CN111763257B - anti-GITR antibodies and uses thereof - Google Patents
anti-GITR antibodies and uses thereof Download PDFInfo
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- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2878—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
- A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
- A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
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- A61P35/00—Antineoplastic agents
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
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- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/565—Complementarity determining region [CDR]
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- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
- C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
Abstract
The invention provides an anti-GITR antibody or an antigen-binding fragment thereof, and a preparation method and application thereof. The anti-GITR antibody or the antigen binding fragment thereof can effectively block the binding of GITR and a ligand, has strong specificity and high affinity when being bound with human GITR, and in vivo experiments also show that the anti-GITR antibody is well tolerated by non-human animals, is nontoxic and obviously inhibits the growth of tumors.
Description
Technical Field
The invention relates to the technical field of anti-GITR antibodies, in particular to an anti-GITR antibody or an antigen binding fragment thereof, a preparation method and application thereof.
Background
Cancer is one of the most common diseases in human life. According to the statistical data of the world health organization, the number of cancer onset and death cases in the whole world in 2012 reaches 1400 ten thousand and 820 ten thousand respectively. In China, the number of newly diagnosed cancer cases is 307 million and the number of deaths is 220 million.
The recent clinical and commercial success of anti-cancer antibodies has led to a great interest in antibody-based therapeutics. There is a need to develop anti-cancer antibodies for use in various antibody-based therapies to treat cancer.
GITR (glucocorticoid-induced tumor necrosis factor receptor protein) molecule is a type I transmembrane protein, belongs to a member of the Tumor Necrosis Factor Receptor (TNFR) superfamily, and includes an extracellular region, a transmembrane region, and a cytoplasmic region, and is expressed at high levels in Treg cells and also expressed at low levels in CD4+ and CD8+ T cells, and the expression is significantly increased after stimulation and activation. GITRL, a ligand for GITR, is a member of the Tumor Necrosis Factor (TNF) superfamily, and is predominantly expressed on Antigen Presenting Cells (APC). After GITR is combined with GITRL, a costimulatory signal on the surface of a T cell is transmitted, and the effect of the T Cell Receptor (TCR) is assisted by CD28 and CD3, so that the T cell is stimulated to activate, proliferate and secrete cytokines (but the auxiliary stimulation effect is not as strong as that of CD 28); inhibit Foxp3+ Tregs immunosuppressive activity; inducing and activating macrophage. Studies have shown that GITR plays an important role in a variety of immunological processes and has great application value in the fields of infection, tumor and autoimmune/inflammatory disease treatment.
Accordingly, there is a great need in the art for more techniques for inhibiting the binding of GITR to its ligand, and the present invention provides a novel anti-GITR antibody or antigen-binding fragment thereof, which is useful for the treatment of infection, tumor, autoimmune disease, or inflammatory disease.
Disclosure of Invention
The invention provides an antibody or an antigen binding fragment thereof specifically binding GITR, which can effectively inhibit the binding of the GITR and a ligand thereof, has strong specificity and high affinity when being bound with human GITR, and in vivo experiments also show that the antibody or the antigen binding fragment thereof is well tolerated by non-human animals, is nontoxic and can obviously inhibit the growth of tumors. The method comprises the following specific steps:
in a first aspect of the invention, there is provided an anti-GITR antibody or antigen-binding fragment thereof comprising VHCDR1, VHCDR2 and VHCDR3 of the heavy chain variable region and VLCDR1, VLCDR2 and VLCDR3 of the light chain variable region, wherein the amino acid sequence of VHCDR1 comprises SEQ ID NO:1 or 7, the amino acid sequence of VHCDR2 comprises SEQ ID NO: 2 or 8, the amino acid sequence of VHCDR3 comprises SEQ ID NO: 3, the amino acid sequence of VLCDR1 comprises SEQ ID NO:4, the amino acid sequence of VLCDR2 comprises SEQ ID NO: 5, the amino acid sequence of VLCDR3 comprises SEQ ID NO: and 6.
Further preferably, the amino acid sequence of the heavy chain variable region comprises SEQ ID NO 13 or has more than 80% homology with SEQ ID NO 13 and retains the ability to bind to GITR.
Further preferably, the amino acid sequence of the light chain variable region comprises SEQ ID NO. 14 or has more than 80% homology with SEQ ID NO. 14 and retains the ability to bind to GITR.
Preferably, the anti-GITR antibody or antigen-binding fragment thereof is a monoclonal antibody or a polyclonal antibody.
