CN108101990B - Monoclonal antibody for blocking human Tim-3 function and coding gene and application thereof - Google Patents

Abstract

The invention belongs to the technical field of bioengineering, and particularly relates to a monoclonal antibody capable of blocking the function of human Tim-3 and a coding gene thereof, wherein the antibody can be combined with an extracellular region of human Tim-3, can specifically block the combination of Tim-3 and a ligand thereof, and inhibits signal transduction, so that the monoclonal antibody can be used as a blocking agent of a Tim-3 signal path, thereby becoming a novel antibody medicament for tumor immunotherapy, chronic virus infectious diseases and autoimmune diseases.

Description

Monoclonal antibody for blocking human Tim-3 function and coding gene and application thereof

Technical Field

The invention belongs to the technical field of bioengineering, and particularly relates to a monoclonal antibody for blocking the function of human Tim-3, and a coding gene and application thereof.

Background

McIntire et al discovered a Tim gene family in 2001, and research found that Tim gene encodes protein molecules containing immunoglobulin and mucin domains, is widely expressed on immune cells, participates in the regulation of the differentiation of various T cells including regulatory T cells, and plays an important role in immune homeostasis and the clearance of apoptotic cells. The Tim family has only 3 genes in human, and is located in 5q33.2 of human chromosome, and encodes protein Tim-1, 3 and 4 respectively. Tim-3 is an important member of the Tim family, consisting of 301 amino acids, and its structure includes extracellular immunoglobulin V-region, mucin region, transmembrane region, and cytoplasmic region [ FreemanGJ, casinnovas JM, Umetsu DT, et a1.Tim genes: a family of cell surface phosphor receptors which are regulated in and adaptive immunity [ J ]. Immunol Rev, 2010, 235 (1): 172-189; umetsu SE, Lee WL, McInterie JJ, et a1.TIM-1 indices T cell activation and inhibition of the degradation of the coherence [ J ]. Nat Immunol, 2005, 6 (5): 447454, respectively; Rodriguez-Manzanet R, Dekruyf R, Kuchroo VK, et a1.the costimulatory role of TIM molecules [ J ]. Immunol Rev, 2009, 229 (1): 259-270.].

It has now been found that Tim-3 is expressed on the surface of differentiated Th1 cells, CD8⁺Immune cells such as T cells, NK cells, macrophages, DC cells, Treg cells, T17 cells and mast cells, and some tumor cells [ Anderson AC, Anderson DE, Bregoli L et a1. movement of tissue inflammation by the immune receptor TIM-3expressed on animal cells, science 2007; 318(5853) 1141 and 1143; freeman GJ, casarnova JM, Umetsu DT, DeKruyff RH. TIM genes a family of cell motility receptor that is regulated in and adaptive immunity. immunological reviews 2010; 235:172-89.]。

Negative regulation of CD4 after Tim-3 is combined with ligand Galectin-9⁺Function of Th1 cell, induction of CD4⁺Apoptosis of Th1 cells. The abnormal Tim-3/galectin-9 pathway is involved in the occurrence of various immune diseases, such as autoimmune encephalomyelitis, type I diabetes and acute graft-versus-host disease. Studies have shown that the animal condition worsens after inhibiting Tim-3/Gal-9 signaling in vivo in an animal model of multiple sclerosis. More studies have shown that Tim-3 plays an important regulatory role in autoimmune diseases [ Monney L, Sabases CA, Gaglia JL, et a1.Th 1-specific cell surface protein Tim-3 modulators pathological activation and maintenance of an autoimmune disease. Nature, 2002; 415: 536-; zhu C, et a1.the Tim-3ligand and galectin-9 ligand ligands T helper type 1immunity. nat Immunol.2005; 6: 1245-1252; boenisch O, D' Addio F, Watanabe T, Elyaman W, et a1.TIM-3 a novel regulation of expression of immune activity.J Immuno1.2010Nov 15; 185(10):5806-19]。

