antibodies can be antibodies of different isotypes, such as IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies. In some embodiments, the antibody can be IgG, IgA, IgM, IgD, or IgE. Preferably, in some embodiments, the type of the antibody can be IgG. Further, in some embodiments, the antibody can be one or more selected from IgG1, IgG2, IgG3, or IgG4. Preferably, the antibody can be IgG1.

[0042] Preparation of antibodies:

[0043] In some embodiments, mammalian cells are used to produce the antibody. For example, monoclonal antibodies are produced in mammalian cells using hybridoma technology. The monoclonal antibodies can be prepared by the hybridoma preparation method reported by Kohler et al. in Nature 256:495 (1975). First, a mouse or other suitable host animal is immunized with an immunogen (optionally with an adjuvant).

[0044] The immunogen or adjuvant is usually injected subcutaneously at multiple points or intraperitoneally. Adjuvants can include Freund's adjuvant (Freund's complete adjuvant or Freund's incomplete adjuvant) or MPL-TDM, etc. After the animal is immunized, lymphocytes that secrete antibodies specifically binding to the immunogen are generated in the body. The target lymphocytes are collected and fused with myeloma cells using a suitable fusogen (such as PEG4000) to obtain hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103, Academic Press, 1996).

[0045] The hybridoma cells prepared above are inoculated into a suitable medium for growth, and the medium contains one or more substances that can inhibit the growth of unfused, parental myeloma cells. For example, for parental myeloma cells lacking hypoxanthine-guanine phosphoribosyltransferase (HGPRT or HPRT), adding substances such as hypoxanthine, aminopterin, and thymidine (HAT medium) to the medium can inhibit the growth of HGPRT-deficient cells.

[0046] Preferred myeloma cells should have high fusion rates, stable antibody secretion ability, sensitivity to HAT medium, etc. Among them, murine myeloma is preferred, such as MOP-21 and MC-11 mouse tumor-derived strains (THE Salk Institute Cell Distribution Center, San Diego, Calif., USA), and SP-2 / 0 or X63-Ag8-653 cell lines (American Type Culture Collection, Rockville, Md., USA). In addition, human myeloma and human-mouse heteromyeloma cell lines can also be used to prepare human monoclonal antibodies (Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63, Marcel Dekker, Inc., New York, 1987).

[0047] The medium for hybridoma cell growth is used to detect the production of monoclonal antibodies against specific antigens. The following methods can be used to determine the binding specificity of monoclonal antibodies produced by hybridoma cells: immunoprecipitation or in vitro binding assays, such as radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA). For example, the Scatchard analysis described by Munson et al. in Anal. Biochem. 107:220 (1980) can be used to determine the affinity of monoclonal antibodies.

[0048] After determining the specificity, affinity, and reactivity of the antibodies produced by the hybridoma, the target cell line can be subcloned by the limited dilution method described by Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103, Academic Press, 1996. Suitable media can be DMEM or RPMI-1640, etc. In addition, hybridoma cells can also grow in the form of ascites tumors in animals.

[0049] Using traditional immunoglobulin purification methods, such as protein A agarose gel, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography, etc., the monoclonal antibodies secreted by subcloned cells can be separated from cell culture supernatants, ascites, or sera, and thus the monoclonal antibodies can be obtained.

[0050] In other embodiments, the anti-human CD47 antibody can also be produced by known recombinant methods. For example, a recombinant antibody library in a phage or similar vector can be selected. See, for example, the description in Smith GP. Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface. Science. 1985;228:1315–17.

[0051] Modification and alteration of the antibody:

[0052] In some embodiments, the isolated antibody can be a humanized antibody. Humanizing the antibody may improve the affinity or other characteristics of the antibody. Descriptions and methods of humanized antibodies can be referred to Riechmann, L., Clark, M., Waldmann, H., & Winter, G. (1988). Reshaping human antibodies for therapy. Nature, 332(6162), 323–327.

[0053] In some embodiments, the Fc (fragment crystallizable region, Fc) of the antibody is modified to enhance its effector functions by binding to Fc receptors or complement. These functions can include: complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), and antibody-dependent cellular phagocytosis (ADCP). The above modifications can include: 1) Modifying glycosylation. For example, asparagine 297 (N297) in the Fc region can be modified by N-acetylglucosamine. Mutating N297 to alanine (A), glutamine (Q), or glycine (G) will all hinder the glycosylation of the antibody, thereby reducing the Fc-mediated effector functions. After the antibody is deglycosylated, the ability to induce ADCC or CDC activity will decrease; sialic acid modification will reduce the binding affinity to FcγRIIIa, thus resulting in a decrease in CDC and ADCC activities.