Preferably, the anti-GITR antibody or antigen-binding fragment thereof can be a single chain antibody (scFv), Fv antibody, Fd, dAb, bispecific antibody, bispecific single chain antibody, linear antibody, single chain antibody molecule, multispecific antibody formed from antibody fragments, and any polypeptide comprising an antibody binding domain or homologous antibody binding domain. The antibody binding domain may comprise, among other things, an intact heavy and/or light chain CDR, an intact heavy and/or light chain variable region of an antibody, an intact full-length heavy and/or light chain, or a single, two, three, four, five or six CDR from the antibody. Single chain antibodies comprise a heavy chain variable region and a light chain variable region.
Preferably, the anti-GITR antibody or antigen-binding fragment thereof further comprises a constant domain from a human IgG1 antibody. Further preferred are CL, CH1, CH2 and/or CH3 domains.
Preferably, the anti-GITR antibody or antigen-binding fragment thereof specifically binds to human, chimeric or non-human animal GITR.
In a specific embodiment of the invention, the anti-GITR antibody or antigen-binding fragment thereof specifically binds human GITR (e.g., SEQ ID NO: 12), monkey GITR (e.g., SEQ ID NO: 10), or chimeric GITR (e.g., SEQ ID NO: 11).
In a second aspect of the invention, there is provided a nucleic acid encoding the above-described anti-GITR antibody, or antigen-binding fragment thereof.
In a third aspect of the invention, there is provided a nucleic acid comprising a polynucleotide encoding a polypeptide comprising:
(1) an immunoglobulin heavy chain or fragment thereof comprising a heavy chain variable region comprising VHCDRs 1, 2 and 3 of SEQ ID NOs 1, 2 and 3, and wherein the heavy chain variable region binds GITR when paired with a light chain variable region comprising the amino acid sequence set forth in SEQ ID No. 14; and/or the presence of a gas in the gas,
(2) an immunoglobulin light chain or fragment thereof comprising a light chain variable region comprising the VLCDRs 1, 2, and 3 of SEQ ID NOS: 4, 5, and 6, and wherein the light chain variable region binds GITR when paired with a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 13.
In a fourth aspect of the invention, there is provided a vector comprising a nucleic acid according to the invention.
Preferably, the vector is capable of expression in vivo or in vitro or ex vivo. Further preferably, the expression vector is expressed at a high level in vivo in cells. Preferably, the expression vector is a prokaryotic expression vector or a lentiviral expression vector. Further preferably, the prokaryotic expression vector is an escherichia coli series.
In a fifth aspect of the invention, there is provided a cell comprising a nucleic acid or vector as described above.
Preferably, the cell may be eukaryotic or prokaryotic. More preferably, the cell may be a yeast cell, 293 cell, CHO cell, escherichia coli, or the like.
In a sixth aspect of the present invention, there is provided a hybridoma cell producing the above-described anti-GITR antibody or antigen-binding fragment thereof.
In a seventh aspect of the present invention, there is provided a method for producing a hybridoma, comprising immunizing a non-human animal with human GITR, collecting splenocytes from the non-human animal after immunization with human GITR, and fusing the collected splenocytes with SP2/0 cells to obtain a hybridoma.
In an eighth aspect of the present invention, there is provided a method for producing an anti-GITR antibody or an antigen-binding fragment thereof, comprising culturing a cell comprising the above-described nucleic acid to obtain the anti-GITR antibody or the antigen-binding fragment thereof.
In a ninth aspect of the present invention, there is provided a method for preparing an anti-GITR antibody or an antigen-binding fragment thereof, the method comprising a protein immunization method or a DNA immunization method.
Preferably, the method comprises immunizing a non-human animal with human GITR.
Preferably, the method further comprises collecting splenocytes from the non-human animal after immunization with human GITR.
Preferably, the method further comprises the step of fusing the collected spleen cells with SP2/0 cells to obtain hybridoma cells.
Preferably, the hybridoma cells are introduced into a non-human animal, and ascites fluid is collected from the non-human animal.
Preferably, the human GITR may be a human GITR protein (preferably, the amino acid sequence shown in SEQ ID NO: 12) or a nucleotide sequence (preferably, a cDNA sequence, more preferably, a nucleotide sequence coding for the amino acid sequence shown in SEQ ID NO: 12) encoding the human GITR protein.
Preferably, the human GITR protein is His-tagged.
Preferably, the non-human animal is a non-human mammal. Further preferably a rodent.
In one embodiment of the invention, the non-human animal is a rat or a mouse.
In a tenth aspect of the invention, there is provided an antibody-drug conjugate comprising said antibody or antigen-binding fragment thereof covalently bound to a therapeutic agent.
