CN110563848A

CN110563848A - bispecific antibody and application thereof

Info

Publication number: CN110563848A
Application number: CN201910678208.1A
Authority: CN
Inventors: 杨彪
Original assignee: Individual
Current assignee: Individual
Priority date: 2019-07-25
Filing date: 2019-07-25
Publication date: 2019-12-13

Abstract

The invention provides a bispecific antibody, which comprises a first antigen-binding functional region capable of specifically binding to PD-1 and a second antigen-binding functional region capable of specifically binding to TIM-3, wherein the antibody is in an IgG form, can simultaneously bind to two target molecules, has better effect on treating complex diseases, and is not easy to generate off-target effect.

Description

Bispecific antibody and application thereof

Technical Field

The invention relates to the field of antibody medicaments, in particular to a bispecific antibody and application thereof.

Background

At present, dozens of monoclonal antibody medicines are certified by related organizations and applied to clinical antitumor therapy, and although the monoclonal antibody medicines bring breakthrough effect to tumor immunotherapy, the curative effect is limited, for example, some monoclonal antibody medicines can generate drug resistance, so that partial patients cannot respond after being used, or relapse and progress after initial response. The common tumor therapeutic antibody can only combine with a single antigen, has relatively low combination specificity and is easy to generate off-target effect.

The Bispecific antibody (BsAb) can respectively recognize and combine two different antigens, so that it can connect immune cells, virus molecules and the like to tumor cells to further enhance the killing effect on target cells, and can also combine different antigens on the same tumor cells to enhance the combination specificity, thereby reducing the side effects such as off-target toxicity and the like. The recombinant antibody with double functions has higher curative effect than that of a monoclonal antibody drug when being used as a drug for treating tumors.

In 1986, Staerz and Bevan used bispecific antibodies for the first time to apply cytotoxic T lymphocytes to cytolytic experiments. In 12 months 2014, a bispecific antibody immunotherapy drug Blinatumomab developed by ann incorporated was used for the treatment of relapsed or refractory philadelphia chromosome-negative pre-B cell Acute Lymphoblastic Leukemia (ALL).

with the deterioration of global environment and the change of living habits of people, the incidence rate of tumors is higher and higher, the mortality rate of tumor patients is increased gradually, and the market of anti-tumor drugs is expanded continuously. With the development of scientific research technology, the plasma clearance rate, tissue permeability, pharmacokinetics and the like of the bispecific antibody can be improved by changing the structure, relative molecular mass and valence of the bispecific antibody, so that the stability and clinical curative effect of the bispecific antibody are improved.

Disclosure of Invention

In order to solve the above technical problems, the present invention provides a bispecific antibody and use thereof.

The invention is realized by the following technical scheme:

A bispecific antibody comprising a first antigen-binding domain capable of specifically binding to PD-1 and a second antigen-binding domain capable of specifically binding to TIM-3.

Further, the amino acid sequence of the heavy chain variable region of the second antigen-binding functional region is a variant sequence having at least 95% identity to any one of the amino acid sequences shown in SEQ ID nos. 7, 9 and 10 and retaining the corresponding biological activity or a variant sequence obtained by deletion, substitution and/or addition of one or more amino acid residues and retaining the corresponding biological activity.

Further, the amino acid sequence of the light chain variable region of the second antigen-binding functional region is a variant sequence having at least 95% identity to any one of the amino acid sequences shown in SEQ ID nos. 8, 11 and 12 and retaining the corresponding biological activity or a variant sequence obtained by deletion, substitution and/or addition of one or more amino acid residues and retaining the corresponding biological activity.

Further, the amino acid sequence of the heavy chain variable region of the second antigen binding functional region is shown in any one of SEQ ID No.7, 9 and 10 and/or the amino acid sequence of the light chain variable region of the second antigen binding functional region is shown in any one of SEQ ID No.8, 11 and 12.

Preferably, the amino acid sequence of the heavy chain variable region of the second antigen binding functional region is shown as SEQ ID No.9, and the amino acid sequence of the light chain variable region of the second antigen binding functional region is shown as SEQ ID No. 11.

Further, the Fc chain of the bispecific antibody is in the IgG format.

Further, the antigen binding fragment is selected from the group consisting of Fab, Fab ', F (ab)'2, single chain Fv (scFv), and bispecific antibodies.

Further, the antibody production step comprises: culturing the host cell containing the corresponding nucleotide coding sequence for the antibody in a culture medium under suitable culture conditions, collecting the cells, and purifying the bispecific antibody or other equivalent production protocol.

Further, the bispecific antibody can be conjugated to at least one therapeutic agent to form an immunoconjugate.

