CN111763257B - Anti-GITR antibodies and their uses - Google Patents

Abstract

The present invention provides an anti-GITR antibody or an antigen-binding fragment thereof, as well as a preparation method and use thereof. The anti-GITR antibody or its antigen-binding fragment can effectively block the binding of GITR to ligands, and has strong specificity and high affinity for human GITR binding. Inhibit tumor growth.

Description

Anti-GITR antibodies and their uses

technical field

The present invention relates to the technical field of anti-GITR antibodies, in particular to anti-GITR antibodies or antigen-binding fragments thereof, as well as preparation methods and uses.

Background technique

Cancer is currently one of the diseases with the highest mortality rate in humans. According to statistics from the World Health Organization, in 2012, the number of global cancer incidence and deaths reached 14 million and 8.2 million, respectively. In China, there were 3.07 million newly diagnosed cancer cases and 2.2 million deaths.

Recent clinical and commercial successes of anti-cancer antibodies have generated 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 that belongs to the tumor necrosis factor receptor (TNFR) superfamily member, including extracellular domain, transmembrane domain and cytoplasmic domain. It is expressed at a high level on Treg cells, and also at a low level on CD4+ and CD8+ T cells, and the expression is significantly increased after stimulation and activation. The ligand GITRL of GITR, a member of the tumor necrosis factor (TNF) superfamily, is mainly expressed in antigen-presenting cells (APCs). After GITR is combined with GITRL, it transmits co-stimulatory signals on the surface of T cells, and together with CD28 and CD3, it acts as a T cell receptor (TCR) to stimulate T cell activation, proliferation and secretion of cytokines (but its co-stimulatory effect is not as strong as that of CD28. ); inhibits the immunosuppressive activity of Foxp3+ Tregs; induces activation of macrophages. Studies have shown that GITR plays an important role in various immune processes and has great application value in the fields of infection, tumor and autoimmune/inflammatory disease treatment.

Therefore, there is an urgent need in the art to obtain more technologies for inhibiting the binding of GITR to its ligands, so the present invention provides a novel anti-GITR antibody or an antigen-binding fragment thereof for use in infections, tumors, autoimmune diseases or inflammatory diseases disease treatment.

SUMMARY OF THE INVENTION

The present invention provides an antibody or its antigen-binding fragment that specifically binds to GITR. The antibody or its antigen-binding fragment can effectively inhibit the binding of GITR and its ligand, and has strong specificity and high affinity for binding to human GITR. It was also shown to be well tolerated by non-human animals, non-toxic, and significantly inhibited tumor growth. details as follows:

A first aspect of the present invention provides an anti-GITR antibody or an antigen-binding fragment thereof, the anti-GITR antibody or antigen-binding fragment thereof comprising VHCDR1, VHCDR2 and VHCDR3 of the heavy chain variable region, and the light chain variable region VLCDR1, VLCDR2 and VLCDR3, 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, and the amino acid sequence of VHCDR3 comprises SEQ ID NO: 3. The amino acid sequence of VLCDR1 is shown in SEQ ID NO: 4, the amino acid sequence of VLCDR2 is shown in SEQ ID NO: 5, and the amino acid sequence of VLCDR3 is shown in SEQ ID NO: 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 its antigen-binding fragment 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 antibodies formed from antibody fragments, as well as any polypeptide comprising an antibody binding domain or a homologous antibody binding domain. Wherein, the antibody binding domain may comprise the complete heavy chain and/or light chain CDRs, the heavy chain and/or light chain variable regions of the complete antibody, the complete full length heavy chain and/or light chain, or from the antibody single, two, three, four, five or six CDRs. Single chain antibodies contain a heavy chain variable region and a light chain variable region.

Preferably, the anti-GITR antibody or its antigen-binding fragment further comprises a constant domain derived from a human IgG1 antibody. It is further preferred to comprise 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 (eg SEQ ID NO: 12), monkey GITR (eg SEQ ID NO: 10) or chimeric GITR ( eg SEQ ID NO: 11).

The second aspect of the present invention provides a nucleic acid encoding the above-mentioned anti-GITR antibody or an antigen-binding fragment thereof.

A third aspect of the present invention provides a nucleic acid comprising a polynucleotide encoding a polypeptide, the polypeptide comprising:

(1) An immunoglobulin heavy chain or a fragment thereof comprising a heavy chain variable region comprising VHCDR1, 2 and 3 of SEQ ID NOs: 1, 2 and 3, and wherein the heavy chain variable region The 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,

(2) an immunoglobulin light chain or a fragment thereof comprising a light chain variable region comprising VLCDR1, 2 and 3 of SEQ ID NOs: 4, 5 and 6, and wherein the light chain is variable The region binds GITR when paired with a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:13.

The fourth aspect of the present invention provides a vector comprising the nucleic acid of the present invention.

Preferably, the vector can be expressed in vivo or in vitro or in vitro. Further preferably, the expression vector is continuously expressed at a high level in cells in vivo. Preferably, the expression vector is a prokaryotic expression vector or a lentiviral expression vector. Further preferably, the prokaryotic expression vector is Escherichia coli series.