Preferably, the therapeutic agent may be a chemical synthetic drug, an antibiotic, or various biological drugs.
In an eleventh aspect of the invention, there is provided a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of the invention, or the antibody-drug conjugate, and a pharmaceutically acceptable carrier.
Preferably, the pharmaceutically acceptable carrier may be one or more, and includes, but is not limited to, diluents, binders, humectants, surfactants, lubricants, or disintegrants, and the like.
More preferably, the pharmaceutical composition may be administered comprised in a nanocarrier, a viral vector, a microcapsule, a liposome, or the like.
In a twelfth aspect of the invention, there is provided a composition, a test kit, a chip or an antibody conjugate comprising any one of the following:
1) an anti-GITR antibody or antigen-binding fragment thereof according to the present invention;
2) a nucleic acid according to the present invention;
3) a vector of the present invention;
4) a cell according to the invention;
5) a hybridoma cell according to the invention; alternatively, the first and second electrodes may be,
6) the T cell antigen receptor or CAR molecule of the invention.
Preferably, the composition further comprises other agents which assist or cooperate with any of the products 1) to 5) above.
Preferably, the agent may be a pharmaceutically acceptable carrier, which may be one or more, including but not limited to diluents, binders, humectants, surfactants, lubricants, disintegrants, and the like.
More preferably, the composition may be administered in a nanocarrier, a viral vector, a microcapsule, a liposome, or the like.
In a thirteenth aspect of the present invention, there is provided a use of the above-mentioned anti-GITR antibody or antigen-binding fragment thereof, the above-mentioned nucleic acid, the above-mentioned vector, the above-mentioned cell, the above-mentioned composition, the detection kit, the chip, or the antibody conjugate, the use comprising:
A) use in the manufacture of a product for the treatment of a GITR/GITRL related disease; or the like, or, alternatively,
B) use in a GITR assay.
Preferably, the GITR/GITRL-associated disease is an infection, a tumor, an autoimmune disease, or an inflammatory disease.
In a fourteenth aspect of the invention, there is provided a T cell antigen receptor or CAR molecule comprising the above anti-GITR antibody or antigen binding fragment thereof.
In a fifteenth aspect of the present invention, there is provided a method of treating a tumor or killing tumor cells, said method comprising administering to an individual an effective amount of an anti-GITR antibody or antigen-binding fragment thereof, composition as described above, a test kit, a chip or an antibody conjugate according to the present invention.
Preferably, the method comprises treating the disease by combining the above composition with other drugs or treatments.
More preferably, the present invention provides a method of combined treatment of a malignant tumor in a subject, comprising administering to the subject a therapeutically effective amount of the above pharmaceutical composition, and further comprising administering to the subject a therapeutically effective amount of another agent for treating a malignant tumor or administering another method for treating a malignant tumor, such as chemotherapeutic drugs, surgical treatments, radiation treatments, biological treatments, chinese traditional medicine treatments, minimally invasive treatments, and the like.
In a sixteenth aspect of the invention, there is provided a method of inducing an immune response, the method comprising administering to an individual an anti-GITR antibody or antigen-binding fragment thereof of the invention, a composition, a test kit, a chip or an antibody conjugate as described above.
In a seventeenth aspect of the present invention, there is provided a method for detecting GITR, comprising contacting a sample to be detected with the anti-GITR antibody or antigen-binding fragment thereof according to the present invention, and then detecting a complex formed between GITR and the anti-GITR antibody or antigen-binding fragment thereof.
Preferably, the detection method is detecting the presence or amount of GITR. Wherein the presence or absence is indicated, and the content may be an expression amount, a protein concentration, or the like.
In an eighteenth aspect of the present invention, there is provided a method for diagnosing a GITR/GITRL-associated disease, which comprises sampling, contacting the sample with the anti-GITR antibody or antigen-binding fragment thereof according to the present invention, and then detecting a complex formed between GITR and the anti-GITR antibody or antigen-binding fragment thereof.
In a nineteenth aspect of the present invention, there is provided a method of reducing the rate of tumor growth or killing tumor cells, the method comprising: contacting a tumor cell with an effective amount of a product comprising an antibody or antigen-binding fragment thereof, or an antibody-drug conjugate, or a pharmaceutical composition of the invention.
In a twentieth aspect of the present invention, there is provided a use of the anti-GITR antibody or antigen-binding fragment thereof of the present invention for the preparation of a medicament for treating a tumor.