[0054] In addition to the above functions, glycosylation modification of antibodies can also affect the conformation and stability of antibodies. For example, the sugar chains in glycosylation modification can maintain the conformation of antibodies and prevent them from aggregating or unfolding. For example, the sugars on the α1-3 arm do not contact the antibody surface but penetrate into the space formed by the Fc segments of the two heavy chains. There is an interaction between the mannoses on the α1-3 arms of the two sugar chains, which is very important for maintaining the conformation of antibodies. Without sugar chains, the CH2 domain of the Fc segment will expand slightly, resulting in an earlier elution time of the antibody in size exclusion chromatography and being more sensitive and prone to aggregation in thermal acceleration stability experiments. At the same time, glycosylation modification can also affect the binding of antibodies to receptors on the cell membrane to form complexes, thus playing an important role in the signal transduction process. This regulation of signal transduction is crucial for physiological processes such as cell proliferation, differentiation, and apoptosis.

[0055] The above-mentioned modification can also include: 2) point mutation. For example, the LALA mutation (L234A / L235A) will cause a change in the affinity of the antibody for FcγR (eliminating the binding to low-affinity FcγR and reducing the binding to FcγRI), thus significantly reducing its ADCC and CDC activities. In addition, the effector function of the antibody can also be regulated by the combination of cross-subtype antibodies.

[0056] In some embodiments, the point mutation results in the substitution of some conserved amino acids, thereby obtaining a "variant with conserved amino acid substitution". The change results in the substitution of some amino acids with other amino acids that are chemically and / or functionally similar. Conservative substitution tables providing chemically and / or functionally similar amino acids are well known in the art. Typical examples of mutually conservative substitutions are, for example, (1) alanine (A), glycine (G); (2) aspartic acid (D), glutamic acid (E); (3) asparagine (N), glutamine (Q); (4) arginine (R), lysine (K); (5) isoleucine (I), leucine (L), methionine (M), valine (V); (6) phenylalanine (F), tyrosine (Y), tryptophan (W); (7) serine (S), threonine (T); (8) cysteine (C), methionine (M).

[0057] The above-mentioned modifications may also include: 3) Phosphorylation modification. Phosphorylation modification refers to the process of adding phosphate groups to the amino acids of proteins within cells. Phosphorylated antibodies can specifically recognize specific phosphorylation sites, thereby detecting the enhancement or reduction of the phosphorylation level of proteins when cells are stimulated. These antibodies play an important role in life science research fields such as cell signaling, apoptosis, and cancer. 4) Methylation modification: Methylation modification is an important dynamic modification and biological phenomenon catalyzed by methyltransferases on specific residues of proteins. Methylated antibodies can specifically recognize specific methylated amino acid sites to distinguish between methylated and non-methylated forms of proteins. They have a wide range of applications in research fields such as epigenetics, cancer, Alzheimer's disease, and aging. 5) Acetylation modification. Acetylation is one of the most common types of acylation modifications. Acetylated antibodies can specifically recognize the acetylated form of the target protein and specific acetylated amino acid sites to detect the activity level of the protein. These antibodies are widely used in research on the regulation of the cell cycle, signal transduction, neurodegenerative diseases, metabolic diseases, and the occurrence and development of cancer.

[0058] The above-mentioned modifications may also include: 6) Label modification. Antibodies can be cross-linked with different chemical reagents to be linked to substances such as enzymes, fluorescent dyes, biotin, or colloidal gold, so as to change their detection or analysis performance. For example, enzyme labeling: Antibodies can be cross-linked to enzymes, such as horseradish peroxidase (HRP), alkaline phosphatase, etc. It is commonly used in experiments such as immunohistochemistry and ELISA. Through the catalytic action of the enzyme, a color reaction is generated to detect the presence of antibodies. For example, an HRP-labeled antibody can produce a color precipitate after binding to the antigen by adding a substrate, which is convenient for observation and quantification. Fluorescent dye labeling: Antibodies can also be combined with fluorescent dyes (such as FTC, PE, APC, etc.) for detection methods such as flow cytometry and fluorescence microscopy. Fluorescent-labeled antibodies can localize specific antigens in cells or tissue sections and judge the expression level of antigens according to the intensity of the fluorescent signal. Biotin labeling: Biotin is a small molecule compound that can bind to antibodies without affecting their antigen-binding ability. Biotin-labeled antibodies can achieve signal amplification and detection by binding to avidin (such as streptavidin). It is commonly used in multiplex immunolabeling experiments to detect multiple antigens simultaneously.

