Anti-human IFN-γ antibody and its application

Abstract

The present invention discloses anti-human IFN-γ antibodies and their applications. The anti-human IFN-γ monoclonal antibodies provided by the present invention have a strong affinity for human IFN-γ protein, can be used to detect the expression of human IFN-γ protein, and can also be used alone or in combination with other methods to diagnose and treat tumors, autoimmune diseases or infections.

Description

Technical Field

[0001] The present disclosure relates to the field of biomedicine, and in particular, to anti-human IFN-γ antibodies and applications thereof. Background Art

[0002] IFN-γ is a soluble dimeric cytokine and the only member of type II interferons. It is mainly secreted by natural killer (NK) and natural killer T cells (NKT) cells, playing a role in innate immunity; in antigen-specific immunity, it is secreted by CD4 Th1 and CD8 cytotoxic T cells. The expression of IFN-γ is induced by mitogens and cytokines, such as IL-12, IL-15, IL-18, and type I IFN. Signaling is transduced by binding to IFN-γ receptors (IFN-γR, IFN-γR1, and IFN-γR2).

[0003] The biological response of IFN-γ is caused by activating intracellular molecular signaling pathways, mainly through the JAK / STAT pathway, which regulates the transcription of hundreds of genes and mediates a variety of biological responses. After IFN-γ binds to the receptor, it activates downstream signals JAK1 and JAK2, and STAT1 is phosphorylated to form a homodimer complex, which translocates to the nucleus to directly activate the transcription of interferon-stimulated genes (ISGs). ISGs encode many products with direct effector immune functions, such as chemokines, antigen presenting molecules (including MHC molecules), phagocytic receptors, and various antiviral and antibacterial factors. IFN-γ can exert its immunomodulatory effects by enhancing antigen processing and presentation, increasing the transport of leukocytes, inducing antiviral states, enhancing antimicrobial functions, and affecting cell proliferation and apoptosis. IFN-γ can be used as a disease marker in many different pathological conditions, including infection, autoimmune diseases, transplant rejection, and allergic reactions. Therefore, targeting IFN-γ has become a strategy for treating the above diseases. Immunotherapy based on antibody technology has developed rapidly in fields such as tumor treatment. However, there is currently no ideal antibody drug targeting human IFN-γ that has been used in clinical practice, so there is a need to further develop molecules that specifically bind to human IFN-γ to treat IFN-γ-mediated diseases. Summary of the invention

[0004] Technical issues solved:

[0005] One aspect of the present invention is to provide an anti-human IFN-γ antibody and its application in view of the lack of an ideal molecule that specifically binds to human IFN-γ in the prior art.

[0006] Technical solution:

[0007] An isolated antibody or antigen-binding portion that specifically binds to human IFN-γ 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:

[0010] 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.

[0011] 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.

[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 that selectively binds to at least human IL-10, and the multispecific antibody comprises the above-mentioned antibody or antigen-binding portion.

[0019] Another aspect of the present disclosure is to provide an isolated polynucleotide encoding the above-mentioned antibody or antigen-binding portion. In one embodiment, the nucleotide sequence of the heavy chain variable region of the polynucleotide is shown in SEQ ID No.9, and the nucleotide 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 method for producing an anti-human IL-10 antibody or antigen-binding portion thereof, wherein the anti-human IL-10 antibody or antigen-binding portion thereof is obtained by expressing the protein in the above-mentioned cells.

[0023] 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.

[0024] 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 multispecific antibody, the above-mentioned isolated polynucleotide, the above-mentioned vector or the above-mentioned cell in the preparation of a drug for treating tumors, autoimmune diseases or infections.

[0025] Another aspect of the present disclosure is to provide the use of the above-mentioned antibody or antigen-binding portion in the preparation of a product for detecting the presence or level of human IFN-γ molecules in a sample.