An "antigen-binding fragment" as described herein is a portion of an antibody that retains the specific binding activity of an intact antibody, i.e., any portion of the antibody is capable of specifically binding to an epitope on a target molecule of an intact antibody. It includes, for example, Fab ', F (ab')2 and variants of these fragments. For example, a heavy and/or light chain CDR of a whole antibody, a heavy and/or light chain variable region of a whole antibody, a full length heavy or light chain of a whole antibody, or a single CDR from a heavy or light chain of a whole antibody.
"administration" as used herein includes, but is not limited to, oral, enteral, subcutaneous, intradermal, intramuscular, intraarterial, intravenous, nasal, transdermal, subconjunctival, intraperitoneal, intrabulbar, orbital, retrobulbar, retinal, choroidal, intrathecal administration and the like.
An "effective amount" as described herein refers to an amount or dose of a product described herein (preferably an anti-GITR antibody or antigen-binding fragment thereof) that provides the desired treatment after administration to a patient or organ or individual in a single or multiple doses.
"diagnosis" as used herein refers to the determination of whether a patient has suffered from a disease or condition in the past, at the time of diagnosis, or in the future, or the determination of the progression or likely progression of a disease in the future, or the assessment of a patient's response to a therapy.
"treating" as referred to herein means slowing, interrupting, arresting, controlling, stopping, reducing, or reversing the progression or severity of one sign, symptom, disorder, condition, or disease, but does not necessarily involve the complete elimination of all disease-related signs, symptoms, conditions, or disorders, and refers to therapeutic intervention that ameliorates the signs, symptoms, etc. of a disease or pathological state after the disease has begun to develop.
The term "and/or" as used herein includes a list of items in the alternative as well as any number of combinations of items.
The terms "comprises" and "comprising" as used herein are intended to be open-ended terms that specify the presence of the stated elements or steps, as well as any other elements or steps that do not materially affect the technical effects specified. When used herein to describe the sequence of a protein or nucleic acid, the protein or nucleic acid may be composed of the sequence, or may have additional amino acids or nucleotides at one or both ends of the protein or nucleic acid, but still have the activity described herein.
An "individual" according to the invention may be a human or non-human animal. The non-human animal can be a non-human mammal, such as a monkey, a mouse, a rabbit, and the like.
In one aspect, the non-human animal is a mammal. In one aspect, the non-human animal is a small mammal, such as a muridae or superfamily murinus. In one embodiment, the genetically modified animal is a rodent. In one embodiment, the rodent is selected from a mouse, a rat, and a hamster. In one embodiment, the rodent is selected from the murine family. In one embodiment, the genetically modified animal is from a family selected from the family of the family. In a particular embodiment, the genetically modified rodent is selected from a true mouse or rat (superfamily murinus), a gerbil, a spiny mouse, and a crowned rat. In one embodiment, the genetically modified mouse is from a member of the murine family. In one embodiment, the animal is a rodent. In a particular embodiment, the rodent is selected from a mouse and a rat. In one embodiment, the non-human animal is a mouse.
In a particular embodiment, the non-human animal is a rodent, a strain of C57BL, C58, a/Br, CBA/Ca, CBA/J, CBA/CBA/mouse selected from BALB/C, a/He, a/J, A/WySN, AKR/A, AKR/J, AKR/N, TA1, TA2, RF, SWR, C3H, C57BR, SJL, C57L, DBA/2, KM, NIH, ICR, CFW, FACA, C57BL/A, C57BL/An, C57BL/GrFa, C57BL/KaLwN, C57BL/6, C57BL/6J, C57BL/6ByJ, C57BL/6NJ, C57BL/10, C57BL/10 sn, C57BL/10Cr and C57 BL/Ola.
The "inflammatory disease" according to the present invention is selected from acute inflammation, also including chronic inflammation. Specifically, it includes, but is not limited to, degenerative inflammation, exudative inflammation (serous inflammation, cellulolytic inflammation, suppurative inflammation, hemorrhagic inflammation, necrotizing inflammation, catarrhal inflammation), proliferative inflammation, specific inflammation (tuberculosis, syphilis, leprosy, lymphogranuloma, etc.).
The infection of the invention includes but is not limited to plague, cholera, AIDS, atypical pneumonia, influenza A, tuberculosis, avian influenza, viral hepatitis, rabies, poliomyelitis, measles, epidemic cerebrospinal meningitis, epidemic encephalitis B, epidemic hemorrhagic fever, leptospirosis, typhoid fever, schistosomiasis and the like.