Preferably, the therapeutic agent is selected from one or more of a cytotoxic agent, a radionuclide, a boron atom, an immunomodulator, an immunoconjugate, an oligonucleotide.

The invention also provides the use of the bispecific antibody, which comprises the use of the antibody and the antigen-binding fragment thereof recovered and produced from a culture medium or cultured host cells and the use of the antibody and the antigen-binding fragment thereof and a pharmaceutically acceptable carrier for preparing a medicament for treating tumors, wherein the tumors comprise but are not limited to gastric cancer, pancreatic cancer, gallbladder cancer, liver cancer, colorectal cancer, leukemia, breast cancer, ovarian cancer, cervical cancer, endometrial cancer, uterine sarcoma, prostate cancer, bladder cancer and renal cell carcinoma. It is well known in the art that an antigen binding domain refers to a region that can specifically interact with a target molecule, such as an antigen, with a high degree of selectivity of action, and that sequences recognizing one target molecule are generally unable to recognize other molecular sequences.

Representative antigen binding domains include: a variable region of an antibody, a structural variant of a variable region of an antibody, a binding domain of a receptor, a ligand binding domain, or an enzyme binding domain.

the binding specificity and avidity of an antibody are determined primarily by the CDR sequences, and variants with similar biological activity can be obtained by readily altering the amino acid sequence of the non-CDR regions according to well-established and well-known techniques of the art.

By "variable region" is meant that certain segments of the variable region differ significantly in sequence between antibodies, the hypervariable regions being 3-12 amino acids each, which predominantly adopt a beta-sheet configuration, joined by three hypervariable regions, which form loops connecting and in some cases forming part of the beta-sheet structure. The hypervariable regions in each chain are bound together by the FR immediately, while the hypervariable regions with the other chains contribute to the formation of the antigen binding site of the antibody (see Kabat et al, Sequences of Proteins of Immunological Interest, 5 th edition, Public Health service, National Institutes of Health, Bethesda, Md. (1991)), the constant regions are not directly involved in the binding of the antibody to the antigen.

"antigen-binding fragment of an antibody" refers to a fragment, portion, region or domain of an antibody that is capable of binding to an epitope, and thus the terms "antigen-binding" and "epitope-binding" and "antigen-binding fragment of an antibody" are the same as "epitope-binding fragment of an antibody". Antigen-binding fragments may contain 1, 2, 3, 4, 5 or all 6 CDR domains of such antibodies and, although capable of binding to the epitope, may exhibit different specificities, affinities or selectivities. Preferably, the antigen binding fragment contains all 6 CDR domains of the antibody.

An "antigen-binding fragment of an antibody" can be part of or comprise a single polypeptide chain (e.g., an scFv), or can be part of or comprise two or more polypeptide chains (each having an amino-terminus and a carboxy-terminus, e.g., a diabody, a Fab fragment, a Fab2 fragment, etc.).

"IgG" is an abbreviation for Immunoglobulin G (IgG), which is the major antibody component of serum, and human IgG has four subtypes based on the antigenic difference of the r chain in IgG molecules: IgG1, IgG2, IgG3, IgG 4.

"monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts in individual antibodies. Monoclonal antibodies are highly specific, meaning directed against a single antigenic site. Furthermore, each monoclonal antibody is directed against a single determinant on the antigen, as opposed to a polyclonal antibody preparation comprising different antibodies directed against different determinants (epitopes). In addition to the specificity of a monoclonal antibody, a monoclonal antibody is advantageous in that it can be synthesized without contamination by other antibodies.

By one or more disulfide interchain linkages is meant that the first Fc chain and the second Fc chain are linked by one or more disulfide interchain linkages, forming a heterodimeric fragment. In the present invention, the formation of the one or more disulfide bonds may be performed when the first Fc chain and the second Fc chain or the first Fc chain and the second Fc chain and the antigen-binding domain linked thereto are synthesized in the same cell, or may be performed by synthesizing the first Fc chain and the second Fc chain or the first Fc chain and the second Fc chain and the antigen-binding domain linked thereto in different cells, respectively, and then performing in vitro reduction and oxidation.

Host cells of the invention include, but are not limited to, E.coli, phage display systems, yeast, plant cells, animal cells.

"treatment" includes, but is not limited to, one or more of the following assay characterizations: alleviating one or more symptoms caused by the disease; attenuation of the extent of disease; preventing or delaying the progression of the disease; preventing or delaying the spread of the disease; preventing or delaying the recurrence of the disease; delay or slow the progression of the disease; improving the disease condition; providing remission of the disease; reducing the dose of one or more other drugs required to treat the disease; delay of progression of the disease; increase or improve quality of life; increase body weight gain and/or prolong survival. In the present invention "treatment" may be interpreted as a pathological consequence of cancer (e.g. reduction of tumor volume).

