CD47 antibody and application thereof

Abstract

The invention discloses a CD47 antibody and application thereof. The CD47 monoclonal antibody 47-F provided by the invention has relatively strong affinity with human CD47 protein, and the Kd value reaches 0.4068 * 10 <-9 > M; the antibody is a mouse IgG1 subtype, and a light chain is a kappa chain; the antibody B6H12.2 can compete with the commodity CD47 antibody B6H12.2 to bind to target cells; mouse macrophages can be obviously induced to phagocytize B lymphoma cells Raji in vitro; the kit not only can be used for detecting the expression of human CD47 protein, but also can be applied to immunotherapy independently or in combination with other methods, and has diagnosis and treatment 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 applications thereof. 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, it forms a "don't eat me" signal that inhibits the phagocytic function of macrophages. It has been reported that CD47 is overexpressed in a variety of hematological malignancies (such as acute myeloid leukemia, myelodysplastic syndrome) and solid tumors (such as breast cancer, lung cancer, and colorectal cancer). Tumor cells escape phagocytosis of macrophages by highly expressing CD47. Studies have shown that the expression level of CD47 in tumors is significantly positively correlated with poor prognosis of treatment. Therefore, the CD47-SIRPα pathway is considered to be a highly promising 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 showed good efficacy in patients with high-risk myelodysplastic syndrome and acute myeloid leukemia. It is the first CD47 target drug to enter Phase III clinical trials. However, in February 2024, the Phase III clinical trial of Magrolimab (NCT05079230) was terminated early due to increased mortality. Not only Magrolimab, but current antibody drugs targeting CD47 (such as B6H12.2, CC90002, etc.) generally face serious side effects such as anemia and thrombocytopenia, and no drugs have been officially used in clinical practice. 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 issues solved:

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

[0006] Technical solution:

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

[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: a heavy chain variable region as shown in SEQ ID No.7 and a light chain variable region as shown in SEQ ID No.8.

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

[0011] In some embodiments, the antibody may be IgG, IgA, IgM, IgD or IgE. Preferably, in some embodiments, the type of the antibody may be IgG. Further, in some embodiments, the antibody may 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 modification includes 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 a single-chain antibody scFv.

[0016] Another aspect of the present disclosure provides a multivalent antibody comprising 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 using a suitable method 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, which selectively binds to at least human CD47, and comprises the above-mentioned 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 encoding the above-mentioned antibody or antigen-binding portion, or encoding the above-mentioned multivalent antibody. In one embodiment, the sequence of the heavy chain variable region of the polynucleotide is shown in SEQ ID No.9, and the sequence of the light chain variable region of the polynucleotide is shown in SEQ ID No.10.

[0020] Another aspect of the present disclosure provides a vector comprising the above-mentioned polynucleotide.

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

[0022] Another aspect of the present disclosure is to provide a pharmaceutical composition comprising the above-mentioned antibody or antigen-binding portion, the above-mentioned multivalent antibody, the above-mentioned polynucleotide, the above-mentioned vector or the above-mentioned cell, and a pharmaceutically acceptable carrier. In order to achieve a better therapeutic effect, in some embodiments, the pharmaceutical composition may also contain other therapeutic drugs.

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

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

[0025] Beneficial effects:

[0026] The CD47 monoclonal antibody 47-F provided by the present invention has a strong affinity with human CD47 protein, and the Kd value is 0.4068×10 -9 M; it is a mouse IgG1 subtype, and its light chain is a kappa chain; it can compete with the commercial CD47 antibody B6H12.2 for binding to target cells; it can significantly induce mouse 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, and has diagnostic and therapeutic value in tumors, autoimmune diseases, and inflammatory diseases. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0028] Figure 2 This is a diagram showing the competitive binding results between antibody 47-F and commercial antibody B6H12.2 measured by flow cytometry in the disclosed embodiment;

[0029] Figure 3 This is a result diagram of high-content imaging of antibody 47-F promoting mouse macrophages to phagocytize lymphoma cell line Raji in the disclosed embodiment, wherein B6H12.2 is a commercial positive control group, red in the figure represents mouse macrophages, and green represents phagocytosed Raji cells;

[0030] Figure 4 This is a dose-dependency curve of the antibody 47-F in the disclosed example that promotes mouse macrophages to phagocytize the lymphoma cell line Raji.