The "autoimmune disease" described in the present invention includes, but is not limited to, allergy, asthma, myocarditis, nephritis, hepatitis, systemic lupus erythematosus, rheumatoid arthritis, scleroderma, hyperthyroidism, idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, ulcerative colitis, autoimmune liver disease, diabetes, pain, or neurological disorder, etc.
The "tumor" according to the present invention is selected from leukemia, lymphoma, ovarian cancer, breast cancer, endometrial cancer, colon cancer, rectal cancer, stomach cancer, bladder cancer, lung cancer (e.g., non-small cell lung cancer, etc.), bronchial cancer, bone cancer, prostate cancer, pancreatic cancer, liver and bile duct cancer, esophageal cancer, kidney cancer, thyroid cancer, head and neck cancer, testicular cancer, glioblastoma, astrocytoma, melanoma, myelodysplastic syndrome, and sarcoma. Wherein said leukemia is selected from the group consisting of: acute lymphocytic (lymphoblastic) leukemia, acute myelogenous leukemia, chronic lymphocytic leukemia, multiple myeloma, plasma cell leukemia, and chronic myelogenous leukemia; the lymphoma is selected from the group consisting of: hodgkin's lymphoma and non-hodgkin's lymphoma, including B-cell lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, marginal zone B-cell lymphoma, T-cell lymphoma, and waldenstrom's macroglobulinemia; and said sarcoma is selected from the group consisting of: osteosarcoma, ewing's sarcoma, leiomyosarcoma, synovial sarcoma, alveolar soft tissue sarcoma, angiosarcoma, liposarcoma, fibrosarcoma, rhabdomyosarcoma, and chondrosarcoma.
Kabat numbering is used by default in this disclosure unless specifically noted otherwise in this disclosure.
Drawings
Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:
FIG. 1 is a diagram showing a process for preparing anti-hGITR antibody, specifically, a step of immunization into serum collection, and detection of antibody titer of the collected serum.
FIG. 2 is a flowchart showing the procedure for preparing anti-hGITR antibody, specifically, the steps of boosting immunization, collecting spleen cells, preparing hybridoma cells, and screening the hybridoma cells, and producing and purifying the antibody using the hybridoma cells.
FIG. 3 is a set of flow cytometry plots showing the binding between anti-hGITR antibody (2D 3-mHvKv-IgG 1) and hGITR, with INCAGN01876 as a positive control and NC as a negative control.
FIG. 4 is a set of graphs showing the flow cytometry results of analyzing the cross-reactivity of anti-hGITR antibody (2D 3-mHvKv-IgG 1) with monkey GITR (rmGITR), mouse GITR (mGITR) and human-mouse chimeric GITR (chiGITR), where NC represents a negative control and INCAGN01876 is a positive control.
FIG. 5 is a graph showing body weight over time of a humanized GITR mouse having MC-38 tumor cells (B-hGITR) treated with an anti-hGITR antibody (2D 3-mHvKv-IgG 1) and a humanized anti-hGITR antibody INCAGN 01876.
FIG. 6 is a graph showing the percentage change of body weight over time of humanized GITR mice (B-hGITR) having MC-38 tumor cells treated with anti-hGITR antibody (2D 3-mHvKv-IgG 1) and humanized anti-hGITR antibody INCAGN 01876.
FIG. 7 is a graph showing tumor volume over time of humanized GITR mice (B-hGITR) having MC-38 tumor cells treated with anti-hGITR antibody (2D 3-mHvKv-IgG 1) and humanized anti-hGITR antibody INCAGN 01876.
Detailed Description
The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.
Example 1: generation of mouse anti-hGITR antibodies
To generate mouse antibodies against human GITR (hGITR; SEQ ID NO: 12), 6-8 week old female BALB/c mice were immunized with human GITR. The anti-hGITR antibody was collected by the methods described below and shown in fig. 1 and 2.
Immunization of mice
Female BALB/c mice 6-8 weeks old were immunized with His-tagged human GITR protein at a concentration of 100. mu.g/mL at 20. mu.g/mouse. His-tagged human GITR protein was emulsified with adjuvant and injected at four locations on the back of mice. For the first subcutaneous (s.c.) injection, the diluted antigen was emulsified with an equal volume of Complete Freund's Adjuvant (CFA). In a subsequent subcutaneous injection, the protein was emulsified with an equal volume of Incomplete Freund's Adjuvant (IFA). Three days after the third injection or booster immunization, blood (serum) was collected and analyzed for antibody titer using ELISA.