[0026] Beneficial effects:

[0027] The anti-human IFN-γ monoclonal antibody provided by the present invention has a strong affinity for human IFN-γ protein, can be used to detect the expression of human IFN-γ protein, and can also be used alone or in combination with other methods to diagnose and treat tumors, autoimmune diseases or infections. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is a graph showing the results of the determination of the antiserum titer of mice after secondary immunization in the embodiments of the present disclosure;

[0029] Figure 2 This is the electrophoresis diagram of the light and heavy chain gene clones of the anti-human IFN-γ monoclonal antibody in the embodiment of the present disclosure;

[0030] Figure 3 The diagram is a construction map of the heavy chain and light chain vectors of the anti-human IFN-γ genetically engineered antibody in the embodiment of the present disclosure, wherein: Figure 3 A is the heavy chain expression vector, Figure 3 B is a light chain expression vector;

[0031] Figure 4 The electrophoresis diagram of the expression results of the anti-human IFN-γ genetically engineered antibody in the embodiment of the present disclosure, wherein the red box on the left is the sample that has not been reduced, the red box on the right is the reduced sample, and the sample that has not been added is mixed with the loading buffer without mercaptoethanol;

[0032] Figure 5 : is a graph showing the activity identification results of the anti-human IFN-γ genetically engineered antibody in the embodiments of the present disclosure, wherein: Figure 5 A is the result of ELISA comparing the activity of genetically engineered antibodies and commercial anti-human IFN-γ antibodies. Figure 5 B is a graph showing the specificity of antigen recognition of genetically engineered antibodies and commercial antibodies analyzed by western blotting;

[0033] Figure 6 The specificity analysis result diagram of the anti-human IFN-γ genetically engineered antibody in the embodiment of the present disclosure, wherein: Figure 6 A shows that only the anti-human IFN-γ genetically engineered antibody can react with the commercialized human IFN-γ antigen. Figure 6 B shows that the genetically engineered antibody does not react with commercial human IL-10 and IL-6 antigens. Figure 6 C indicates that the genetically engineered antibody can detect human IFN-γ antigen in patient specimens.

[0034] Sequence description.

[0035] DETAILED DESCRIPTION

[0036] The present invention discloses an anti-human IFN-γ 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.

[0037] 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.

[0038] 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.

[0039] 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.

[0040] 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.

[0041] 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.

[0042] definition:

[0043] 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.

[0044] 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.

[0045] Preparation of antibodies:

[0046] 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).

[0047] 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).

[0048] 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.

[0049] 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).

[0050] 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).

[0051] 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.

[0052] 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.

[0053] In other embodiments, anti-human IL-10 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.

[0054] Antibody modification and transformation:

[0055] 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.

[0056] 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.

[0057] 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.

[0058] 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.

[0059] 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).

[0060] 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.

[0061] 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.

[0062] Isolated antigen binding fraction:

[0063] 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.

[0064] Among them, the term "Fab fragment" means an antibody fragment consisting of VL, VH, CL and CH1 domains; the term "F(ab')2 fragment" means an antibody fragment comprising two Fab fragments connected by a disulfide bridge on the hinge region. The term "Fd fragment" means an antibody fragment consisting of 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.

[0065] 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.

[0066] 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.

[0067] The term "vehicle" in the present disclosure refers to a polynucleotide molecule capable of transporting and / or expressing one or more target genes. Examples of vectors may include viral vectors, naked DNA or RNA expression vectors, plasmids, cosmids or phage vectors, DNA or RNA expression vectors associated with a cationic condensing agent, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells such as production cells.