[0031] Sequence Description

[0032] DETAILED DESCRIPTION

[0033] The present invention discloses an anti-human CD47 antibody and its application. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be particularly pointed out 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 modify or appropriately change and combine the contents 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 the present disclosure, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Unless otherwise expressly stated, throughout the specification and claims, the term "comprising" or its variations such as "including" or "including" etc. will be understood to include the elements or components stated, without excluding other elements or other components. The term "a", "an" and "the" include plural indicators. The term "multiple" refers to two or more. The terms "such as", "for example", etc. are intended to refer to exemplary embodiments, and are not intended to limit the scope of the present disclosure.

[0035] In the present disclosure, when a range of values ​​is provided, it should be understood that the endpoints are included in the range and each intervening value between the upper and lower limits of the range and any other specified value or intervening value in the specified range and any value in the smaller range between the specified values ​​are included unless the context clearly dictates otherwise.

[0036] In this disclosure, the term "about" generally refers to variations within a range of 0.5%-10% above or below a specified value, for example, 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 a specified value.

[0037] In the present disclosure, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. The definition 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. The definition of common terms in biochemistry can be found in Lehninger Principles of Biochemistry, Eighth Edition, David L. Nelson, Michael M. Cox, Publisher: WH Freeman. The definition 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. The definition 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, the 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 the following: Molecular Cloning: A Laboratory Manual; Cell Biology: A Laboratory Handbook, etc.

[0039] definition:

[0040] The term "isolated" in this disclosure refers to a substance or entity that is separated from its natural environment or the environment that existed before separation 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 separation ratio 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 relative to their substances before separation.

[0041] The term "antibody" in the present disclosure refers to an immunoglobulin molecule that is generally composed of two pairs of polypeptide chains, each pair having a "light" (L) chain and a "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 define the isotype of the antibody as IgM, IgD, IgG, IgA and IgE, respectively. Within the light and heavy chains, the variable and constant regions are connected by a "J" region of about 12 or more amino acids, and the heavy chain also contains a "D" region of about 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 immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The VH and VL regions can also be subdivided into regions with high variability (called complementary determining regions (CDRs)), interspersed with more conservative regions called framework regions (FRs). Each VH and VL consists of 3 CDRs and 4 FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions (VH and VL) of each heavy chain / light chain pair form antibody binding sites, respectively. The term "antibody" is not limited by any particular method for producing antibodies. For example, it includes, in particular, recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. The antibody can be an antibody of different isotypes, for example, IgG (e.g., IgG1, IgG2, IgG3 or IgG4 subtype), IgA1, IgA2, IgD, IgE or IgM antibody. 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, hybridoma technology is used to produce monoclonal antibodies in mammalian cells. The hybridoma preparation method reported by Kohler et al. in Nature 256:495 (1975) can be used to prepare the monoclonal antibody. First, mice or other suitable host animals are immunized with an immunogen (adding an adjuvant when necessary).

[0044] Immunogens or adjuvants are usually injected subcutaneously at multiple points or intraperitoneally. Adjuvants can be Freund's adjuvant (Freund's complete adjuvant or Freund's incomplete adjuvant) or MPL-TDM. After being immunized, animals will produce lymphocytes that secrete antibodies that specifically bind to immunogens. The target lymphocytes are collected and fused with myeloma cells using a suitable fusion agent (such as PEG4000) to obtain hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, pp.59-103, Academic Press, 1996).

[0045] The hybridoma cells prepared as described above are inoculated into a suitable culture medium for growth, wherein the culture 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 phosphotransferase (HGPRT or HPRT), the addition of substances such as hypoxanthine, aminopterin and thymidine to the culture medium (HAT medium) will inhibit the growth of HGPRT-deficient cells.