The fifth aspect of the present invention provides a cell comprising the above nucleic acid or vector.

Preferably, the cells may be eukaryotic or prokaryotic. More preferably, the cells can be yeast cells, 293 cells, CHO cells, Escherichia coli and the like.

The sixth aspect of the present invention provides a hybridoma cell that produces the above-mentioned anti-GITR antibody or an antigen-binding fragment thereof.

A seventh aspect of the present invention provides a method for preparing hybridoma cells, the method comprising: obtaining by immunizing a non-human animal with human GITR, collecting spleen cells of the non-human animal immunized with human GITR, mixing the collected spleen cells with SP2 /0 cells were fused to obtain hybridoma cells.

The eighth aspect of the present invention provides a method for preparing an anti-GITR antibody or an antigen-binding fragment thereof, which comprises culturing cells containing the above nucleic acid to obtain an anti-GITR antibody or an antigen-binding fragment thereof.

The ninth aspect of the present invention provides 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 obtaining by immunizing non-human animals with human GITR.

Preferably, it also comprises collecting spleen cells of the non-human animal immunized with human GITR.

Preferably, hybridoma cells are obtained by fusing the collected splenocytes with SP2/0 cells.

The steps of introducing the hybridoma cells into the non-human animal, and collecting the ascites fluid of the non-human animal are preferred.

Preferably, the human GITR can be a human GITR protein (preferably the amino acid sequence shown in SEQ ID NO: 12) or a nucleotide sequence encoding a human GITR protein (preferably a cDNA sequence, more preferably encoding SEQ ID NO: 12 the nucleotide sequence of the amino acid sequence shown).

Preferably, the human GITR protein is His-tagged.

Preferably, the non-human animal is a non-human mammal. More preferred are rodents.

In a specific embodiment of the present invention, the non-human animal is a rat or a mouse.

A tenth aspect of the present invention provides an antibody-drug conjugate comprising the 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.

The eleventh aspect of the present invention provides a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of the present invention, or the antibody-drug conjugate, and a pharmaceutically acceptable carrier.

Preferably, the pharmaceutically acceptable carrier may be one or more, including but not limited to diluents, binders, wetting agents, surfactants, lubricants or disintegrating agents and the like.

More preferably, the pharmaceutical composition may be administered in nanocarriers, viral vectors, microcapsules, liposomes, and the like.

The twelfth aspect of the present invention provides a composition, detection kit, chip or antibody conjugate, comprising any of the following groups:

1) The anti-GITR antibody or antigen-binding fragment thereof of the present invention;

2) the nucleic acid of the present invention;

3) The carrier of the present invention;

4) The cells of the present invention;

5) the hybridoma cell of the present invention; or,

6) The T cell antigen receptor or CAR molecule of the present invention.

Preferably, it also contains other auxiliary or synergistic reagents with any of the products 1) to 5).

Preferably, the agent can be a pharmaceutically acceptable carrier, which can be one or more, and the carrier includes but is not limited to diluents, binders, humectants, surfactants, lubricants agent, disintegrant, etc.

More preferably, the composition may be administered in nanocarriers, viral vectors, microcapsules, liposomes, and the like.

The thirteenth aspect of the present invention provides the 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, detection kit, chip or antibody conjugate, said Uses include:

A) use in the manufacture of a product for the treatment of GITR/GITRL-related disorders; or,

B) Use in GITR detection.

Preferably, the GITR/GITRL-related disease is infection, tumor, autoimmune disease or inflammatory disease.

A fourteenth aspect of the present invention provides a T cell antigen receptor or CAR molecule, the T cell antigen receptor or CAR molecule comprising the above-mentioned anti-GITR antibody or an antigen-binding fragment thereof.

The fifteenth aspect of the present invention provides a method for treating tumors or killing tumor cells, the method comprising administering to an individual an effective amount of the anti-GITR antibody or antigen-binding fragment thereof of the present invention, the above-mentioned composition , detection kits, chips or antibody conjugates.

Preferably, the method includes combining the above composition with other drugs or treatment methods for disease treatment.

More preferably, the present invention provides a method for combined treatment of malignant tumors in a subject, comprising administering to the subject a therapeutically effective amount of the above-mentioned pharmaceutical composition, and further comprising administering to the subject a treatment An effective amount of other agents for treating malignant tumors or other methods for treating malignant tumors, such as chemotherapeutic drugs, surgical treatment, radiotherapy, biological therapy, traditional Chinese medicine treatment, minimally invasive treatment and the like.

The sixteenth aspect of the present invention provides a method for inducing an immune response, the method comprising applying the anti-GITR antibody or antigen-binding fragment thereof, the above-mentioned composition, detection kit, and chip of the present invention to an individual or antibody conjugates.