Pharmaceutically acceptable carrier means a pharmaceutical carrier that is conventional in the pharmaceutical art, including but not limited to diluents, excipients, water, and the like; including but not limited to adhesives such as gelatin and polyvinylpyrrolidone; humectants such as glycerol; including but not limited to absorption enhancers such as quaternary ammonium compounds; including but not limited to surfactants such as cetyl alcohol, sodium lauryl sulfate, and the like.

Drawings

FIG. 1 shows the structure of an anti-PD-1/TIM-3 bispecific antibody;

FIG. 2 shows the simultaneous binding activity of PD-1, TIM-3 of an anti-PD-1/TIM-3 bispecific antibody.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in further detail below.

example 1:

Female BALB/c mice (purchased from Jersey laboratory animals Co., Ltd., Shanghai) of 6-8 weeks old were used as experimental animals for the mouse immunization test. For the primary immunization, 50 μ g of human TIM-3 protein (purchased from Beijing Hokkaiyuan Biotechnology Co., Ltd.) was mixed with complete Freund's adjuvant to form an emulsion, the emulsion was injected into the abdominal cavity of a mouse at an injection rate of 0.5 ml/injection amount, 25 μ g of human TIM-3 protein was sufficiently mixed with incomplete Freund's adjuvant every 2 weeks to form an emulsion for boosting, the boosting was performed three times, after 1 week of the final immunization, venous blood and serum of the mouse were collected and separated, and the titer of the antibody was measured by ELISA method and mouse cells having high titer were selected to prepare a hybridoma to prepare a monoplasmatic cell suspension.

Collecting logarithmic growth myeloma cells (SP2/0) to prepare immune spleen cell suspension, mixing myeloma cells and spleen cells at a ratio of 1:5, washing with incomplete culture solution, centrifuging for 8 minutes, discarding supernatant, preheating cell precipitate in 40 deg.C water bath, adding 1ml of PEG-4000 solution preheated to 40 deg.C to the cell precipitate, and adding 25ml of incomplete culture medium preheated to 40 deg.C to reaction solution within 1min after the occurrence of particles to terminate the reaction. After standing, addadding 2ml HAT culture medium, slightly blowing the precipitated cells to make them suspended and uniformly mixed, then supplementing HAT culture medium until the spleen cell concentration in the centrifugal tube is reached to 1.5X10⁷And/ml, subpackaging the cell suspension into a 96-well plate for culture and observation, and sucking out a supernatant sample for antibody detection when the cell surface area reaches more than 1/2 of the area of the well plate.

Example 2:

Hybridoma culture supernatants were screened for anti-human TIM-3 antibodies. Specifically, human TIM-3 (purchased from Hitachi bioscience, Inc., Beijing) was coated with a buffer solution on a 96-well high-adsorption enzyme-labeled plate in an amount of 100. mu.L per well, and then washed with the buffer solution 3 times; blocking with a buffer solution containing 1% BSA and incubating at 25 ℃ for 1h, wherein the blocking amount is 280. mu.L/well, after the incubation is completed, washing with the buffer solution for 3 times, respectively adding 75. mu.L of a supernatant sample (S1-S85) and positive serum (control, CK1-5) into wells 1-90, incubating at 25 ℃ for 1 hour, and washing with the buffer solution for 5 times; adding 100 mu L of anti-mouse IgG antibody diluted by 1/10000 in a buffer solution containing 1% BSA (bovine serum albumin) into each hole, labeling the anti-mouse IgG antibody by horseradish peroxidase, incubating for 1 hour at 25 ℃, and washing for 5 times by the buffer solution; adding 100 mu L of colorimetric substrate 3,3',5,5' -Tetramethylbenzidine (TMB) into each well, developing for 10min at 30 ℃, stopping the color development reaction, reading the absorbance at 450nm on an enzyme-linked immunosorbent assay, and selecting a positive clone capable of secreting human TIM-3 binding antibody according to the strength of OD450 nm.

Example 3:

The clones obtained by screening and having both the antigen-binding activity and the antigen-neutralizing activity were subjected to the measurement of the antibody DNA sequence. Cellular mRNA was first extracted using the RNA prep Pure kit (Tiangen) according to the instructions. First strand cDNA was then synthesized using the Quant Script RT kit (Tiangen). The first chain of cDNA generated by reverse transcription is used for subsequent PCR reaction, a target band obtained by PCR amplification is cloned into a pGEM-T vector, and single clone is selected to complete sequencing by Nanjing Jinsry Biotech Co.