High expression of Tim-3 in chronic viral infection state to make CD4⁺T and CD8⁺T cell dysfunction loses immune effects. Studies have shown that Tim-3 is responsible for HIV, HBV and HCV viral infections⁺CD8⁺Increased ratio of T and reduced immune effector function, CD8 after treatment with blocking agents such as Tim-3 antibody⁺The T cell efficacy is restoredJones RB,Ndhlovu LC,Barbour JD,et a1.TIM-3expression defines a novel population of dysfunctionalT cells with highly elevated frequencies in progressive HIV-1infection.ExpMed 2008；205:2763-2779；Wei Wu,Yu Shi,Shuping Li,et al.Blockade of Tim-3signaling restore the virus-specific CD8+T-cell response in patients withchronic hepatitis B.Eur.J.Immunol.2012.42:1180-1191.]。

CD4 in tumor infiltration⁺T and CD8⁺T cells highly express Tim-3, and the cell effector function is damaged, and has correlation with tumor progression. In acute myeloid leukemia, most of the leukemia stem cells express Tim-3, and the use of Tim-3 antibody can effectively inhibit the regeneration of leukemia stem cells, and complement-dependent and antibody-dependent toxicity reactions are also enhanced without affecting normal hematopoietic stem cells [ Kikushige Y, Akashi K.TIM-3as a therapeutic target for a mammalian cell in acute myelogenous leukemia Leukemia.Ann N Y Acad Sci,2012,1266: 118-.]。

The research shows that the Tim-3 pathway has important immunoregulation function and plays an important role in eliminating apoptotic cells and pathogenic bacteria, regulating allergic diseases and autoimmune diseases, generating and developing tumors and the like. Therefore, the development of a neutralizing antibody with high affinity with Tim-3 and the functional blocking of the signal pathway of Tim-3 are of great significance for the mechanism research, disease diagnosis and treatment of diseases such as autoimmunity, infectivity and tumor.

The existing Tim-3 antibody is low in affinity, and a blocking Tim-3 antibody with high affinity is still absent.

Disclosure of Invention

In order to overcome the problems in the prior art, the invention aims to provide a monoclonal antibody for blocking the function of human Tim-3, a coding gene and application thereof.

The monoclonal antibody or the fragment thereof is combined with an extracellular region containing T cell immunoglobulin mucin molecule 3 (hereinafter referred to as Tim-3) and has the function of blocking the Tim-3 function.

In order to achieve the above objects and other related objects, the present invention adopts the following technical solutions:

in a first aspect of the present invention, a monoclonal antibody is provided, which comprises a light chain and a heavy chain, wherein the light chain comprises a CDR region having an amino acid sequence as shown in SEQ ID NO. 1-3, and the heavy chain comprises a CDR region having an amino acid sequence as shown in SEQ ID NO. 4-6.

The heavy and light chains are linked by disulfide bonds.

Preferably, the amino acid sequence of the variable region of the light chain of the monoclonal antibody is SEQ ID No.7 or a conservative variant thereof, and the amino acid sequence of the variable region of the heavy chain of the monoclonal antibody is SEQ ID No.8 or a conservative variant thereof.

Preferably, the coding nucleotide sequence of the light chain variable region is SEQ ID NO.9 or a conservative variant sequence thereof, and the coding nucleotide sequence of the heavy chain variable region is SEQ ID NO.10 or a conservative variant sequence thereof.

Preferably, the monoclonal antibody is of murine origin.

More preferably, the monoclonal antibody is an immunoglobulin of the IgG2a heavy chain and Kappa type light chain subtypes.

The monoclonal antibody provided by the invention is used for resisting (combining or acting) differentiated Th1 cell surface and CD8⁺A Tim-3 antigen (or a receptor or an epitope) expressed on immune cells such as T cells, NK cells, macrophages, DC cells, Treg cells, T17 cells and mast cells, or a monoclonal antibody partially against (binding to or acting on) the Tim-3 antigen or partially against (binding to or acting on) the Tim-3 antigen.

The monoclonal antibody or a derivative thereof may be a single-chain antibody, a double-chain antibody, a chimeric antibody, a humanized antibody, as long as it can bind to human Tim-3 protein and has a functional effect of blocking the Tim-3 pathway.