The seventeenth aspect of the present invention provides a method for detecting GITR, which comprises contacting a sample to be detected with the anti-GITR antibody or its antigen-binding fragment of the present invention, and then detecting the GITR and the anti-GITR antibody. or complexes formed by antigen-binding fragments thereof.

Preferably, the detection method is to detect the presence or content of GITR. Wherein, the presence indicates presence or absence, and the content may be the expression amount or protein concentration.

The eighteenth aspect of the present invention provides a method for diagnosing GITR/GITRL-related diseases, the method comprising sampling, contacting the sample with the anti-GITR antibody or antigen-binding fragment thereof of the present invention, and then detecting GITR Complexes with anti-GITR antibodies or antigen-binding fragments thereof.

The nineteenth aspect of the present invention provides a method for reducing tumor growth rate or killing tumor cells, the method comprising: contacting tumor cells with an effective amount of a product comprising the antibody of the present invention or its Antigen-binding fragments, or antibody-drug conjugates, or pharmaceutical compositions.

The twentieth aspect of the present invention provides an application of the anti-GITR antibody or its antigen-binding fragment of the present invention in preparing a medicament for treating tumors.

The "antigen-binding fragment" of the present invention is a part of an antibody that retains the specific binding activity of the intact antibody, ie any part of the antibody is capable of specifically binding to an epitope on the target molecule of the intact antibody. It includes eg Fab, Fab', F(ab')2 and variants of these fragments. For example, heavy and/or light chain CDRs of an intact antibody, heavy and/or light chain variable regions of an intact antibody, full-length heavy or light chains of an intact antibody, or heavy or light chains from an intact antibody a single CDR.

"Applying" in the present invention includes, but is not limited to, oral administration, enteral administration, subcutaneous injection, intradermal injection, intramuscular injection, intraarterial injection, intravenous injection, intranasal administration, transdermal administration, subconjunctival administration, intraperitoneal administration Intraocular injection, intraocular administration, orbital administration, retrobulbar administration, retina administration, choroidal administration, intrathecal injection, etc.

The "effective amount" of the present invention refers to the amount of the product of the present invention (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 single or multiple doses. amount or dose.

"Diagnosing" as used herein means to ascertain whether a patient has a disease or condition in the past, at the time of diagnosis or in the future, or to ascertain the progression or possible future progression of a disease, or to assess a patient's response to treatment.

"Treatment" as used herein means slowing, interrupting, preventing, controlling, stopping, alleviating, or reversing the progression or severity of a sign, symptom, disorder, condition, or disease, but not necessarily all disease-related signs, Complete elimination of a symptom, disorder, or disorder, and refers to a therapeutic intervention that ameliorates the signs, symptoms, etc. of a disease or pathological state after the disease has begun to develop.

The references herein to "and/or" include any number of combinations of the listed items in alternative.

The "comprising" or "comprising" described in the present invention is an open-ended description, including the specified components or steps described, as well as other specified components or steps that will not substantially affect the technical effect. When it is used in this application to describe the sequence of a protein or nucleic acid, the protein or nucleic acid may consist 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 in the present invention.

The "individual" referred to in the present invention can be a human or a non-human animal. Wherein, the non-human animals can be non-human mammals, such as monkeys, mice, rabbits 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 the Jerboidae or Murine superfamily. In one embodiment, the genetically modified animal is a rodent. In one embodiment, the rodent is selected from the group consisting of mice, rats and hamsters. In one embodiment, the rodent is selected from the murine family. In one embodiment, the genetically modified animal is from a group selected from the group consisting of hamsteridae (eg, mouse-like hamsters), hamsteridae (eg, hamsters, New World rats and mice, voles), murine superfamily (true mice) and rats, gerbils, spiny rats, crested rats), falciparum (climbing mice, rock mice, tailed rats, Madagascar rats and mice), dormouse (e.g. dormouse) and mole rats (such as the mole rat, bamboo rat and zokor) family. In a specific embodiment, the genetically modified rodent is selected from the group consisting of true mice or rats (Muridae), gerbils, spiny rats and crested rats. 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 specific embodiment, the rodent is selected from mice and rats. In one embodiment, the non-human animal is a mouse.

In a specific embodiment, the non-human animal is a rodent selected from the group consisting of BALB/c, A, A/He, A/J, A/WySN, AKR, 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/10ScSn, C57BL/10Cr and C57BL/Ola C57BL, C58, CBA/Br, CBA/Ca, CBA/J, CBA/st, CBA/H strain mice.

The "inflammatory disease" of the present invention is selected from acute inflammation, and also includes chronic inflammation. Specifically, including but not limited to degenerative inflammation, exudative inflammation (serous inflammation, fibrinoid inflammation, suppurative inflammation, hemorrhagic inflammation, necrotizing inflammation, catarrhal inflammation), proliferative inflammation, specific inflammation ( tuberculosis, syphilis, leprosy, lymphogranuloma, etc.).

The "infection" in the present invention includes but is not limited to plague, cholera, AIDS, atypical pneumonia, influenza A, tuberculosis, avian influenza, viral hepatitis, rabies, polio, measles, meningococcal meningitis, epidemic Japanese encephalitis, epidemic hemorrhagic fever, leptospirosis, typhoid and schistosomiasis.