[0059] In the present disclosure, modifications and alterations to antibodies generally occur in the Fc region of the antibody and in the framework regions (FRs) of the antibody variable region, rather than in the complementarity-determining regions (CDRs) of the antibody variable region. The framework regions of the antibody variable region have relatively conserved amino acid sequences, provide stable support for the hypervariable region structure, and are involved in maintaining the three-dimensional conformation of the antigen-binding groove. Therefore, modifications and alterations to it do not affect the binding ability of the antibody. In the present disclosure, the term "having more than 90% identity" means having approximately 90%, approximately 91%, approximately 92%, approximately 93%, approximately 94%, approximately 95%, approximately 96%, approximately 97%, approximately 98%, approximately 99% identity compared to the sequences shown in SEQ ID No. 1-16 under the above-described modification or alteration methods.

[0060] Isolated antigen-binding portion:

[0061] The term "antigen-binding portion" in the present disclosure refers to a polypeptide comprising a fragment of a full-length antibody that retains the ability to specifically bind the same antigen to which the full-length antibody binds and / or competes with the full-length antibody for specific binding to the antigen, and is also referred to as an "antigen-binding fragment". See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed., Raven Press, N.Y. (1989)). Antigen-binding fragments of antibodies can be generated by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. In some cases, antigen-binding fragments include Fab, Fab′, F(ab′) 2 , Fd, Fv, etc.

[0062] Among them, the term "Fab fragment" means an antibody fragment consisting of the VL, VH, CL, and CH1 domains; the term "F(ab′) 2 fragment" means an antibody fragment comprising two Fab fragments linked by a disulfide bridge in the hinge region. The term "Fd fragment" means an antibody fragment consisting of the VH and CH1 domains; the term "Fv fragment" means an antibody fragment consisting of the VL and VH domains of a single arm of the antibody.

[0063] In some embodiments, the above antigen-binding portion is prepared by protease digestion using proteases such as papain, pepsin, etc. In other embodiments, the above antigen-binding portion is prepared by treatment with chemical reagents. In still other embodiments, the above antigen-binding portion is prepared by genetic engineering methods. That is, a fragment containing all or part of the gene sequence of the antigen-binding portion is ligated to a suitable vector and expressed. Examples of the expression vectors include bacterial plasmids, phages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors.

[0064] The term "polynucleotide" in the present disclosure is also interchangeably used as "nucleic acid" and refers to a chain of nucleotides of any length and includes DNA or RNA. It may include any known nucleotide analogs or modified nucleotides or bases.

[0065] Examples:

[0066] To enable those skilled in the art to better understand the technical solutions of the present invention, the present invention will be further described in detail below with reference to specific embodiments.

[0067] Example 1: Screening of mouse hybridoma monoclonal antibodies

[0068] A six-week-old Balb / c female mouse was immunized by intraperitoneal injection three times with an hCD47-positive cell line, 1×10 7 cells each time, with a two-week interval between each immunization. On the eighth day after the third immunization, the mouse tail blood was taken and the titer was identified by flow cytometry with CD47-positive cells Jurkat. And 1×10 7 hCD47-positive cells were intraperitoneally injected for booster immunization 1-2 weeks after the three immunizations.

[0069] The steps for identifying the titer are as follows:

[0070] The mouse tail blood was dissolved in PBS and left standing at room temperature for 1 hour. Centrifuged at 12000 rpm at 4°C for 10 minutes, the supernatant was collected, the precipitate was discarded, and diluted with PBS in half to different concentrations, diluted from 1:200 to 1:12800. Jurkat cells were collected, washed once with PBS, and 1×10 6cells, added 100 μl of mouse serum of different dilutions to the cells, mixed well, used non-immune mouse serum as negative control group, incubated at 4°C for 30 min, washed twice with PBS, added 0.2 μl of anti-mouse IgG antibody (APC marker) to each well, incubated at 4°C in the dark for 30 min, resuspended the cells in 500 μl PBS buffer, and detected the binding of antibodies in serum to cells by flow cytometry, analyzed the percentage and fluorescence intensity, when the average fluorescence intensity of the test serum test tube was more than twice that of the secondary antibody control tube, the corresponding dilution was the effective titer, and when the titer was higher than 6400, the corresponding immunized mice could be used as fusion mice.