Obtaining an antibody light chain variable region and an antibody heavy chain variable region through PCR amplification, and obtaining a complementary determining region sequence after eliminating a framework region sequence; wherein the three complementarity determining regions CDR-L1 amino acid sequences of the light chain are shown in SEQ ID is 1; the amino acid sequence of CDR-L2 is shown as SEQ ID NO. 2, and the amino acid sequence of CDR-L3 is shown as SEQ ID NO. 3; the amino acid sequences of three complementarity determining regions CDR-H1 of the heavy chain are shown as SEQ ID NO. 4, CDR-H2 is shown as SEQ ID NO. 5, and CDR-H3 is shown as SEQ ID NO. 6; the antibody light chain constant region amino acid sequence is derived from murine IgVH4-21 x 07, antibody heavy chain constant region sequence murine IgVH2-09 x 01, and the light chain full-length sequence is obtained by connecting the antibody light chain variable region with the light chain constant region; the heavy chain full-length sequence is obtained by connecting an antibody heavy chain variable region with a heavy chain constant region, and the variable region sequence and the constant region sequence are respectively cloned into a eukaryotic cell expression vector TL10-11 (a vector framework pEGFP-N1 purchased from Chimana Shanghai). Antibody light chain and antibody heavy chain expression vectors were transfected into 293F cell line (purchased from shanghai jingning biotechnology). Cells were seeded the day before transfection, harvested by centrifugation the day of transfection, resuspended in fresh expression medium at a cell density of 1.5X10⁷cells/mL. Plasmid was added to a final concentration of 39.1. mu.g/mL and linear polyethyleneimine was added to a final concentration of 45. mu.g/mL, according to the transfection volume. And (3) putting the mixture into a cell culture box for culturing at 37 ℃ for 1 hour, then adding a fresh culture medium into the culture solution until the final volume is 20 times of the transfection volume, continuing culturing for 5-6 days, and collecting the supernatant.

Example 4:

the kinetic constants of the binding of the anti-human TIM-3 murine monoclonal antibody (hereinafter abbreviated as OM-anti-TIM-3) obtained in example 1 to the antigen were determined. The instrument optical surface plasma resonance technology is used for detecting the combination and dissociation between the molecules coupled and coated on the biochip and the molecules to be detected. Briefly, OM-anti-TIM-3 was dissolved in sodium acetate buffer (pH 5.0) and coupled to CM chips, blocked with 1M ethanolamine. Different concentrations of OM-anti-TIM-3 were injected at a rate of 27. mu.L/min for 2.5min during the binding phase and 27. mu.L/min for 8min during the dissociation phase, and the binding kinetic constants and dissociation kinetic constants were calculated by analysis using Biacore 3000 software. OM-anti-TIM-3 has a binding kinetic constant of 4.77E +04(1/Ms), a dissociation kinetic constant of 1.64E-05(1/s), and a dissociation equilibrium constant of 0.01 (nM).

Example 5: detection of in vivo neutralization Activity of murine monoclonal antibodies

The in vivo neutralizing activity of OM-anti-TIM-3 was determined. Briefly, 6-8 week old female BALB/c mice (purchased from Shanghai Jitsie laboratory animals Co., Ltd.) were selected. The composition was randomly divided into five groups of 6 animals, administered intravenously and in a single dose, each group at a dose level of 0.5nmol/kg,5nmol/kg, 15nmol/kg, 25nmol/kg, 50nmol/kg OM-anti-TIM-3. One hour after administration, injecting 15 μ g human TIM-3 protein subcutaneously into each mouse, after 2 hours of injection, performing orbital blood collection on each mouse without anticoagulation, standing the blood sample at room temperature for about 40 minutes until the blood sample is coagulated, centrifuging to obtain a serum sample, and measuring the concentration of mouse CCK in the serum by using a CCK ELISA kit according to the instruction, wherein the result shows that 25nmol/kg OM-anti-TIM-3 can inhibit the level of CCK secreted by the mouse stimulated by the human TIM-3, and the level of the mouse CCK can be basically reduced to an unstimulated state.