The invention further provides a derivative of the monoclonal antibody, wherein the derivative can be the monoclonal antibody fragment or a fusion protein containing the monoclonal antibody or the monoclonal antibody fragment, and the fragment of the monoclonal antibody can be Fab, Fab ', F (ab') 2, Fv, scFv or the like. So long as it binds to human Tim-3 protein and has a functional effect of blocking the Tim-3 pathway.

In a second aspect, the invention provides an isolated DNA molecule encoding the variable region or full length amino acids of the heavy and/or light chain of said monoclonal antibody.

In a third aspect of the invention, a construct is provided comprising the isolated DNA molecule.

Preferably, the DNA vector expression construct is constructed by inserting the isolated antibody DNA molecule into a multiple cloning site of an expression vector.

The expression vector may be, in particular, an expression vector commonly used in the art, which is well known to those skilled in the art, and particularly employable is an expression vector including, but not limited to: pET series expression vector, pGEX series expression vector, pcDNA series expression vector, etc.

The fourth aspect of the invention provides an expression system of the monoclonal antibody, which is constructed by transfecting the construct into a host cell.

Any cell suitable for expressing the antibody described herein in an expression vector (construct) may be used as the host cell. For example, cells of yeast, insects, plants, and the like. Preferably, the host cell is a eukaryotic cell, and mammalian host cell lines that do not produce antibodies can be used, including but not limited to: ovary cells of Chinese Hamster (CHO), kidney cells of baby hamster (BHK, ATCC CCL 10), Sertoli cells of baby mouse (Sertoli cells), kidney cells of monkey (COS cells), kidney CVI cells of monkey transformed by SV40(COS-7, ATCC CRL 1651), human embryonic kidney cells (HEK-293), kidney cells of monkey (CVI, ATCC CCL-70), kidney cells of African green monkey (VERO-76, ATCCRL-1587), human cervical cancer cells (HELA, ATCC CCL-2), and the like.

The fifth aspect of the present invention provides a method for preparing the monoclonal antibody, comprising the following steps: culturing said monoclonal antibody expression system under conditions suitable for expression of said antibody, thereby expressing said monoclonal antibody, and purifying and isolating said monoclonal antibody.

After obtaining a nucleic acid sequence encoding the antibody of the present invention, the antibody of interest can be produced according to the following method. For example, a vector containing a nucleic acid encoding an antibody of interest is directly introduced into a host cell, and the cell is cultured under appropriate conditions to induce expression of the encoded antibody. The expression vector and the host cell used in the invention are the prior art and can be directly obtained from commercial sources, the 1640 culture medium of 10% FBS used in the culture is various conventional mammalian cell culture media, and the skilled person can select the suitable 1640 culture medium according to experience and culture the suitable condition for the growth of the host cell. After the host cells have been grown to an appropriate cell density, the selected promoter is induced by suitable means (e.g., temperature shift or chemical induction) and the cells are cultured for an additional period of time. The recombinant polypeptide in the above method may be expressed intracellularly or on the cell membrane, or secreted extracellularly. Once the monoclonal antibody of the present invention is obtained, it can be isolated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of such methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (such as salt precipitation), centrifugation, cell lysis by osmosis, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, High Performance Liquid Chromatography (HPLC), and other various liquid chromatography techniques, and combinations thereof.

A sixth aspect of the invention provides the use of the monoclonal antibody in the manufacture of a medicament having the effect of any one or more of:

(1) activation of immune cell function; (2) treatment of cancer; (3) treatment of immune diseases; (4) treatment of infectious diseases.

In some embodiments of the invention, monoclonal antibodies of the invention have been shown to promote CD8⁺Efficacy of T cell proliferation. Therefore, the invention also provides the application of the monoclonal antibody in preparing a medicine capable of promoting CD8⁺Use in the manufacture of a medicament for the proliferation of T cells.

According to the test data provided by the invention, the technical personnel in the field can know that the monoclonal antibody can be combined with the human Tim-3 extracellular region, can specifically block the combination of Tim-3 and the ligand thereof, and inhibits signal transduction, so that the monoclonal antibody can be used as a blocking agent of a Tim-3 signal path, thereby becoming a novel antibody medicament for treating tumor immunotherapy, chronic virus infectious diseases and autoimmune diseases.