[0071] Three days before fusion, the mice were immunized by tail vein injection of immune cells. Two days before fusion, trophoblast cells were plated on a flat-bottom 96-well plate for fusion. The steps are as follows: Healthy eight-week-old Balb / c mice were killed by dragging their necks, and after disinfection of the body surface, a small incision was made in the center of the lower abdomen to open the peritoneum.

[0072] The fusion steps are as follows: Take the spleen cells of the successfully immunized mice and myeloma SP2 / 0 cells and mix them in a 37°C water bath. After centrifugation, discard the supernatant. Add 50% PEG to the mixed cell mass within 1 minute, shake in a 37°C water bath for 1 minute; add 10 ml of serum-free 1640 medium within 2 minutes; centrifuge at 500g for 5 minutes, discard the supernatant; resuspend the cells with 1640 medium containing HAT, mix and pipette into a flat-bottom 96-well plate. The number of cells is 2.5x10 7 / plate; at 37°C, 5% CO 2 Cells were cultured under these conditions.

[0073] One week after fusion, when the clones were observed to be large enough (at least 1 / 8 of the area of ​​a single well of a flat-bottom 96-well plate), culture supernatant screening was performed. That is, 100 μl of supernatant was taken from each well and mixed with 2×10 5 hCD47 positive cells were co-cultured and flow cytometry was used to detect the positive situation. The operation method was the same as the titer detection method. The average immunofluorescence intensity of the negative wells was more than twice that of the negative wells as positive wells, and the corresponding hybridomas were regarded as positive hybridomas for the next step. Otherwise, they were regarded as negative hybridomas and discarded.

[0074] The positive hybridoma clones were expanded from 96-well plates to 24-well plates and cultured for 3-5 days. The culture supernatant was screened again and the clones that were positive were then subcloned and the remaining cells were frozen.

[0075] The 24-well plates with positive results were subcloned by limiting dilution. The hybridoma cells in the 24-well plate were evenly blown, the cell density was adjusted to 10 cells / mL, and the cells were plated into a flat-bottom 96-well plate with 200 μl per well at 37°C and 5% CO.2 Cultured in an incubator for about 10 days, visible clone formation can be observed. Select the wells with only single clones, aspirate 100 μl of the culture supernatant, and the detection method is the same as before. Select the positive clones, expand them to a 24-well plate for culture. After detecting the supernatant again, select the positive clones for the second round of subcloning culture. At least 3 rounds of subcloning culture are carried out until all the detection wells randomly selected from the well plate are positive, that is, a stable hybridoma cell line is obtained. In this example, a positive hybridoma strain was obtained, and the clone number is 47-F. Select the culture supernatant of hybridoma 47-F, and use an antibody subtype detection test strip (Roche, #11493027001) to operate according to the instructions. The detected antibody 47-F subtype is murine IgG1 subtype, and the light chain is κ chain.

[0076] Example 2: Ascites preparation and purification

[0077] Inject 0.5 ml of liquid paraffin intraperitoneally into 8 - 10-week-old Balb / c female mice. One week later, harvest the hybridoma cells, wash them twice with PBS, and centrifuge at 500 g for 5 min. Inject the cells into the pre-sensitized Balb / c mice intraperitoneally at a cell amount of 5×10 6 / 0.5 ml. Observe the state and abdominal condition of the mice closely 7 days later. When it is observed that the activity of the mice is restricted, decapitate the mice and take out the ascites.

[0078] Collect the ascites, centrifuge at 3000 rpm for 10 min at RT, and collect the supernatant. Crudely purify the antibody with saturated ammonium sulfate at a final concentration of 33%. Add ammonium sulfate while stirring, and place it at 4°C overnight. The next day, centrifuge at 10000 rpm for 10 min, discard the supernatant, dissolve it with a small amount of PBS and put it into a pretreated dialysis bag, tie the dialysis bag tightly, and dialyze against PBS to remove salts at 4°C for 2 days, changing the liquid 3 times a day during this period.

[0079] The crudely purified antibody is purified using an AKTA protein purification system according to the purification manual provided by GE Company, and concentrated using an ultrafiltration tube. After purification and concentration, a high-concentration pure antibody product can be obtained.