[0046] The preferred myeloma cells should have high fusion rate, stable antibody secretion ability, and sensitivity to HAT culture medium. Among them, the preferred myeloma cells are mouse myeloma, such as MOP-21 and MC-11 mouse tumor derivatives (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 heterologous myeloma 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 culture medium in which the hybridoma cells are grown is used to detect the production of monoclonal antibodies against specific antigens. The binding specificity of the monoclonal antibodies produced by the hybridoma cells can be determined using the following methods: immunoprecipitation or in vitro binding assays, such as radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA). For example, the affinity of the monoclonal antibody can be determined using the Scatchard analysis method described by Munson et al. in Anal. Biochem. 107:220 (1980).

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

[0049] The monoclonal antibody secreted by the subclone cells can be separated from the cell culture medium, ascites or serum using conventional immunoglobulin purification methods, such as protein A agarose gel, hydroxyapatite chromatography, gel electrophoresis, dialysis or affinity chromatography, thereby obtaining the monoclonal antibody.

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

[0051] Antibody modification and transformation:

[0052] In some embodiments, the isolated antibody can be a humanized antibody. Antibody humanization may improve the affinity or other characteristics of the antibody. The description and method of humanized antibodies can be found in Riechmann, L., Clark, M., Waldmann, H., & Winter, G. (1988). Reshaping human antibodies for therapy. Nature, 332(6162), 323–327.

[0053] In some embodiments, the antibody Fc (crystallizable region fragment, Fc) is modified to enhance its effector functions by binding to Fc receptors or complement triggering. These functions may include complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), and antibody-dependent cellular phagocytosis (ADCP). The above-mentioned modification may include: 1) modification of glycosylation, for example, aspartic acid (N297) at position 297 in the Fc region can be modified by N-acetylglucosamine. Mutation of N297 to alanine (A), glutamine (Q), or glycine (G) will hinder the glycosylation of the antibody, thereby reducing the effector function mediated by Fc. After deglycosylation of the antibody, the ability to induce ADCC or CDC activity will decrease; sialic acid modification will reduce the binding affinity with FcγRIIIa, thereby leading to a decrease in CDC and ADCC activity.

[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 agglomeration or unfolding. For example, the sugar on the al-3 arm does not contact the surface of the antibody, but penetrates into the space formed by the two heavy chain Fc segments. There is an interaction between the mannose on the al-3 arms of the two sugar chains, which is very important for maintaining the conformation of the antibody. If there is no sugar chain, the CH2 domain of the Fc segment will be slightly enlarged, resulting in an earlier elution time of the antibody in molecular exclusion chromatography, and it will be more sensitive and easy to aggregate in the thermal accelerated stability experiment. At the same time, glycosylation modification can also affect the binding of antibodies to receptors on the cell membrane to form a complex, thereby 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 modifications can also include: 2) point mutations, for example, LALA mutation (L234A / L235A) will lead to changes in the affinity of the antibody for FcγR (eliminating the binding to low-affinity FcγR and reducing the binding to FcγRI), thereby significantly reducing its ADCC and CDC activities. In addition, the combination of cross-subtype antibodies will also regulate the effector function of the antibody.

[0056] In some embodiments, the point mutation results in the substitution of some conservative amino acids, thereby obtaining a "conservative amino acid substitution variant". The change results in some amino acids being replaced by other amino acids with similar chemical properties and / or functions. It is well known in the art to provide a conservative substitution table for amino acids with similar chemical properties and / or functions. Typical examples of mutually conservative substitutions include, 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 modifications may also include: 3) Phosphorylation modification. Phosphorylation modification refers to the process of adding phosphate groups to the amino acids of intracellular proteins. Phosphorylation antibodies can specifically recognize specific phosphorylation sites, thereby detecting the increase or decrease in 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 methylases on specific residues of proteins. Methylation antibodies can specifically recognize specific methylated amino acid sites and are used to distinguish between methylated and unmethylated forms of proteins. They are widely used 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. Acetylation 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 cell cycle regulation, signal transduction, neurodegenerative diseases, metabolic diseases and the occurrence and development of cancer.