Example 6:

The pharmacokinetics of OM-anti-TIM-3 in rats was determined. Briefly, 6-8 week old female SD rats (purchased from Shanghai Jiesi laboratory animals Co., Ltd.) were selected. 5 rats were given 25nmol/kg OM-anti-TIM-3. At 0 point, 5 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 6 hours, 9 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 120 hours, 168 hours, 216 hours and 264 hours after administration, the orbital blood collection is not anticoagulated, the blood sample is placed at room temperature for 45 minutes until the blood coagulation, the blood sample is obtained by centrifugation, and the blood sample is frozen at-80 ℃ for testing. The content of OM-anti-TIM-3 in serum is measured by ELISA, and the test result shows that the drug-induced parameters of OM-anti-TIM-3 with the dosage of 25nmol/kg for single intravenous injection are as follows: half-life of 498 hours; the area under the curve is 43721 nM.hr; zero concentration was estimated to be 391 nM; the apparent distribution volume is 119 mL/Kg; the clearance rate is 0.178 mL/hr/kg; the average residence time was 163 hours.

Example 7:

Humanized versions of anti-human TIM-3 antibodies were prepared by reference to the preparation of molecular Immunol, selecting the humanized template best matched to OM-anti-TIM-3 non-CDR region in the Germinle database, wherein the template for heavy chain variable region was human IgVH 4-28A 03, the template for light chain variable region was human IGKV 1-16A 02, grafting the murine antibody CDR region onto the selected humanized template, and substituting to obtain the humanized antibody heavy chain variable region, whose amino acid sequence is shown in SEQ ID NO.7, and the humanized antibody light chain variable region, whose amino acid sequence is shown in SEQ ID NO. 8. The amino acid sequences of the heavy chain variable region (VH) and the light chain variable region (VL) obtained by selecting suitable sites for back mutation by sequence alignment are shown in table 1.

TABLE 1 amino acid sequences obtained by back-mutation

The heavy chain variable region (SEQ ID NO: 9) of the humanized anti-human TIM-3 monoclonal antibody was ligated to the heavy chain constant region (SEQ ID NO:13) of human antibody IgG1 to obtain the corresponding heavy chain full-length sequences, respectively. The light chain variable region (SEQ ID NO: 11) of the humanized anti-human TIM-3 monoclonal antibody is connected with the constant region (SEQ ID NO:14) of the light chain of the human antibody Kappa to respectively obtain corresponding full-length sequences of the light chain, all the full-length sequences of the heavy chain and the full-length sequences of the light chain are combined to obtain the full-length sequence of the humanized antibody, and the full-length sequence of the humanized antibody is connected into a TL10-11 (vector framework pEGFP-N1) vector through enzyme digestion.

example 8:

constructing TL10-11 expression vectors respectively containing heavy chains and light chains of antibodies (Pembrolizumab) against human PD-1, wherein the sequences of variable regions of the antibodies are derived from http:// www.imgt.org/3 Dstructure-DB/cgi/tails. cgi.

TL10-11 expression vectors of heavy chain and light chain of an anti-human TIM-3 antibody are respectively constructed (the amino acid sequence of the heavy chain is shown as SEQ ID NO:9, and the amino acid sequence of the light chain is shown as SEQ ID NO: 11), and the expression vectors of the heavy chain and the light chain of the anti-human TIM-3 antibody are obtained according to a conventional method and are respectively used for expressing the heavy chain and the light chain of the anti-human TIM-3 antibody in eukaryotic cells.

Example 9:

The present invention refers to a method for large scale production of heterodimeric bispecific antibodies described in WO2013060867 by reducing two mixed homodimeric forms of the antibodies, then by introducing asymmetric amino acid mutations in the Fc regions of the two homodimeric antibodies, thereby facilitating the exchange of the Fab arms of the different antibodies, and finally by oxidizing the interchain disulfide bonds of the hinge region to form stable bispecific antibodies.

Expression vectors containing the heavy and light chains of anti-human PD-1 antibody were transfected into 293F cells, respectively, and expression vectors containing the heavy and light chains of anti-human TIM-3 antibody were also transfected into 293F cells, respectively. Cells were seeded the day before transfection, harvested by centrifugation the day of transfection, resuspended in fresh 293 expression medium at a cell density of 1.5X10⁷cells/mL. Adding plasmid according to the transfection volume, wherein the final concentration is 31.12ug/mL, and gently mixing; linear PEI was then added to a final concentration of 45ug/mL and gently mixed. Then, the cells were placed in a cell incubator and incubated for 1 hour at 37 ℃ on a shaker. Then, 20 times of the transfection volume of fresh medium was added, the shaking culture was continued at 37 ℃ and the expression level of the bispecific antibody against PD-1/TIM-3 was determined by ELISA and the protein purification was carried out by Wuhanpu's healthcare, the structure of the bispecific antibody is shown in FIG. 1.