In another experiment, 6-8 week old female BALB/c mice were immunized by injection of an expression plasmid encoding human GITR into the mice. The plasmid encoding the antigen was injected into the tibialis anterior muscle of the mouse at a concentration of 1000. mu.g/. mu.L by using a gene gun at 60. mu.g per mouse (intramuscular injection; i.m. injection). At least four injections were performed with at least 14 days between each injection. Blood (serum) was collected 7 days after the last immunization and the serum was tested for antibody titer by ELISA.
Procedures to enhance immunization (either by injection of plasmids or by injection of proteins) were also performed at least fourteen days after the previous immunization. CHO cells expressing GITR antigen on the surface were injected into mice via tail vein. Spleens were then collected four days after injection.
Fusion of SP2/0 cells with splenocytes
Spleen tissue was ground. Splenocytes were first selected by CD3 microbeads and anti-mouse IgM microbeads, and then fused to SP2/0 cells. The cells were then plated in 96-well plates with hypoxanthine-aminopterin-thymidine (HAT) medium.
Preliminary screening for hybridomas
Hybridoma supernatants from 96-well plates were initially screened using Fluorescence-Activated Cell Sorting (FACS) according to standard procedures. Chinese Hamster Ovary (CHO) cells were added to 96-well plates (2X 10 per well) prior to screening4Individual cells). 50 μ L of supernatant was used. The antibodies used in the experiments were:
(1) fluorescein (FITC) conjugated AffiniPure F (ab)2Fragment goat anti-mouse IgG, specific for Fc γ fragments; and
(2) alexa Fluor 647 conjugated AffiniPure F (ab)2Fragment goat anti-human IgG, Fc γ fragment specific.
Subcloning
Subcloning was performed using ClonePix 2. Briefly, positive wells identified in the primary screen were transferred to semi-solid medium and IgG positive clones were identified and tested. FITC anti-mouse IgG Fc antibody was used.
Ascites antibody
Will be 1 × 106The individual positive hybridoma cells were intraperitoneally injected into B-NDG mice (Beijing Baiosao chart, China Beijing). Monoclonal antibodies were produced by growing hybridoma cells intraperitoneally in mice. Hybridoma cells expand in the abdomen of mice and produce ascites fluid. Ascites fluid contains high concentrations of antibodies and can be harvested for later use.
Antibody purification
Antibodies in the ascites were purified using GE AKTA protein chromatography (GE Healthcare, Chicago, Illinois, United States) to give 61-2D3 ("2D 3") mouse antibodies.
The VH, VL and CDR regions of the antibody were determined. The heavy chain CDR1, CDR2, and CDR3 of 2D3 and the light chain CDR1, CDR2, and CDR3 amino acid sequences are shown in SEQ ID NOs 1-6 (Kabat numbering) or SEQ ID NOs 7, 8, 3, 4, 5, 6 (Chothia numbering). The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO 13 and the amino acid sequence of the light chain variable region is shown in SEQ ID NO 14.
Example 2: in vitro testing of mouse anti-hGITR antibodies: binding to human GITR (hGITR)
In vitro assays were performed to determine whether anti-hGITR antibodies could bind to hGITR.
anti-hGITR antibodies were collected from the mouse ascites and purified by chromatography. 25 μ L of CHO cells transiently transfected with human GITR were added to each well in the plate. The purified antibody was titrated to final concentrations of 10, 1, 0.1, 0.01, 0.001. mu.g/mL. Titrated antibody was added to each well at 25 μ Ι per well at 4 ℃ and incubated for 30 minutes.
2D3 is the mouse anti-hGITR antibody described in example 1. Based on 2D3, a 2D3-mHvKv-IgG1 chimeric anti-hGITR antibody was generated. The chimeric antibody has a heavy chain variable domain and a light chain variable domain from a corresponding mouse anti-hGITR antibody, and a constant domain (including, e.g., CL, CH1, CH2 and CH3 domains) from a human IgG1 antibody.
After washing twice with Phosphate Buffered Saline (PBS), 50. mu.L of FITC-labeled Anti-Human IgG Fc antibody (Fluoroscein (FITC) -AffiniPure F (ab')2 Fragment Goat Anti-Human IgG, Fc. gamma. Fragment specificity, from Jackson, cat # 109-. The signal of FITC was determined by flow cytometry.
As shown in FIG. 3, when the concentrations of the chimeric anti-hGITR antibody 2D3-mHvKv-IgG1 and the humanized anti-hGITR antibody INCAGN01876 antibody (as a positive control drug) were increased, the signal of FITC was increased, indicating that the anti-hGITR antibody 2D3-mHvKv-IgG1 could bind to human GITR, and that the binding ability to human GITR was significantly stronger than that of the positive control group.