In a seventh aspect of the invention, there is provided a pharmaceutical composition comprising a therapeutically effective amount of said monoclonal antibody or immunoconjugate thereof.

The immunoconjugates include, but are not limited to, conjugates formed by binding the monoclonal antibody or fragment thereof to drugs, toxins, cytokines, radionuclides, enzymes, or other diagnostic agents, and the like.

The pharmaceutical composition takes Tim-3expressed by the focus as an antigen, combines or acts on the antigen, specifically blocks the combination of Tim-3 and a ligand thereof, and inhibits signal transduction, thereby treating diseases.

The monoclonal antibodies of the invention may be used by formulating pharmaceutical compositions by any means known in the art. Such compositions comprise the monoclonal antibody as an active ingredient, together with one or more pharmaceutically acceptable carriers or excipients which are compatible with the monoclonal antibody.

Compared with the prior art, the invention has the following beneficial effects:

the invention newly finds a monoclonal antibody capable of blocking the function of human Tim-3 and a coding gene thereof, the antibody can be combined with the extracellular region of human Tim-3, can specifically block the combination of Tim-3 and a ligand thereof, and inhibits signal transduction, so that the monoclonal antibody can be used as a blocking agent of a Tim-3 signal path, thereby becoming a novel antibody medicament for treating tumor immunotherapy, chronic virus infectious diseases and autoimmune diseases.

Drawings

FIG. 1 is an agarose gel electrophoresis of the light and heavy chain variable region gene of anti-human Tim-3 monoclonal antibody CDT1 amplified by RT-PCR.

FIG. 2 shows the anti-Tim-3 antibodyIn vivo blocking Effect assay, CDT1 antibody-treated CD8⁺Multiple peaks can be detected by the T cell flow, the cell has obvious proliferation effect, and the PBS and the isotype control are single peak images and have no effect of promoting cell proliferation.

FIG. 3: CDT1 antibody was compared to a commercial Tim-3 functional antibody, where A was CDT1 antibody, B was isotype control, C was PBS, and D was a commercial functional antibody.

FIG. 4: CDT1 antibody and a simultaneously prepared antibody without functional effect are compared, wherein A is CDT1 antibody; b is isotype control; c is PBS, D and E are two non-functional antibodies, named: CPT1CPT 2.

Detailed Description

Before the present embodiments are further described, it is to be understood that the scope of the invention is not limited to the particular embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention. Test methods in which specific conditions are not specified in the following examples are generally carried out under conventional conditions or under conditions recommended by the respective manufacturers.

When numerical ranges are given in the examples, it is understood that both endpoints of each of the numerical ranges and any value therebetween can be selected unless the invention otherwise indicated. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, and materials used in the examples, any methods, devices, and materials similar or equivalent to those described in the examples may be used in the practice of the invention in addition to the specific methods, devices, and materials used in the examples, in keeping with the knowledge of one skilled in the art and with the description of the invention.

Unless otherwise indicated, the experimental methods, detection methods, and preparation methods disclosed herein all employ techniques conventional in the art of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related arts. These techniques are well described in the literature, and may be found in particular in the study of the MOLECULAR CLONING, Sambrook et al: a LABORATORY MANUAL, Second edition, Cold Spring harbor LABORATORY Press, 1989and Third edition, 2001; ausubel et al, Current PROTOCOLS Inmolecular BIOLOGY, John Wiley & Sons, New York, 1987and periodic updates; the series METHODS IN ENZYMOLOGY, Academic Press, San Diego; wolffe, CHROMATINSTRUCUTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998; (iii) Methods Inenzymolygy, Vol.304, Chromatin (P.M. Wassarman and A.P.Wolffe, eds.), academic Press, San Diego, 1999; and METHODS in the study of molecular BIOLOGY BIOLOGY, Vol.119, chromatography protocols (P.B. Becker, ed.) Humana Press, Totowa, 1999, etc.

Example 1 construction of anti-human Tim-3 monoclonal antibody CDT1 hybridoma cell line

1. Material

DNA immunoadjuvants were purchased from Sigma; fetal bovine serum and PRMI1640 medium were purchased from Gibco; SP2/0 cells were purchased from ATCC; BALB/c mice were provided by the Experimental animals center of university of Zhejiang.