Example 10:

The kinetic constants of the binding of the bispecific antibody obtained in example 9 to the antigen TIM-3 and the antigen PD-1 were determined in the same manner as in example 4, and the binding kinetic constants, dissociation kinetic constants and dissociation equilibrium constants of the bispecific antibody are shown in Table 2.

TABLE 2 kinetic constants for bispecific antibodies and their binding to their antigens

The pharmacokinetics of the bispecific antibody in rats was determined, briefly, 6-8 week old female SD rats were randomly divided into 2 groups (test group 1, test group 2, 5 per group), test group 1 was given 25nmol/kg of bispecific antibody; test group 2 was given 50nmol/kg bispecific antibody. At 0 point, 5 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 6 hours, 9 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 120 hours, 168 hours, 216 hours and 264 hours after administration, the orbital blood collection is not anticoagulated, the blood sample is placed at room temperature for 45 minutes until the blood coagulation, the blood sample is obtained by centrifugation, and the blood sample is frozen at-80 ℃ for testing.

The pharmacokinetic parameters for a single intravenous dose of 50nmol/kg of bispecific antibody are as follows: half life t_1/243 hours, area under the time-of-drug curve AUC_lastHr is 32823nM, estimated zero concentration C0 is 101nM, apparent distribution volume Vd is 41mL/Kg, clearance CL is 0.89mL/hr/Kg, average residence time MRT_lastIt was 83 hours.

The pharmacokinetic parameters for a single intravenous dose of 25nmol/kg of bispecific antibody are as follows: half life t_1/2The area under the time-of-drug curve AUC at 36 hours_lastHr was 29123nM, estimated zero concentration C0 was 97nM, apparent volume of distribution Vd was 32mL/Kg, clearance CL was 0.81mL/hr/Kg, average residence time MRT_lastIt was 72 hours.

Example 11:

The binding activity of the dual targets of the anti-PD-1/TIM-3 bispecific antibody was determined by enzyme-linked immunosorbent assay (ELISA) as in example 2. Briefly, recombinant human TIM-3 (concentration 1. mu.g/mL, 100. mu.L) was coated with carbonate buffer (pH9.3) on a 96-well high adsorption microplate and left overnight at 4 ℃; PBST was washed 5 times and then wells (280. mu.L/well) were blocked with PBST (containing 1% BSA) and incubated for 1h at 25 ℃; PBST was washed 5 times, then a sample of bispecific antibody diluted in PBST (containing 1% BSA) and a control (100. mu.L) were added, incubated at 25 ℃ for 1h, PBST washed 5 times, then diluted biotin-labeled PD-1-Fc (0.5. mu.g/mL, 100. mu.L per well) was added, incubated at 25 ℃ for 1h, streptavidin-horseradish peroxidase conjugate (1:1000) diluted in PBST was added, incubated at 25 ℃ for 1h, PBST washed 5 times, colorimetric substrate TMB was added, 100. mu.L/well, and developed at room temperature for 10 min. Addition of H₂SO₄(1M, 100. mu.L/well) and the absorbance at 450nm was read on a microplate reader, and the results are shown in FIG. 2, in which the combination of PD-1 monoclonal antibody and TIM-3 monoclonal antibody could not bind to PD-1 and TIM-3 simultaneously, and only the anti-PD-1/TIM-3 bispecific antibody had the activity of binding to both antigens simultaneously.

Example 12:

The growth inhibition effect of anti-human TIM-3 humanized monoclonal antibody (MeH-037), anti-human PD-1 (Pembrolizumab) and bispecific antibody on inoculated mouse tumor graft was tested, and the experimental material was selected from 8-week-old female mice (C57BL/6 background, purchased from Beijing Baioecker Gene biotechnology, Inc.). Taking 15 mice, inoculating MC38 cells, verifying the obvious tumor-bearing section of the mice, observing the tumor growth and recording the tumor volume after successful construction, administering 2 times per week (intraperitoneal injection), continuously administering for 5 weeks, measuring the tumor volume 1 time per week from the administration date, and measuring the major diameter a and the minor diameter b, wherein the tumor volume calculation formula is as follows: tumor volume ═ a x b²)/2。

When the tumor volume of the mice is increased to the required volume, 5 mice are divided into groups according to the volume: y1, Y2, Y3, Y4, Y5 (volume about 150 mm)³) (ii) a Y6, Y7, Y8, Y9, Y10 (volume about 100 mm)³) (ii) a Y1 and Y6 are set as a solvent group (equal volume of physiological saline is injected), 25nmol/kg of anti-human TIM-3 humanized monoclonal antibody is administered to Y2 and Y7, 25nmol/kg of anti-human PD-1 (Pembrolizumab) is administered to Y3 and Y8, Y4 and Y9 are administered at 25nmol/kg of bispecific antibody, Y5 and Y10 are administered at 50nmol/kg of bispecific antibody, and the test results are shown in Table 3.