Example 3: cross-reactivity of anti-hGITR antibodies against monkey, mouse and human-mouse chimeric GITR
In each experiment, CHO cells were transfected with the coding sequences of mouse GITR (mGITR, SEQ ID NO: 9), monkey (rhesus) GITR (rmGITR, SEQ ID NO: 10) or chimeric (mouse and human) GITR (chiGITR, SEQ ID NO: 11).
25 μ L CHO cells were added to each well. 25 μ L of purified anti-hGITR antibody (10 μ g/mL) was added to each well and incubated at 4 ℃ for 30 minutes.
After washing twice with PBS (1200 rmp, 5 min), 50 μ L of FITC-labeled anti-human IgG Fc antibody was added to each well at a dilution of 1:500 and incubated at 4 ℃ for 30 min, followed by washing with PBS (1200 rmp, 5 min). The signal of FITC was determined by flow cytometry.
As shown in FIG. 4, both the chimeric anti-hGITR antibody 2D3-mHvKv-IgG1 and the humanized anti-hGITR antibody INCAGN01876 did not cross-react with mouse GITR, but strongly cross-reacted with rmGITR and chiGITR.
Example 4: binding affinity of anti-hGITR antibodies
The binding affinity of anti-hGITR antibodies was measured by Surface Plasmon Resonance (SPR) with a Biacore (Biacore, INC, Piscataway n.j.) T200 biosensor equipped with a pre-immobilized protein a sensor chip.
anti-hGITR antibody 2D3-mHvKv-IgG1 or incagnn 01876 (1 μ g/mL) was injected into the Biacore T200 biosensor at 10 μ L/min for 30 seconds to reach the desired protein density (approximately 67 Response Units (RU)). Histidine-tagged human GITR protein (source ACRO, cat # GIR-H5228) was then injected at a concentration of 200, 100, 50, 25, 12.5, 6.25, 3.125, 1.5625nM for 100 seconds at 30 μ L/min. Dissociation was monitored for 400 seconds. The chips were regenerated with glycine (pH 2.0, 30 μ L/min for 12 seconds) after the last injection of each titer. The kinetic association rate (kon) and dissociation rate (koff) were obtained simultaneously by fitting the data globally to a 1:1 Langmuir binding model using Biacore T200 evaluation software 3.0 (Karlsson, r. Roos, h. fagerstat, l. Petersson, b., 1994. Methods Enzymology 6.99-110). Affinity was deduced from the quotient of kinetic rate constants (KD = koff/kon).
Table 1 below shows the results for 2D3-mHvKv-IgG1 and INCAGN01876 antibodies.
TABLE 1
Example 5: in vivo testing of anti-GITR antibodies
To test anti-hGITR antibodies in vivo and predict the effect of these antibodies in humans, a humanized GITR mouse model was generated. The humanized GITR mouse model was engineered to express a chimeric GITR protein (SEQ ID NO: 11) in which a portion of the extracellular region of the mouse GITR protein was replaced with the corresponding extracellular region of human GITR. Amino acid residues 1 to 130 of mouse GITR (SEQ ID NO: 9) are substituted with amino acid residues 1 to 142 of human GITR (SEQ ID NO: 12). The humanized mouse model (B-hGITR) provides a new tool for testing new therapeutic treatments in a clinical setting by significantly reducing the difference between clinical outcomes in human and ordinary mice expressing mouse GITR. A detailed description of a humanized GITR mouse model can be found in PCT/CN2018/091844, which is incorporated herein by reference in its entirety.
The effect of anti-hGITR antibodies on tumor growth in vivo was tested in a colon cancer model. Murine MC-38 cells (colon adenocarcinoma cells) were injected subcutaneously in B-hGITR mice. When tumors in mice reached 100. + -.50 mm3At volume (c), mice were randomly assigned to different groups (5 mice per group) depending on the tumor volume. Mice were then injected with Physiological Saline (PS) and anti-hGITR antibody, respectively, by intraperitoneal administration. Antibodies were administered on days 2 and 5 of each week, i.e., twice a week for 3 weeks (6 total injections).
The injection volume was calculated at 3mg/kg based on the body weight of the mice. The lengths of the major and minor axes of the tumor were measured and counted as 0.5 (major axis) x (minor axis)2The volume of the tumor was calculated. Body weights of mice were also measured prior to injection at the time of dividing the mice into different groups (before the first antibody injection), twice per week during the antibody injection period, and before euthanasia was performed.