[0068] Pharmaceutical composition:

[0069] The term "pharmaceutical composition" in the present disclosure refers to a composition that contains at least one other substance in addition to the antibody, the antigen-binding portion, the multivalent antibody, the polynucleotide, the vector, the cell, or the multispecific antibody described in the present disclosure. In some embodiments, the other substance can be, for example, a pharmaceutically acceptable carrier (a carrier that does not affect the effect of the mesenchymal stem cells and has no effect on the patient's physical condition, such as physiological saline, cell culture medium, glucose, water for injection, glycerol, ethanol, and a combination thereof), an excipient, a stabilizer, a surfactant, a preservative, an isotonic agent, and the like. It can also be other therapeutic agents, such as chemotherapeutic drugs: melphalan, doxorubicin, cyclophosphamide, vincristine, and the like; glucocorticoid drugs: prednisone, dexamethasone, betamethasone, and the like; immunomodulatory drugs: thalidomide, lenalidomide, pomalidomide, and the like.

[0070] In some embodiments, the above pharmaceutical composition can be prepared into any suitable formulation. For example, pills, tablets, creams, gelatin capsules, capsules, suppositories, soft gelatin capsules, gels, films, tubules, solutions or suspensions. The above pharmaceutical composition can be administered in any suitable manner, for example, intralesional, intravenous, topical, rectal, parenteral, topical, inhaled or subcutaneous, submuscular, intrathecal, abdominal, oral and intracerebral.

[0071] Example:

[0072] 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.

[0073] Example 1: Preparation of anti-human IFN-γ antibody

[0074] 1. Mouse hybridoma monoclonal antibody screening

[0075] Human IFN-γ gamma (E coli) was used as the immunogen and Balb / c mice were immunized by intraperitoneal injection. The secondary immunization and booster immunization were performed at the 2nd and 4th weeks after the primary immunization, respectively. One week after the secondary immunization, the tail blood of the mice was collected and allowed to stand at room temperature for 1 hour. The serum was collected by centrifugation at 12000 rpm for 10 minutes at 4°C and the tail blood supernatant was diluted with PBS to different concentrations: 1:1000, 1:2000, 1:4000, 1:8000, 1:16000, and 1:32000. The titer was detected by ELISA method. The main process was as follows: 1 μL (1 μg) of human IFN-γ protein + 10 mL of coating solution, 100 μL / well, overnight at 4°C; blocked with 5% FBS, and allowed to stand at room temperature for 1 hour; discarded the supernatant, added the primary antibody (the above mouse antiserum at different dilutions), 100 μL / well, set up a positive control (IFN-γ antibody, 2G1, Invitrogen) and a negative control (non-immunized normal mouse tail vein serum), and shook at room temperature for 2 hours; discarded the supernatant, washed 3 times, added the secondary antibody-HRP (1:5000 dilution), 100 μL / well, and shook at room temperature for 1 hour; discarded the supernatant, washed 3 times, added the colorimetric solution 100 μL / well, protected from light at room temperature for 10 minutes, and added the stop solution 100 μL / well; detected on the machine, the wavelength was 450nm, and the OD value (such as Figure 1 as shown).

[0076] Take the spleen cells of the successfully immunized mice and myeloma SP2 / 0 cells for cell fusion (at a ratio of 10:1). During the fusion, add 50% PEG to the spleen cells and myeloma cell clusters that have been mixed and the supernatant discarded within 1 minute in a 37°C water bath. Shake in a 37°C water bath for 90 seconds, and then add 10mL of serum-free 1640 medium within 5 minutes. Centrifuge at 800rpm for 5 minutes, discard the supernatant, resuspend the cells with a semi-solid medium containing HAT, and spread them into a 96-well plate (200μL / well). Culture the cells at 37°C and 5% CO2.