[0080] Example 3: Detection of monoclonal antibody titer

[0081] PE fluorescence label the pure antibody product. The labeled antibody is mixed with 2.5×10 at final concentrations of 479.50 nM, 239.75 nM, 119.87 nM, 59.94 nM, 29.97 nM, 14.98 nM, 7.49 nM, 3.75 nM, 1.87 nM, 0.94 nM, 0.47 nM, 0.23 nM respectively 5Incubate Jurkat at room temperature in the dark for 30 min. Centrifuge at 500 g for 5 min, discard the supernatant, wash the cells with PBS, repeat three times, resuspend the cells in 400 μl of PBS, measure the fluorescence intensity by FACS, and calculate the average value. Analyze the Kd value of the antibody using GraphPad Prism 5 software. The Kd value of antibody 47-F is 0.4068×10 -9 M. (The results are shown in Figure 1 )

[0082] Example 4: Cloning of Ig variable region genes by RT-PCR

[0083] Total RNA extraction and single-stranded cDNA synthesis are carried out as follows:

[0084] Collect the 47-F hybridoma cell line, extract the total cellular RNA with Trizol (Invitrogen), and reverse transcribe the total RNA into a cDNA library using M-MLV reverse transcriptase (Invitrogen). Amplify the variable region gene fragments of the heavy chain (VH) and light chain (VL) of the anti-human CD47 antibody by RT-PCR.

[0085] Prepare a 50 μl PCR reaction system as follows (TAKARA): PrimeSTAR Max Premix (2×) 25 μl, upstream primer (10 μM) 2 μl, downstream primer (10 μM) 2 μl, cDNA 2 μl, ddH 2 O to make up to 50 μl. The PCR reaction conditions are as follows: 95°C for 5 min; 95°C for 30 s, 58°C for 30 s, 72°C for 1 min, cycle 35 times; 72°C for 10 min.

[0086] Recover the VL and VH fragments using a gel extraction kit.

[0087] Ligate the recovered fragments with the pMD19-T (simple) vector (Takara) using ligase (Takara). The ligation system is as follows: 1 μl of PCR product, 1 μl of T-Vector pMD19 (Simple), and make up ddH 2 O to 3 μl.

[0088] Add an equal volume (5 μl) of DNA Ligation Kit <Mighty Mix> (CodeNo. 6023) to the above DNA solution, mix well, and react at 16°C for 30 minutes.

[0089] Add the whole volume (10 μl) to 50 μl of E. coli competent cells, gently mix, and incubate on ice for 30 minutes. After a 45-s water bath at 42 °C, quickly transfer to ice and place for 1 minute. Add 890 μl of 2-YT medium and incubate with shaking at 37 °C for 60 min. Take 100 μl of the transformation solution and spread it on an agar plate medium containing Amp, and incubate overnight at 37 °C. Select white colonies and verify by PCR.

[0090] Prepare a 25-μl bacterial liquid PCR reaction system as follows: 1 μl of bacterial liquid, 1 μl of upstream primer (10 μM), 1 μl of downstream primer (10 μM), 2 μl of dNTP Mixture, 0.5 μl of Taq DNA polymerase (5 U / μl), 2.5 μl of 10× Taq Buffer (Mg 2+ plus), and make up to 10 μl with ddH2O. The reaction conditions are as follows: 95 °C for 5 min; 95 °C for 30 s, 58 °C for 30 s, 72 °C for 1 min, cycle 30 times; 72 °C for 10 min.

[0091] Select clones with positive PCR for bacteria for enlarged culture, extract the plasmids of the positive clones using a plasmid extraction kit (Takara), and send them for sequencing. For each chain of each antibody, at least 8 cloned samples should be submitted for testing until the sequencing results of at least three samples are the same. Successfully clone the variable region sequences of the heavy and light chains of 47-F that conform to the typical antibody variable region sequence characteristics.

[0092] Example 5: Detection of the competitive relationship between antibody 47-F and commercial Anti-CD47 antibody B6H12.2 by FACS

[0093] The hCD47-positive cell line Raji cells was selected as the detection cells. The commercial CD47 antibody B6H12.2 (abcam: ab3283; APC direct label) was used as the antibody to be competed, and the dosage was fixed at 0.625 ng / 50 μl. The maximum dosage of antibody 47-F was 1280 ng / 50 μl, and it was serially diluted to 0.3125 ng / 50 μl. Different concentrations of 47-F were mixed with 0.625 ng / 50 μl of the commercial antibody B6H12.2, and then mixed with 2×10 5 / 50 μl of Raji cells, and incubated in the dark at room temperature for 30 min (final volume 150 μl / tube). Centrifuge at 500 g for 5 min, discard the supernatant, wash twice with PBS, resuspend the cells in 400 μl of PBS, and measure the fluorescence intensity by FACS. As the dosage of 47-F increased, the fluorescence intensity of the binding of the commercial antibody B6H12.2 to Raji cells in the same tube gradually decreased, that is, antibody 47-F could effectively compete with the commercial CD47 antibody B6H12.2, and showed a gradient dependence (the results are shown in Figure 2 ).