2. The method comprises the following steps:

(1) plasmid construction

A primer is designed according to the Tim-3 gene sequence searched on NCBI, a DNA sequence of the Tim-3 gene is amplified by taking human cDNA as a template, and the DNA sequence is constructed on pcDNA3.1 plasmid by utilizing a molecular biology method.

(2) Immunization of mice

Selecting BALB/c mice of 4-6 weeks old, dissolving the constructed Tim-3 gene recombinant plasmid in PBS and adjuvant, mixing and emulsifying in equal volume, and performing subcutaneous and abdominal multipoint immunization. Every 2 weeks after the first immunization, the plasmid and the adjuvant with the same dose are mixed and emulsified respectively, and then injected for boosting immunization. After 7 days of 3 rd immunization, tail vein blood was collected and subjected to gradient dilution by ELISA method to detect the antibody production. 3 days before fusion, the same dose of plasmid was mixed with adjuvant and boosted 1 time.

(3) Cell fusion

After the immunized mice were sacrificed, spleens of the mice were aseptically harvested, milled to prepare single cell suspensions, and washed with RPMI1640 mediumCounting the number of cells after the next time; mixing mouse spleen lymphocyte and myeloma cell SP2/0 at a ratio of 1:5-1:10, slowly dripping 50% PEG solution 1ml in short time, placing in 37 deg.C water bath for 1min, adding RPMI1640 culture solution, centrifuging at 800r/min for 5min, discarding supernatant, adding 1640 culture medium suspension containing 20% FBS, mixing the cell suspension with equal volume of feeder cell (mouse abdominal cavity macrophage), subpackaging in 96-well cell culture plate, placing at 37 deg.C, 5% CO₂Culturing in an incubator. After 3d of culture, HAT 1640 containing 20% FBS was added for selection medium culture.

(4) Screening of hybridoma cells

When the cell colony of the 96-well plate grows to a proper size, the cell culture supernatant is extracted and detected by an indirect ELISA method, and positive hybridoma cells are screened.

(5) Hybridoma cell cloning

When the hybridoma cells identified as positive were cultured to good condition, the cells were diluted and plated into 96-well plates by limiting dilution so that only one cell was present in each well, and the cells were incubated at 37 ℃ in 5% CO₂Culturing in an incubator. Observing the cell growth condition in 5d-10d, taking the supernatant in the hole forming single cell clone, detecting by using an indirect ELISA method, screening and identifying positive clone; and repeating the subcloning for 3-5 times until the positive porosity of the hybridoma cells reaches 100%, and determining the stability of the cells.

Example 2: anti-human Tim-3 monoclonal antibody CDT1 antibody subclass identification and stability test

The subclass of the antibody is identified by referring to the specification of the antibody subclass identification kit, and as a result, the heavy chain class is IgG2a type, and the light chain is Kappa type.

Continuously subculturing the hybridoma cell strain in vitro for 3 months, and measuring the titer of the antibody in the supernatant; the cryopreserved hybridoma cell strain is recovered after 4 months, and the antibody titer in the supernatant is detected without obvious change, which shows that the obtained hybridoma cell strain generating the antibody has stable performance.

Example 3: heavy and light chain clones of anti-human Tim-3 monoclonal antibody CDT1

1. Material

Designing monoclonal antibody heavy chain and light chain gene amplification primers according to literature data, and synthesizing the primers by Shanghai biological engineering Co., Ltd; the DNA fragment purification kit and the plasmid extraction kit are purchased from QIAGEN company; pMD-18T kit and reverse transcriptase were purchased from TAKARA; competent cells were purchased from promega; KOD plus fidelity PCR enzyme was purchased from TOYOBO, and Trizol reagent was purchased from Invitrogen.