Percent tumor growth inhibition (TGI%) was calculated using the following formula:
. Ti is the mean tumor volume in the treatment group on day i. T0 is the mean tumor volume at day 0 in the treatment group. Vi is the mean tumor volume of the control group at day i. V0 is the mean tumor volume at day 0 in the control group.
A t-test was performed for statistical analysis. TGI% above 60% indicates significant inhibition of tumor growth. p <0.05 is a threshold indicating a significant difference.
In each of the three groups (G1, G2, G3), B-hGITR mice were injected with Physiological Saline (PS) as a control (G1), chimeric anti-hGITR antibody 2D3-mHvKv-IgG1 (G2) or humanized anti-hGITR antibody INCAGN01876 (G3), respectively. The body weight of the mice was monitored throughout the treatment period. The body weight of the mice in the different groups was all increased (fig. 5 and 6). No significant difference in body weight was observed between the three groups. The results show that 2D3-mHvKv-IgG1 and INCAGN01876 are well tolerated and are non-toxic to mice.
The degree of tumor volume increase was smaller in the group treated with INCAGN01876 and 2D3-mHvKv-IgG1 compared to the control group (FIG. 7). In particular, the tumor volume in G2 was less than G3.
As shown in the table below, TGI% at day 24 (24 days after the group) was also calculated.
TABLE 2
The results show that both the chimeric antibody 2D3-mHvKv-IgG1 and the humanized antibody INCAGN01876 inhibit tumor growth. Among them, 2D3-mHvKv-IgG1 had a higher TGI% than humanized antibody INCAGN 01876.
It will be appreciated by persons skilled in the art that while the invention has been described in conjunction with specific embodiments thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.
Sequence listing
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Claims (10)
1. An anti-GITR antibody or antigen-binding fragment thereof comprising VHCDR1, VHCDR2 and VHCDR3 of the heavy chain variable region and VLCDR1, VLCDR2 and VLCDR3 of the light chain variable region, wherein the amino acid sequence of VHCDR1 is as set forth in SEQ ID NO:1, the amino acid sequence of VHCDR2 is shown in SEQ ID NO: 2, the amino acid sequence of VHCDR3 is set forth in SEQ ID NO: 3, the amino acid sequence of VLCDR1 is set forth in SEQ ID NO:4, the amino acid sequence of VLCDR2 is set forth in SEQ ID NO: 5, the amino acid sequence of VLCDR3 is shown in SEQ ID NO: 6, the anti-GITR antibody or the antigen-binding fragment thereof is a monoclonal antibody or a polyclonal antibody.
2. The anti-GITR antibody or antigen-binding fragment thereof of claim 1, wherein the amino acid sequence of the heavy chain variable region is set forth in SEQ ID No. 13 and the amino acid sequence of the light chain variable region is set forth in SEQ ID No. 14.
3. The anti-GITR antibody or antigen-binding fragment thereof according to claim 1, wherein said anti-GITR antibody or antigen-binding fragment thereof is a single chain antibody, Fv antibody, Fd, dAb, bispecific antibody, bispecific single chain antibody, linear antibody, or multispecific antibody.
4. The anti-GITR antibody or antigen-binding fragment thereof of any one of claims 1-3, wherein the anti-GITR antibody or antigen-binding fragment thereof specifically binds human GITR.
5. A nucleic acid comprising a polynucleotide encoding a polypeptide comprising:
(1) an immunoglobulin heavy chain comprising a heavy chain variable region comprising VHCDRs 1, 2 and 3 of SEQ ID NOs 1, 2 and 3, and wherein the heavy chain variable region binds GITR when paired with a light chain variable region of an amino acid sequence set forth in SEQ ID No. 14; and the combination of (a) and (b),
(2) an immunoglobulin light chain comprising a light chain variable region comprising the VLCDRs 1, 2, and 3 of SEQ ID NOS: 4, 5, and 6, and wherein the light chain variable region binds GITR when paired with the heavy chain variable region of the amino acid sequence set forth in SEQ ID NO: 13.
6. A cell comprising the nucleic acid of claim 5.
7. A method for producing an anti-GITR antibody or antigen-binding fragment thereof, comprising culturing the cell of claim 6 to obtain the anti-GITR antibody or antigen-binding fragment thereof.
8. An antibody-drug conjugate comprising the antibody or antigen-binding fragment thereof of any one of claims 1-4 covalently bound to a therapeutic agent.
9. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of any one of claims 1-4, or the antibody-drug conjugate of claim 8, and a pharmaceutically acceptable carrier.
10. Use of the anti-GITR antibody or antigen-binding fragment thereof of any one of claims 1-4 in the manufacture of a medicament for treating a GITR/GITRL-associated disease.
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