The "autoimmune disease" 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 disorders, etc.

The "tumor" of the present invention is selected from leukemia, lymphoma, ovarian cancer, breast cancer, endometrial cancer, colon cancer, rectal cancer, gastric cancer, bladder cancer, lung cancer (such as non-small cell lung cancer, etc.), bronchial cancer, Bone, prostate, pancreatic, liver and bile duct, esophagus, kidney, thyroid, head and neck, testicular, glioblastoma, astrocytoma, melanoma, myelodysplastic syndrome, and sarcoma. Wherein, the leukemia is selected from the group consisting of acute lymphocytic (lymphoblastic) leukemia, acute myeloid leukemia, myeloid 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 The group consisted of the following: osteosarcoma, Ewing sarcoma, leiomyosarcoma, synovial sarcoma, acinar soft tissue sarcoma, angiosarcoma, liposarcoma, fibrosarcoma, rhabdomyosarcoma, and chondrosarcoma.

Unless specifically stated otherwise in this disclosure, Kabat numbering is used by default in this disclosure.

Description of drawings

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings, wherein:

Figure 1 is a flow chart showing the preparation of anti-hGITR antibodies, specifically the steps from immunization to serum collection, and the detection of antibody titers on the collected serum.

Figure 2 is a flow chart showing the preparation of anti-hGITR antibodies, specifically the steps of boosting immunization, collecting spleen cells, preparing hybridoma cells, and screening hybridoma cells, and using hybridoma cells to produce and purify antibodies.

Figure 3 is a set of flow cytometry graphs showing the binding between the anti-hGITR antibody (2D3-mHvKv-IgG1) and hGITR, where INCAGN01876 is a positive control and NC is a negative control.

Figure 4 is a panel of flow cytometry showing the analysis of cross-reactivity of anti-hGITR antibody (2D3-mHvKv-IgG1) with monkey GITR (rmGITR), mouse GITR (mGITR) and human-mouse chimeric GITR (chiGITR). Graph of the results, where NC represents the negative control and INCAGN01876 is the positive control.

Figure 5 is a graph showing body weight over time of humanized GITR mice (B-hGITR) with MC-38 tumor cells treated with anti-hGITR antibody (2D3-mHvKv-IgG1) and humanized anti-hGITR antibody INCAGN01876.

Figure 6 is a graph showing percent body weight over time of humanized GITR mice (B-hGITR) with MC-38 tumor cells treated with anti-hGITR antibody (2D3-mHvKv-IgG1) and humanized anti-hGITR antibody INCAGN01876 picture.

Figure 7 is a graph showing tumor volume over time in humanized GITR mice (B-hGITR) with MC-38 tumor cells treated with anti-hGITR antibody (2D3-mHvKv-IgG1) and humanized anti-hGITR antibody INCAGN01876 .

Detailed ways

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. Anti-hGITR antibodies were collected as described below and as shown in Figures 1 and 2.

Immunization of mice

6-8 week old female BALB/c mice were immunized with His-tagged human GITR protein at a concentration of 100 μg/mL at 20 μg/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 subsequent subcutaneous injections, 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 titers using ELISA.

In another experiment, 6-8 week old female BALB/c mice were immunized by injecting an expression plasmid encoding human GITR into mice. The antigen-encoding plasmid was injected into the tibialis anterior muscle of mice at a concentration of 1000 μg/μL at 60 μg per mouse by using a gene gun (intramuscular injection; i.m. injection). At least four injections were given with at least 14 days between each injection. Blood (serum) was collected 7 days after the last immunization and serum was tested for antibody titers by ELISA.

A booster immunization program (either by injection of plasmid or by injection of protein) was also performed at least fourteen days after the previous immunization. CHO cells expressing GITR antigen on their surface were injected into mice via tail vein. Spleens were then collected four days after injection.

Fusion of SP2/0 cells and splenocytes

Grind the spleen tissue. Splenocytes were first selected by CD3ε microbeads and anti-mouse IgM microbeads and then fused with SP2/0 cells. Cells were then plated in 96-well plates with hypoxanthine-aminopterin-thymidine (HAT) medium.

Preliminary screening of hybridomas

Preliminary screening of hybridoma supernatants in 96-well plates was performed using Fluorescence-Activated CellSorting (FACS) according to standard procedures. Before screening, Chinese hamster ovary (CHO) cells were added to 96-well plates ( ² × 10 cells per well). Use 50 μL of supernatant. The antibodies used in the experiments were:

(1) Fluorescein (FITC)-conjugated AffiniPure F(ab) ₂ fragment goat anti-mouse IgG, Fcγ fragment specific; and

(2) Alexa Fluor® 647-conjugated AffiniPure F(ab) ₂ fragment goat anti-human IgG, Fcγ fragment specific.

subclone

Subcloning was performed using ClonePix2. Briefly, positive wells identified in the preliminary screening were transferred to semi-solid medium and IgG positive clones were identified and tested. FITC anti-mouse IgG Fc antibody was used.