TABLE 3 tumor ablation ratio in experimental mice

as can be seen from Table 3, the bispecific antibody significantly increased the tumor ablation rate compared to the anti-human TIM-3 humanized monoclonal antibody and PD-1, especially at 5 weeks when higher concentrations of the bispecific antibody (50nmol/kg) were administeredThe tumor ablation ratio is as high as 19.3%, therefore, the bispecific antibody provided by the invention has obvious anti-tumor activity in mouse experiments, obviously inhibits the growth of mouse transplanted tumor, and is directed at medium-volume tumor (about 100 mm)³) The ablation effect is better, namely the inhibition effect in the early stage of cancer is more obvious.

The above disclosure is only for the purpose of illustrating the preferred embodiments of the present invention, and it is therefore to be understood that the invention is not limited by the scope of the appended claims.

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<223> MeH-037 VH

<400> 9

Ala Ile Leu Met Thr Gln Thr Pro Ser Leu Ile Tyr Gln Ala Arg Glu

1 5 10 15

Thr Ala Val Thr Gln Asp Phe Trp Pro Trp Gln Cys Thr Ala Met Met

20 25 30

Trp Thr Gly Thr Trp Tyr Leu Gly Cys Phe Met Gly Ile Asn Cys Gln

35 40 45

Pro Glu His Gly Val Thr Arg Met Gln Tyr Pro Glu Pro His Ser Cys

50 55 60

Glu Arg Ala Pro Val Ser Ile Met Cys Cys His Gln His His Trp Thr

65 70 75 80

Asp Asn Cys Phe Asp Val Thr Trp Ile Ile Arg Asn Val Glu Trp Val

85 90 95

Asp Ala Cys Glu Ala Val Pro Lys Arg Trp Asn Thr Ala His Met His

100 105 110

Thr Pro Gly Asn Lys Leu Glu Trp Met

115 120

<210> 10

<211> 121

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> UNSURE

<223> MeH-012 VH

<400> 10

Asp Ile Leu Met Thr Gln Thr Pro Ser Leu Ile Tyr Gln Ala Arg His

1 5 10 15

Thr Ala Val Thr Gln Asp Phe Trp Pro Trp Gln Cys Thr Ala Met Met

20 25 30

Trp Thr Gly Thr Trp Tyr Leu Trp Cys Phe Leu Ala Ile Asn Cys Gln

35 40 45

Pro Glu His Gly Val Thr Arg Met Gln Tyr Asp Glu Pro His Ser Cys

50 55 60

Glu Arg Ala Pro Val Ser Ile Met Cys Cys His Gln His His Trp Thr

65 70 75 80

Asp Asn Cys Phe Asp Val Thr His Ile Ile Arg Asn Val Glu Trp Val

85 90 95

Asp Ala Cys Glu Ala Val Pro Lys Arg Trp Asn Thr Ala Lys Met Leu

100 105 110

Thr Pro Gly Asn Lys Leu Glu Trp Met

115 120

<210> 11

<211> 116

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> UNSURE

<223> MeH-037 VL

<400> 11

Trp Leu Met Gly Phe Lys Gln Cys Asp Asp Trp Ile Thr Leu Tyr Cys

1 5 10 15

Gly Asp Asp Asn Arg Met Phe Trp Glu Asn Glu Gln His Tyr Pro Met

20 25 30

Phe Gln Leu Met Glu Phe Glu Pro Ala Asp Asp Ala Lys Asn Asn His

35 40 45

Arg Ser Gln Asn Lys His Val Val Gln Phe Ser Ala Trp Pro Phe Trp

50 55 60

Leu Ala Gly Ser Thr Thr Trp Lys Asp Ile Arg Met Ile Ser Arg Lys

65 70 75 80

Cys Arg Cys Ser Ala Lys Ser His Gly Pro Met Glu Asp Arg Pro Cys

85 90 95

Asn Trp Phe Met Tyr Ala Thr Thr Gly Leu Ser Phe Arg Asp Gly Asn

100 105 110

Ile Tyr Ala Asn

115

<210> 12

<211> 116

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<221> UNSURE

<223> MeH-012VL

<400> 12

Asn Leu Met Gly Phe Lys Gln Cys Trp Asp Glu Ile Thr Leu Tyr Cys

1 5 10 15

Gly Asp Asp Asn Arg Met Phe Trp Glu Asn Glu Gln His Tyr Pro Met

20 25 30

Phe Gln Leu Met Glu Phe Glu Pro Ala Asp Asp Ala Trp Asn Asn His

35 40 45

Arg Ser Gln Asn Lys His Val Val Gln Phe Ser Ala Gln Pro Phe Trp

50 55 60

Leu Ala Gly Ser Cys Thr Trp Lys Asp Ile Arg Met Ile Ser Arg Lys

65 70 75 80

Cys Arg Cys Ser Ala Lys Ser His Gly Pro Met Glu Asp Arg Pro Cys

85 90 95

Asn Trp Phe Met Tyr Ala Thr Thr Gly Leu Ser Phe Arg Asp Gly Asn

100 105 110

Ile Tyr Phe Asn

115

<210> 13

<211> 329

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 13

Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser

1 5 10 15

Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe

20 25 30

Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly

35 40 45

Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu

50 55 60

Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr

65 70 75 80

Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys

85 90 95

Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro

100 105 110

Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys

115 120 125

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

130 135 140

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr

145 150 155 160

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

165 170 175

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