[0058] The above modifications may also include: 6) Marker modification. Antibodies can be cross-linked through different chemical reagents to connect to substances such as enzymes, fluorescent dyes, biotin or colloidal gold to change their detection or analytical performance. For example, enzyme labeling: Antibodies can be cross-linked to enzymes such as horseradish peroxidase (HRP), alkaline phosphatase, etc. It is often used in experiments such as immunohistochemistry and ELISA to produce color reactions through the catalytic action of enzymes to detect the presence of antibodies. For example, HRP-labeled antibodies can produce color precipitation by adding substrates after binding to antigens, 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. Fluorescently labeled antibodies can locate specific antigens in cells or tissue sections, and judge the expression level of antigens by the strength of fluorescent signals. Biotin labeling: Biotin is a small molecule compound that can bind to antibodies without affecting their antigen binding ability. Biotin-labeled antibodies can be combined with avidins (such as streptavidin) to achieve signal amplification and detection. Commonly used in multiplex immunolabeling experiments to detect multiple antigens simultaneously.

[0059] In the present disclosure, the modification and transformation of antibodies usually occur in the Fc segment of the antibody and the framework region (Framework Regions, FRs) of the antibody variable region, but not in the complementarity determining region (Complementarity-Determining Regions, CDRs) of the antibody variable region. The framework region of the antibody variable region has a relatively conservative amino acid sequence, provides a stable support for the structure of the hypervariable region, and participates in maintaining the three-dimensional conformation of the antigen binding groove. Therefore, its transformation and modification does not affect the binding ability of the antibody. In the present disclosure, the term "having more than 90% identity" means that under the above-mentioned transformation or modification method, compared with the sequence shown in SEQ ID No.1-16, it has about 90%, about 91%, about 92%, about 93%, about 94, about 95%, about 96%, about 97%, about 98%, about 99% identity.

[0060] Isolated antigen binding fraction:

[0061] The term "antigen binding portion" in the present disclosure refers to a polypeptide comprising a fragment of a full-length antibody, which retains the ability to specifically bind to the same antigen bound by the full-length antibody and / or competes with the full-length antibody for specific binding to the antigen, which is also referred to as an "antigen binding fragment". See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd edition, Raven Press, NY (1989). Antibody antigen binding fragments can be produced by recombinant DNA technology 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] The term "Fab fragment" refers to an antibody fragment consisting of the VL, VH, CL and CH1 domains; the term "F(ab') 2 The term "fragment" means an antibody fragment comprising two Fab fragments linked by a disulfide bridge at 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 an antibody.

[0063] In some embodiments, the antigen binding portion is prepared by a protease digestion method, and the protease used is, for example, papain, pepsin, etc. In other embodiments, the antigen binding portion is prepared by a chemical reagent treatment method. In other embodiments, the antigen binding portion is prepared by a genetic engineering method. That is, a fragment containing all or part of the gene sequence of the antigen binding portion is connected to a suitable vector and expressed. Examples of the expression vector include bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors.

[0064] The term "polynucleotide" in this disclosure is also used interchangeably as "nucleic acid", which 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] Example:

[0066] In order to enable those skilled in the art to better understand the technical solution of the present invention, the present invention is further described in detail below in conjunction with specific embodiments.

[0067] Example 1: Mouse hybridoma monoclonal antibody screening

[0068] Six-week-old Balb / c female mice were immunized with hCD47 positive cell line by intraperitoneal injection for 3 times, with 1×10 7 Each immunization was separated by 2 weeks. On the eighth day after the third immunization, the tail blood of the mice was collected and the titer of CD47 positive cells was determined by Jurkat flow cytometry. 1×10 7 hCD47 positive cells were used for boosting immunization.