Ascites antibodies

1×10 ⁶ positive hybridoma cells were injected intraperitoneally into B-NDG® mice (Beijing Biositu, Beijing, China). Monoclonal antibodies are produced by growing hybridoma cells in the peritoneal cavity of mice. Hybridoma cells expand in the mouse abdomen and produce ascites. Ascites fluid contains high concentrations of antibodies, which can be harvested for later use.

Antibody purification

Antibody in ascites was purified using GE AKTA protein chromatography (GE Healthcare, Chicago, Illinois, United States) to yield 61-2D3 ("2D3") mouse antibody.

The VH, VL and CDR regions of the antibodies were determined. The heavy chain CDR1, CDR2 and CDR3 and light chain CDR1, CDR2 and CDR3 amino acid sequences of 2D3 are shown in SEQ ID NOs: 1-6 (Kabat numbering) or SEQ ID NOs: 7, 8, 3, 4, 5, 6 ( Chothia number). 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 mouse ascites and purified by chromatography. 25 μL of CHO cells transiently transfected with human GITR were added to each well in the plate. The purified antibodies were titrated to final concentrations of 10, 1, 0.1, 0.01, 0.001 μg/mL. The titrated antibody was added to each well at 25 μL per well and incubated for 30 minutes at 4°C.

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 heavy and light chain variable domains from the corresponding mouse anti-hGITR antibody, and constant domains (including, for example, CL, CH1, CH2 and CH3 domains) from a human IgG1 antibody ).

After washing twice with phosphate buffered saline (PBS), 50 μL of FITC-labeled anti-human IgG Fc antibody (Fluorescein (FITC)-AffiniPure F(ab')2 Fragment Goat Anti-Human IgG, FcγFragment Specific, source Jackson, Cat. No. 109-096-170)) was added to each well at a 1:500 dilution, incubated at 4°C for 30 minutes, and washed with PBS. The signal of FITC was determined by flow cytometry.

As shown in Figure 3, when the concentration of chimeric anti-hGITR antibody 2D3-mHvKv-IgG1 and humanized anti-hGITR antibody INCAGN01876 antibody (as a positive control drug) increased, the signal of FITC increased, indicating that anti-hGITR antibody 2D3-mHvKv -IgG1 can bind to human GITR, and its binding ability to human GITR is 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, 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:10) were used ID NO: 11) coding sequence was transfected into CHO cells.

25 μL of CHO cells were added to each well. 25 μL of purified anti-hGITR antibody (10 μg/mL) was added to each well and incubated at 4°C for 30 min.

After washing twice with PBS (1200 rmp, 5 min), 50 μL of FITC-labeled anti-human IgG Fc antibody was added to each well at a 1:500 dilution and incubated at 4°C for 30 min, followed by washing with PBS (1200rmp, 5 minutes). The signal of FITC was determined by flow cytometry.

As shown in Figure 4, neither the chimeric anti-hGITR antibody 2D3-mHvKv-IgG1 nor the humanized anti-hGITR antibody INCAGN01876 cross-reacted with mouse GITR, but had strong cross-reactivity 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 INCAGN01876 (1 μg/mL) was injected into the Biacore T200 biosensor at 10 μL/min for 30 s to achieve the desired protein density (approximately 67 response units (RU)). Histidine-tagged human GITR protein (source ACRO, Cat. No. GIR-H5228) at concentrations of 200, 100, 50, 25, 12.5, 6.25, 3.125, 1.5625 nM was then injected at 30 μL/min for 100 seconds. Dissociation was monitored for 400 seconds. The chip was regenerated with glycine (pH 2.0, 30 μL/min for 12 sec) after the last injection of each titer. Kinetic association rates (kon) and dissociation rates (koff) were obtained simultaneously by globally fitting the data to a 1:1 Langmuir binding model using Biacore T200 evaluation software 3.0 (Karlsson, R. Roos, H. Fagerstam, L. Petersson , B., 1994. Methods Enzymology 6. 99-110). Affinities were derived 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 to predict the effects of these antibodies in humans, a humanized GITR mouse model was generated. A humanized GITR mouse model was engineered to express a chimeric GITR protein (SEQ ID NO: 11) in which a portion of the extracellular domain of the mouse GITR protein was replaced by the corresponding human GITR extracellular domain. Amino acid residues 1-130 of mouse GITR (SEQ ID NO:9) were replaced by amino acid residues 1-142 of human GITR (SEQ ID NO:12). A 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 humans and normal mice expressing mouse GITR. A detailed description of the 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 the tumors in the mice reached a volume of 100 ± 50 mm, the mice were randomly assigned to different groups ( ⁵ mice per group) according to the volume of the tumors. 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 injections in total).