180 185 190

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

195 200 205

Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

210 215 220

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

225 230 235 240

Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro

245 250 255

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

260 265 270

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

275 280 285

Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val

290 295 300

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

305 310 315 320

Lys Ser Leu Ser Leu Ser Pro Gly Lys

325

<210> 14

<211> 110

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 14

Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro

1 5 10 15

Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu

20 25 30

Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn

35 40 45

Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser

50 55 60

Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala

65 70 75 80

Asp Tyr Glu Lys His Lys Leu Tyr Ala Cys Glu Val Thr His Gln Gly

85 90 95

Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

100 105 110

Claims

1. A bispecific antibody comprising a first antigen-binding domain capable of specifically binding to PD-1 and a second antigen-binding domain capable of specifically binding to TIM-3.

2. The antibody according to claim 1, wherein the amino acid sequence of the heavy chain variable region of the second antigen binding domain is a variant sequence having at least 95% identity to any one of the amino acid sequences shown in SEQ ID Nos. 7, 9 and 10 and retaining the corresponding biological activity or a variant sequence obtained by deletion, substitution and/or addition of one or more amino acid residues and retaining the corresponding biological activity.

3. The antibody according to claim 1, wherein the amino acid sequence of the light chain variable region of the second antigen binding domain is a variant sequence having at least 95% identity to any one of the amino acid sequences shown in SEQ ID nos. 8, 11 and 12 and retaining the corresponding biological activity or a variant sequence obtained by deletion, substitution and/or addition of one or more amino acid residues and retaining the corresponding biological activity.

4. The antibody of claim 1, wherein the amino acid sequence of the heavy chain variable region of said second antigen binding domain is represented by any one of SEQ ID nos. 7, 9, and 10 and/or the amino acid sequence of the light chain variable region of said second antigen binding domain is represented by any one of SEQ ID nos. 8, 11, and 12.

5. the antibody of claim 1, wherein the heavy chain variable region of said second antigen binding domain has the amino acid sequence shown in SEQ ID No.9 and the light chain variable region of said second antigen binding domain has the amino acid sequence shown in SEQ ID No. 11.

6. The antibody of any one of claims 1-5, wherein the Fc chain of said bispecific antibody is in the form of an IgG.

7. The antibody of any one of claims 1-6, wherein said antigen binding fragment is selected from the group consisting of Fab, Fab ', F (ab)'2, single chain Fv (scFv), and bispecific antibodies.

8. the antibody of any one of claims 1-7, wherein said antibody is produced by a process comprising: culturing a host cell containing a nucleotide coding sequence corresponding to the antibody of claims 1-7 in a culture medium under suitable culture conditions, harvesting the cells, purifying the bispecific antibody or other equivalent production protocol.

9. The antibody of any one of claims 1-8, wherein said bispecific antibody is conjugated to at least one therapeutic agent selected from one or more of a cytotoxic agent, a radionuclide, a boron atom, an immunomodulator, an immunoconjugate, an oligonucleotide, to form an immunoconjugate.

10. Use of a bispecific antibody comprising recovering the antibody and antigen-binding fragment thereof of claims 1-9 produced from the culture medium or from the cultured host cell and formulating with a pharmaceutically acceptable carrier for the treatment of tumors including, but not limited to, gastric cancer, pancreatic cancer, gallbladder cancer, liver cancer, colorectal cancer, leukemia, breast cancer, ovarian cancer, cervical cancer, endometrial cancer, uterine sarcoma, prostate cancer, bladder cancer, renal cell carcinoma.