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

[0070] Dissolve the tail blood of mice in PBS and let stand at room temperature for 1 hour. Centrifuge at 12000 rpm at 4℃ for 10 minutes, collect the supernatant, discard the precipitate, and dilute with PBS to different concentrations from 1:200 to 1:12800. Collect Jurkat cells, wash once with PBS, and use 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 Incubate for about 10 days, and clone formation can be seen. Select a well with only a single clone, and take 100 μl of culture supernatant. The detection method is the same as before. Select the positive clone and expand it to a 24-well plate for culture. After the supernatant is detected again, select the positive clone for the second round of subclone culture. After at least 3 rounds of subclone culture, until all the test wells in the randomly selected well plate are positive, a stable hybridoma cell line is obtained. In this example, a positive hybridoma strain with a clone number of 47-F was obtained. The culture supernatant of hybridoma 47-F was selected, and the antibody subtype detection strip (Roche, #11493027001) was used according to the instructions. The subtype of antibody 47-F was detected as mouse IgG1 subtype, and the light chain was κ chain.

[0076] Example 2: Preparation and purification of ascites

[0077] 0.5 ml of liquid paraffin was intraperitoneally injected into 8-10 week old Balb / c female mice. One week later, the hybridoma cells were harvested, washed twice with PBS, and centrifuged at 500 g for 5 min. 5 x 10 6 / 0.5ml of cells were intraperitoneally injected into pre-sensitized Balb / c mice. After 7 days, the condition and abdominal condition of the mice were closely observed. When the mice were found to have limited activity, they were killed by dislocation and the ascites was removed.

[0078] Ascites was collected, 3000rpm, 10min, RT, and the supernatant was collected. The antibody was crudely purified with saturated ammonium sulfate at a final concentration of 33%, and the mixture was stirred while adding ammonium sulfate and placed at 4°C overnight. The next day, the mixture was centrifuged at 10000rpm for 10min, and the supernatant was discarded. After being dissolved with a small amount of PBS, the mixture was placed in a pre-treated dialysis bag, and the dialysis bag was tied tightly. The PBS was dialyzed for desalination at 4°C for 2 days, and the solution was changed 3 times a day.

[0079] The crude purified antibody was purified using the AKTA protein purification system according to the purification manual provided by GE, using a 1 ml Protein G purification pre-packed column, and concentrated using an ultrafiltration tube. After purification and concentration, a high-concentration pure antibody product can be obtained.

[0080] Example 3: Monoclonal Antibody Titer Detection

[0081] The antibody was fluorescently labeled with PE. The labeled antibodies were mixed with 2.5×10 5Jurkat cells were incubated at room temperature in the dark for 30 min. Centrifuged at 500 g for 5 min, the supernatant was discarded, and the cells were washed with PBS. This process was repeated three times. The cells were resuspended in 400 μl PBS and the fluorescence intensity was measured by FACS. The average value was calculated. The Kd value of the antibody was analyzed using GraphPad Prism 5 software. The Kd value of antibody 47-F was 0.4068×10 -9 M. (Results see Figure 1 )

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

[0083] Total RNA extraction and single-stranded cDNA synthesis were performed as follows:

[0084] The 47-F hybridoma cell line was collected, and total RNA was extracted with Trizol (Invitrogen), and the total RNA was reversed into a cDNA library with M-MLV reverse transcriptase (Invitrogen). The variable region gene fragments of the heavy chain (VH) and light chain (VL) of the anti-human CD47 antibody were amplified by RT-PCR.

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

[0086] The VL and VH fragments were recovered using a gel recovery kit.

[0087] The recovered fragments were ligated with pMD19-T (simple) vector (Takara) ligase (Takara) respectively. The ligation system was as follows: 1 μl of PCR product, 1 μl of T-Vector pMD19 (Simple), supplemented with ddH 2 O to 3 μl.

[0088] Add an equal volume (5 μl) of DNA Ligation Kit to the above DNA solution.<Mighty Mix> (Code No. 6023), mix thoroughly, and react at 16°C for 30 minutes.

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

[0090] Prepare 25 μl bacterial solution PCR reaction system as follows: 1 μl bacterial solution, 1 μl upstream primer (10 μM), 1 μl downstream primer (10 μM), 2 μl dNTP Mixture, 0.5 μl Taq DNA polymerase (5 U / μl), 10× Taq Buffer (Mg 2+ plus) 2.5μl, add ddH2O to 10μl. The reaction conditions are as follows: 95℃ 5min; 95℃ 30s, 58℃ 30s, 72℃ 1min, 30 cycles; 72℃ 10min.