[0094] Example 6: Ability of Antibody 47-F to Induce Mouse Macrophages to Phagocytose Tumor Cells In Vitro

[0095] Preparation of mouse macrophages: Take peritoneal cells of Balb / c mice, resuspend them in DMEM medium, inoculate them in 96-well plates at a density of 2×10 6 cells / mL, and culture them in an incubator for 3 h. Then gently aspirate the supernatant to remove non-adherent cells, and replace it with complete DMEM culture medium (containing murine M-CSF factor, 25 ng / ml); change the medium after three days, and then change the medium every two days. Perform phagocytosis experiments on the 7th to 10th day of culture.

[0096] Phagocytosis experiment: Before the experiment, change the medium of macrophages to serum-free medium and starve them for 4 h. During this period, prepare a single-cell suspension of tumor cells Raji, wash the cells twice with pre-cooled PBS to wash away the serum. Label the tumor cells with CFSE showing green fluorescence under light protection, incubate them in the dark for 20 min, add 10 mL of complete DMEM medium, and incubate at 37°C for 5 min. Centrifuge and discard the supernatant, 500 g, 10 min. After resuspending with complete medium, adjust the cell density to 2×10 5 cells / mL and inoculate them into the macrophages used in the above experiment (the macrophages are labeled in the same way, and the dye is the red fluorescence Far-Red). Add antibody 47-F or the positive commercial antibody B6H12.2 at final concentrations of 50 nM, 12.5 nM, and 3.125 nM respectively, and set the isotype control group as the negative control. Incubate the tumor cells and pre-starved macrophages in an incubator for 2 h. After phagocytosis, discard the supernatant, wash 3 times with PBS to wash away unphagocytosed tumor cells, and take pictures under a high-content microscope (PerkinElmer) (the results are shown in Figure 3 ), and use the high-content algorithm function to analyze the phagocytosis rate (the results are shown in Figure 4 ). Analyze at least 3000 cells per well.

[0097] The results show that both antibody 47-F and B6H12.2 can effectively induce mouse macrophages to phagocytose Raji cells, and the induction effect is dose-dependent. Moreover, the phagocytosis-promoting effect of antibody 47-F is significantly stronger than that of the commercial CD47 antibody B6H12.2.

[0098] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. An isolated antibody or antigen-binding portion, characterized in that The antibody or antigen binding portion specifically binds to human CD47 protein, and the antibody or antigen binding portion comprises: The heavy chain variable region CDRH1 as shown in SEQ ID No.1, the heavy chain variable region CDRH2 as shown in SEQ ID No.2, the heavy chain variable region CDRH3 as shown in SEQ ID No.3, the light chain variable region CDRL1 as shown in SEQ ID No.4, the light chain variable region CDRL2 as shown in SEQ ID No.5 and the light chain variable region CDRL3 as shown in SEQ ID No.

6.

2. The isolated antibody or antigen-binding portion according to claim 1, characterized in that The antibody or antigen-binding portion comprises: The heavy chain variable region is shown as SEQ ID No.7 and the light chain variable region is shown as SEQ ID No.

8.

3. The antibody or antigen-binding portion according to claim 1 or 2, characterized in that The antibody is a chimeric antibody or a humanized antibody.

4. A multivalent antibody, characterized in that: The multivalent antibody comprises the antibody or antigen-binding portion according to any one of claims 1 to 3.

5. An isolated polynucleotide, characterized in that The polynucleotide encodes the antibody or antigen-binding portion as described in any one of claims 1 to 3.

6. The isolated polynucleotide according to claim 5, characterized in that The sequence of the heavy chain variable region of the polynucleotide is shown as SEQ ID No.9, and the sequence of the light chain variable region of the polynucleotide is shown as SEQ ID No.

10.

7. A carrier, characterized in that The vector comprises the polynucleotide according to claim 5 or 6.

8. A cell, characterized in that The cell comprises the antibody or antigen-binding portion of any one of claims 1 to 3, the multivalent antibody of claim 4, the polynucleotide of claim 5 or 6, or the vector of claim 7.

9. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the antibody or antigen-binding portion as described in any one of claims 1 to 3, the multivalent antibody as described in claim 4, the polynucleotide as described in claim 5 or 6, and a pharmaceutically acceptable carrier.

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