The injection volume was calculated at 3 mg/kg based on the body weight of the mice. The lengths of the long and short axes of the tumor were measured, and the tumor volume was calculated as 0.5×(long axis)×(short axis) ² . The body weight of the mice was also measured prior to injection when the mice were divided into different groups (before the first antibody injection), twice weekly during the antibody injection period, and before euthanasia.

。Ti是处 理组在第i天的平均肿瘤体积。T0是处理组在第0天的平均肿瘤体积。Vi是对照组在第i天的 平均肿瘤体积。V0是对照组在第0天的平均肿瘤体积。 The percent tumor growth inhibition (TGI%) was calculated using the following formula:

. Ti is the mean tumor volume of the treatment group on day i. T0 is the mean tumor volume on day 0 of the treatment group. Vi is the mean tumor volume of the control group on day i. V0 is the mean tumor volume of the control group on day 0.

A t-test was performed for statistical analysis. TGI% higher than 60% indicates significant inhibition of tumor growth. p<0.05 was the threshold to indicate a significant difference.

In each of the three groups (G1, G2, G3), B-hGITR mice were injected with saline (PS) as a control (G1), chimeric anti-hGITR antibody 2D3-mHvKv-IgG1 (G2) or human-derived, respectively Anti-hGITR antibody INCAGN01876 (G3). The body weight of the mice was monitored throughout the treatment period. The weight of the mice in the different groups all increased (Figures 5 and 6). No significant differences in body weight were observed between the three groups. The results showed that 2D3-mHvKv-IgG1 and INCAGN01876 were well tolerated and nontoxic to mice.

Tumor volume increased to a lesser extent in the groups treated with INCAGN01876 and 2D3-mHvKv-IgG1 compared to the control group (Figure 7). In particular, the tumor volume in G2 was smaller than in G3.

As shown in the table below, the TGI% at day 24 (24 days after grouping) was also calculated.

Table 2

The results showed that both the chimeric antibody 2D3-mHvKv-IgG1 and the humanized antibody INCAGN01876 could inhibit tumor growth. Among them, the TGI% of 2D3-mHvKv-IgG1 was higher than that of the humanized antibody INCAGN01876.

It will be understood by those skilled in the art that while the invention has been described in conjunction with specific embodiments thereof, the foregoing description is intended to illustrate rather than 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 appended claims.

sequence listing

<110> Beijing Biositu Gene Biotechnology Co., Ltd.

<120> Anti-GITR antibody and its use

<130> 1

<160> 14

<170> SIPOSequenceListing 1.0

<210> 1

<211> 5

<212> PRT

<213> Artificial Sequence

<400> 1

Asn Tyr Gly Ile Asp

1 5

<210> 2

<211> 17

<212> PRT

<213> Artificial Sequence

<400> 2

Tyr Ile Asn Ile Gly Asn Asn Tyr Thr Tyr Tyr Asn Glu Lys Phe Arg

1 5 10 15

Gly

<210> 3

<211> 10

<212> PRT

<213> Artificial Sequence

<400> 3

Glu Ala Thr Gly Pro Ser Tyr Phe Asp Val

1 5 10

<210> 4

<211> 17

<212> PRT

<213> Artificial Sequence

<400> 4

Lys Ser Ser Gln Ser Leu Leu Tyr Arg Ser Asn Gln Lys Asn Tyr Leu

1 5 10 15

Ala

<210> 5

<211> 7

<212> PRT

<213> Artificial Sequence

<400> 5

Trp Ala Ser Thr Arg Lys Ser

1 5

<210> 6

<211> 9

<212> PRT

<213> Artificial Sequence

<400> 6

Gln Gln Tyr Tyr Ile Tyr Pro Leu Thr

1 5

<210> 7

<211> 10

<212> PRT

<213> Artificial Sequence

<400> 7

Gly Tyr Thr Phe Thr Asn Tyr Gly Ile Asp

1 5 10

<210> 8

<211> 6

<212> PRT

<213> Artificial Sequence

<400> 8

Asn Ile Gly Asn Asn Tyr

1 5

<210> 9

<211> 228

<212> PRT

<213> Mouse (Mus. musculus)