[0091] Select the clones with positive PCR results and expand the culture. Use the plasmid extraction kit (Takara) to extract the positive clone plasmids and send them for sequencing. At least 8 clone samples of each chain of each antibody are sent for testing until the sequencing results of at least three samples are the same. The heavy chain and light chain variable region sequences of 47-F that meet the characteristics of the typical antibody variable region sequence are successfully cloned.

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

[0093] The hCD47 positive cell line Raji cells were used as the detection cells. The commercial CD47 antibody B6H12.2 (abcam: ab3283; APC direct label) was used as the competing antibody, and the dosage was fixed at 0.625ng / 50ul. The maximum dosage of antibody 47-F was 1280ng / 50ul, which was diluted to 0.3125ng / 50ul. Different concentrations of 47-F were mixed with 0.625ng / 50ul B6H12.2 commercial antibody, and then mixed with 2x10 5 / 50ul Raji cells were mixed and incubated at room temperature in the dark for 30min (final volume 150ul / tube). Centrifuge at 500g for 5min, discard the supernatant, wash twice with PBS, resuspend the cells in 400μl PBS, and measure the fluorescence intensity by FACS. As the amount of 47-F increased, the fluorescence intensity of the binding of the commercial antibody B6H12.2 to Raji cells in the same tube gradually weakened, that is, the antibody 47-F can effectively compete with the commercial CD47 antibody B6H12.2, and it is gradient-dependent (see the results). Figure 2 ).

[0094] Example 6: Antibody 47-F induces the ability of mouse macrophages to phagocytose tumor cells in vitro

[0095] Preparation of mouse macrophages: Balb / c mouse peritoneal cells were obtained and resuspended in DMEM medium at 2×10 6 The cells were inoculated at a density of 100 / mL in a 96-well plate and cultured in an incubator for 3 hours. The supernatant was gently aspirated to remove non-adherent cells and replaced with complete DMEM (containing mouse M-CSF factor, 25 ng / ml). The medium was changed after three days, and then every two days. The phagocytosis experiment was performed on the 7th to 10th day.

[0096] Phagocytosis experiment: Before the experiment, the culture medium of macrophages was replaced with serum-free medium and starved for 4 hours. During this period, a single cell suspension of tumor cells Raji was prepared, and the cells were washed twice with pre-cooled PBS to remove the serum. The CFSE light-proof operation that shows green fluorescence was marked on the tumor cells, incubated in a dark room for 20 minutes, and 10 mL of DMEM complete medium was added at 37°C for 5 minutes. The supernatant was discarded by centrifugation, 500g, 10 minutes, and the cell density was adjusted to 2×10 after resuspending in complete medium. 5 1000 cells / mL were inoculated into the macrophages used in the above experiment (macrophages were labeled in the same way, with the red fluorescent Far-Red as the fuel). Antibody 47-F or positive commercial antibody B6H12.2 were added at final concentrations of 50nM, 12.5nM, and 3.125nM, respectively, and an isotype control group was set as a negative control. Tumor cells and pre-starved macrophages were incubated in an incubator for 2h. After the phagocytosis was completed, the supernatant was discarded, and the cells were washed 3 times with PBS to wash away the tumor cells that were not phagocytosed, and the images were taken under a high-content microscope (PerkinElmer) (see the results). Figure 3 ), and applied high-content algorithm to functionally analyze phagocytosis rate (results see Figure 4 ), at least 3000 cells were analyzed per well.

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

[0098] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection 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, a humanized antibody or a fully human 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. A multispecific antibody, characterized in that: The multispecific antibody selectively binds to at least human CD47, and the multispecific antibody comprises the antibody or antigen-binding portion of any one of claims 1 to 3.

6. 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.

7. The isolated polynucleotide according to claim 6, 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.

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

9. A cell, characterized in that The cell 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 multispecific antibody as described in claim 5, the polynucleotide as described in claim 6 or 7, or the vector as described in claim 8.

10. 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 multispecific antibody as described in claim 5, the polynucleotide as described in claim 6 or 7, and a pharmaceutically acceptable carrier.

Images