[0077] When the cell clones in the 96-well plate are large enough under a cell microscope, take 100 μL of the supernatant from the corresponding well and use the ELISA method to determine the antigen binding activity of the anti-human IFN-γ monoclonal antibody. The method is the same as the titer detection method. When the OD value is greater than the OD value of the positive control, the well is considered a positive well and the next cloning culture is carried out. The hybridoma clones that are screened positive are expanded from the 96-well plate to the 24-well plate for 3-5 days, and the culture supernatant is screened again. The clones that are tested positive are then subcloned for the next step, and the remaining cells are frozen. The hybridoma cells in the 24-well plate were collected, and the cells were counted and the cell density was adjusted to 10 cells / mL; the cells were plated in a 96-well plate, 200 μL per well, and cultured in an incubator at 37°C and 5% CO2; after about 10 days of culture, clone formation was observed, and the wells with only a single clone were selected, and the culture supernatant was aspirated. The detection method was the same as before, and the positive clones were selected and expanded to 24-well plates for culture. After the supernatant was detected again, the positive clones were selected for the second round of subclone culture. Generally, after multiple rounds of subclone culture, until all the detection wells were positive, a stable hybridoma cell line was obtained. The positive hybridoma culture supernatant was selected, and the antibody subtype detection paper was used to detect the antibody subtype. A hybridoma cell line secreting anti-human IFN-γ monoclonal antibody obtained in this example: 1H6-F8-H3, the antibody subtype is IgG1 / kappa (as shown in Table 1 below).

[0078] Table 1

[0079]

[0080] 2. Ascites Preparation and Purification

[0081] Hybridoma cells were washed with sterile PBS solution and 5 × 10 6 / 500μL / mouse of cells were intraperitoneally injected into Balb / c mice pre-sensitized with liquid paraffin. After 7 to 10 days, ascites was collected, and the supernatant was collected at 3000rpm at room temperature for 10 minutes. The antibody was crudely purified with saturated ammonium sulfate at a final concentration of 33%. The method was to take 1 part of ascites and add 1 part of PBS, add 1 part of saturated ammonium sulfate dropwise, stir while adding, and centrifuge at 10000rpm for 10 minutes to remove the supernatant, dissolve the precipitate with a small amount of PBS, and dialyze with PBS for desalination at 4℃ for 24 hours, changing the liquid 3 times during the period. The crudely purified antibody was further purified by 1 mL Protein G purification pre-packed column using the AKTA protein purification system according to the purification manual provided by GE. The obtained pure antibody was used for subsequent antibody detection and functional experiments. The protein concentration of the pure antibody was quantitatively detected by BCA.

[0082] 3. Monoclonal antibody light and heavy chain gene cloning

[0083] After RNA extraction, RT-PCR, PCR, and magnetic bead purification, the received hybridoma cells (#3) were subjected to mouse heavy chain BCR and light chain BCR samples (e.g. Figure 2 The library was constructed strictly according to the Thermo Fisher library construction manual, and the Thermo Fisher IonGeneStudio S5 sequencer was used for high-resolution sequencing. The anti-human IFN-γ antibody light and heavy chain variable region genes were cloned into the human IG1 type heavy chain expression vector and light chain expression vector (as shown in Figure 3 The sequence was confirmed to be correct by sequencing.

[0084] Example 2: Expression and activity identification of anti-human IFN-γ genetically engineered antibodies

[0085] The light and heavy chain expression vectors obtained in Example 1 were co-transfected into 293T cells to induce transient expression of anti-human IFN-γ genetically engineered antibodies, with an expression level of about 0.3-0.5 μg / μL (e.g. Figure 4 293T cells co-transfected with anti-human IFN-γ engineered antibody genes (293T-anti-IFN-γ) were cultured in a serum-free medium for hybridomas, and the culture supernatant was collected for activity analysis. The activity of the engineered antibody and the commercial anti-human IFN-γ antibody was compared using ELISA, and the specificity of the genetically engineered antibody and the commercial antibody in recognizing antigens was analyzed using western blotting (as shown in Figure 2). Figure 5 The specificity and reactivity of the genetically engineered antibodies with clinical specimens were analyzed using ELISA (as shown in Figure 6 The results showed that the genetically engineered antibodies had no cross-reaction and good binding specificity.

[0086] 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 IFN-γ 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 isolated 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. The isolated antibody or antigen-binding portion according to claim 1 or 2, characterized in that The antibody or antigen-binding portion is modified, The modification is N-glycosylation modification, O-glycosylation modification or label modification.

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

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 4.

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

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

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