<400> 9

Met Gly Ala Trp Ala Met Leu Tyr Gly Val Ser Met Leu Cys Val Leu

1 5 10 15

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

20 25 30

Lys Val Gln Asn Gly Ser Gly Asn Asn Thr Arg Cys Cys Ser Leu Tyr

35 40 45

Ala Pro Gly Lys Glu Asp Cys Pro Lys Glu Arg Cys Ile Cys Val Thr

50 55 60

Pro Glu Tyr His Cys Gly Asp Pro Gln Cys Lys Ile Cys Lys His Tyr

65 70 75 80

Pro Cys Gln Pro Gly Gln Arg Val Glu Ser Gln Gly Asp Ile Val Phe

85 90 95

Gly Phe Arg Cys Val Ala Cys Ala Met Gly Thr Phe Ser Ala Gly Arg

100 105 110

Asp Gly His Cys Arg Leu Trp Thr Asn Cys Ser Gln Phe Gly Phe Leu

115 120 125

Thr Met Phe Pro Gly Asn Lys Thr His Asn Ala Val Cys Ile Pro Glu

130 135 140

Pro Leu Pro Thr Glu Gln Tyr Gly His Leu Thr Val Ile Phe Leu Val

145 150 155 160

Met Ala Ala Cys Ile Phe Phe Leu Thr Thr Val Gln Leu Gly Leu His

165 170 175

Ile Trp Gln Leu Arg Arg Gln His Met Cys Pro Arg Glu Thr Gln Pro

180 185 190

Phe Ala Glu Val Gln Leu Ser Ala Glu Asp Ala Cys Ser Phe Gln Phe

195 200 205

Pro Glu Glu Glu Arg Gly Glu Gln Thr Glu Glu Lys Cys His Leu Gly

210 215 220

Gly Arg Trp Pro

225

<210> 10

<211> 235

<212> PRT

<213> Rhesus monkey (Macaca mulatta)

<400> 10

Met Cys Ala Ser Gly Thr Leu Cys Cys Leu Ala Leu Leu Cys Ala Ala

1 5 10 15

Ser Leu Gly Gln Arg Pro Thr Gly Gly Pro Gly Cys Gly Pro Gly Arg

20 25 30

Leu Leu Leu Gly Thr Gly Lys Asp Ala Arg Cys Cys Arg Val His Pro

35 40 45

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

50 55 60

Asp Cys Val Cys Val Gln Pro Glu Phe His Cys Gly Asn Pro Cys Cys

65 70 75 80

Thr Thr Cys Gln His His Pro Cys Pro Ser Gly Gln Gly Val Gln Pro

85 90 95

Gln Gly Lys Phe Ser Phe Gly Phe Arg Cys Val Asp Cys Ala Leu Gly

100 105 110

Thr Phe Ser Arg Gly His Asp Gly His Cys Lys Pro Trp Thr Asp Cys

115 120 125

Thr Gln Phe Gly Phe Leu Thr Val Phe Pro Gly Asn Lys Thr His Asn

130 135 140

Ala Val Cys Val Pro Gly Ser Pro Pro Ala Glu Pro Pro Gly Trp Leu

145 150 155 160

Thr Ile Val Leu Leu Ala Val Ala Ala Cys Val Leu Leu Leu Thr Ser

165 170 175

Ala Gln Leu Gly Leu His Ile Trp Gln Leu Arg Ser Gln Pro Thr Gly

180 185 190

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

195 200 205

Ala Ser Ser Cys Gln Phe Pro Glu Glu Glu Arg Gly Glu Arg Leu Ala

210 215 220

Glu Glu Lys Gly Arg Leu Gly Asp Leu Trp Val

225 230 235

<210> 11

<211> 240

<212> PRT

<213> Artificial Sequence

<400> 11

Met Ala Gln His Gly Ala Met Gly Ala Phe Arg Ala Leu Cys Gly Leu

1 5 10 15

Ala Leu Leu Cys Ala Leu Ser Leu Gly Gln Arg Pro Thr Gly Gly Pro

20 25 30

Gly Cys Gly Pro Gly Arg Leu Leu Leu Gly Thr Gly Thr Asp Ala Arg

35 40 45

Cys Cys Arg Val His Thr Thr Arg Cys Cys Arg Asp Tyr Pro Gly Glu

50 55 60

Glu Cys Cys Ser Glu Trp Asp Cys Met Cys Val Gln Pro Glu Phe His

65 70 75 80

Cys Gly Asp Pro Cys Cys Thr Thr Cys Arg His His Pro Cys Pro Pro

85 90 95

Gly Gln Gly Val Gln Ser Gln Gly Lys Phe Ser Phe Gly Phe Gln Cys

100 105 110

Ile Asp Cys Ala Ser Gly Thr Phe Ser Gly Gly His Glu Gly His Cys

115 120 125

Lys Pro Trp Thr Asp Cys Thr Gln Phe Gly Phe Leu Thr Val Phe Pro

130 135 140

Gly Asn Lys Thr His Asn Ala Val Cys Ile Pro Glu Pro Leu Pro Thr

145 150 155 160

Glu Gln Tyr Gly His Leu Thr Val Ile Phe Leu Val Met Ala Ala Cys

165 170 175

Ile Phe Phe Leu Thr Thr Val Gln Leu Gly Leu His Ile Trp Gln Leu

180 185 190

Arg Arg Gln His Met Cys Pro Arg Glu Thr Gln Pro Phe Ala Glu Val

195 200 205

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

210 215 220

Arg Gly Glu Gln Thr Glu Glu Lys Cys His Leu Gly Gly Arg Trp Pro

225 230 235 240

<210> 12

<211> 241

<212> PRT

<213> Homo sapiens

<400> 12

Met Ala Gln His Gly Ala Met Gly Ala Phe Arg Ala Leu Cys Gly Leu

1 5 10 15

Ala Leu Leu Cys Ala Leu Ser Leu Gly Gln Arg Pro Thr Gly Gly Pro

20 25 30

Gly Cys Gly Pro Gly Arg Leu Leu Leu Gly Thr Gly Thr Asp Ala Arg

35 40 45

Cys Cys Arg Val His Thr Thr Arg Cys Cys Arg Asp Tyr Pro Gly Glu

50 55 60

Glu Cys Cys Ser Glu Trp Asp Cys Met Cys Val Gln Pro Glu Phe His

65 70 75 80

Cys Gly Asp Pro Cys Cys Thr Thr Cys Arg His His Pro Cys Pro Pro

85 90 95

Gly Gln Gly Val Gln Ser Gln Gly Lys Phe Ser Phe Gly Phe Gln Cys

100 105 110

Ile Asp Cys Ala Ser Gly Thr Phe Ser Gly Gly His Glu Gly His Cys

115 120 125

Lys Pro Trp Thr Asp Cys Thr Gln Phe Gly Phe Leu Thr Val Phe Pro

130 135 140

Gly Asn Lys Thr His Asn Ala Val Cys Val Pro Gly Ser Pro Pro Ala

145 150 155 160

Glu Pro Leu Gly Trp Leu Thr Val Val Leu Leu Ala Val Ala Ala Cys

165 170 175

Val Leu Leu Leu Thr Ser Ala Gln Leu Gly Leu His Ile Trp Gln Leu

180 185 190

Arg Ser Gln Cys Met Trp Pro Arg Glu Thr Gln Leu Leu Leu Glu Val

195 200 205

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

210 215 220

Arg Gly Glu Arg Ser Ala Glu Glu Lys Gly Arg Leu Gly Asp Leu Trp

225 230 235 240

Val

<210> 13

<211> 119

<212> PRT

<213> Artificial Sequence

<400> 13

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

1 5 10 15

Ser Val Lys Met Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Asn Tyr

20 25 30

Gly Ile Asp Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Ile Gly Asn Asn Tyr Thr Tyr Tyr Asn Glu Lys Phe

50 55 60

Arg Gly Lys Ala Thr Leu Thr Ser Asp Thr Ser Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Gly Asp Ser Ala Ile Tyr Phe Cys

85 90 95

Ala Arg Glu Ala Thr Gly Pro Ser Tyr Phe Asp Val Trp Gly Thr Gly

100 105 110

Thr Thr Val Thr Val Ser Ser

115

<210> 14

<211> 113

<212> PRT

<213> Artificial Sequence

<400> 14

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

1 5 10 15

Gln Lys Val Ile Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Tyr Arg

20 25 30

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

35 40 45

Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Lys Ser Gly Val

50 55 60

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

65 70 75 80

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

85 90 95

Tyr Tyr Ile Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu

100 105 110

Lys

Claims (10)

1. an anti-GITR antibody or its antigen-binding fragment, is characterized in that, described anti-GITR antibody or its antigen-binding fragment comprise VHCDR1, VHCDR2 and VHCDR3 of heavy chain variable region, and the VLCDR1 of light chain variable region, VLCDR2 and VLCDR3, wherein, the amino acid sequence of VHCDR1 is shown 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 shown in SEQ ID NO: 3, and the amino acid sequence of VLCDR1 is shown in SEQ ID NO: 1 ID NO: 4, the amino acid sequence of VLCDR2 is shown in SEQ ID NO: 5, the amino acid sequence of VLCDR3 is shown in SEQ ID NO: 6, and the anti-GITR antibody or its antigen-binding fragment is a monoclonal antibody or a polyclonal antibody. cloned antibodies. 2. The anti-GITR antibody or its antigen-binding fragment according to claim 1, wherein the amino acid sequence of the variable region of the heavy chain is as shown in SEQ ID NO: 13, and the variable region of the light chain is as shown in SEQ ID NO: 13. The amino acid sequence of is shown in SEQ ID NO:14. 3. The anti-GITR antibody or its antigen-binding fragment according to claim 1, wherein said anti-GITR antibody or its antigen-binding fragment 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 according to any one of claims 1-3, wherein the anti-GITR antibody or antigen-binding fragment thereof specifically binds to 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 VHCDR1, 2 and 3 of SEQ ID NOs: 1, 2 and 3, and wherein the heavy chain variable region is in Binds GITR when paired with the light chain variable region of the amino acid sequence set forth in SEQ ID NO: 14; and, (2) an immunoglobulin light chain comprising a light chain variable region comprising VLCDR1, 2 and 3 of SEQ ID NOs: 4, 5 and 6, and wherein the light chain variable region is in Binds GITR when paired with the heavy chain variable region of the amino acid sequence shown in SEQ ID NO:13. 6. A cell comprising the nucleic acid of claim 5. 7 . A method for preparing an anti-GITR antibody or an antigen-binding fragment thereof, wherein the cell according to claim 6 is cultured to obtain an anti-GITR antibody or an antigen-binding fragment thereof. 8 . 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 preparation of a medicament for treating GITR/GITRL-related diseases.

Images