2. Method and results

(1) Take 5X 10⁶-10⁷And centrifuging the monoclonal antibody hybridoma cells to remove supernatant, adding 1ml of Trizol reagent, and repeatedly blowing and beating until the cells are fully lysed. Standing at room temperature for 5min, adding 0.2ml chloroform, mixing by turning upside down, and standing at room temperature for 2-3 min. Centrifuge at 12000 × g for 15min at 4 ℃. Carefully suck the supernatant and mix with another centrifuge tube, add 500. mu.l isopropanol, mix well by inversion, then stand at room temperature for 10min, centrifuge at 12000 × g at 4 ℃ for 10 min. Adding 1ml of precooled 75% ethanol to wash the precipitate, centrifuging 7500 x g for 5min, then discarding the supernatant, drying at room temperature for 5min, and adding RNase-free deionized water to dissolve the precipitate.

(2) 1 mu.L of random primer is added into 1 mu.g of RNA, 6 mu.L of random primer is supplemented by RNase-free water, the mixture is quickly placed on ice for 2min after being incubated for 10min at 70 ℃, 5M-MLV Buffer 2 mu L, dNTP mix 0.5 mu L, RNase-free water is added for 1 mu L of 5M-MLV Buffer, 0.25 mu L, M-MLV enzyme and 1 mu L of RNase-free water, the mixture is incubated for 1h at 42 ℃, the mixture is incubated for 15min at 70 ℃ and then cooled on ice, and the product is the first strand cDNA.

(3) In 25. mu.L of the amplification system, 0.5. mu.L of each of two pairs of heavy chain and light chain PCR amplification primers, 2. mu.L of the reverse transcription product, 0.5. mu.L of kod plus amplilase, 2. mu.L of dNTP, 5. mu.L of Buffer were added, and the mixture was made up to 25. mu.L with water. The PCR amplification parameters were: pre-denaturation at 94 ℃ for 2min, cyclic procedure: 35 cycles of 98 ℃ 10s 55 ℃ 30s 68 ℃ 30 s.

(4) PCR amplification products were run on agarose gel, as shown in FIG. 1, the desired target product was recovered by tapping, and after recovery with a DNA fragment recovery kit, the DNA fragment and the vector were mixed at a molar ratio of 10:1 according to the instructions of the pMD-18T kit, and then ligase and ligation Buffer were added to make a total volume of 10. mu.L, and ligation was performed overnight at 16 ℃.

(5) Adding 10 μ L of the connecting liquid into 200 μ L of TOP 10F' competent bacteria, ice-cooling for 30min, heat-shocking at 42 ℃ for 90s, rapidly placing on ice for 2min, adding 800 μ L of LB culture liquid, shaking and culturing at 37 ℃ and 180rpm/min for 45min, centrifuging at 5000rpm for 2min, discarding about 800 μ L of supernatant, blowing the precipitate uniformly with the rest liquid, spreading on LB solid culture medium plate containing ampicillin, and inversely culturing the plate in a 37 ℃ culture box for 12-16 h. Selecting a single bacterial clone from a plate, inoculating the bacterial clone into an LB liquid culture medium containing ampicillin, carrying out shaking culture at 37 ℃ and 220rpm/min for overnight, removing part of bacterial liquid, cracking, carrying out PCR amplification verification by using light chain and heavy chain primers, and amplifying bacterial liquid with fragments with required sizes to carry out sequencing. After the bacterial liquid with correct sequencing is transferred, a DNA plasmid extraction kit is used for extracting plasmids which carry genes of heavy chains and light chains of variable regions of antibodies.

The identification result shows that the amino acid sequence of the complementarity determining region 1(CDR1) of the light chain variable region of the human Tim-3 monoclonal antibody CDT1 is shown as SEQ ID NO.1, and specifically comprises the following steps:

GlnSerValLeuTyrSerSerAsnGlnLysAsnPhe。

the amino acid sequence of the complementarity determining region 2(CDR2) of the light chain variable region is shown in SEQ ID NO.2, and specifically comprises the following components:

TrpAlaSer。

the amino acid sequence of the complementarity determining region 3(CDR3) of the light chain variable region is shown in SEQ ID NO.3, and specifically comprises the following components:

HisGlnTyrLeuSerLeu ArgThr。

the amino acid sequence of the complementarity determining region 1(CDR1) of the heavy chain variable region is shown in SEQ ID NO.4, and specifically comprises the following components:

GlyPheThrPheThrAspTyrTyr。

the amino acid sequence of the complementarity determining region 2(CDR2) of the heavy chain variable region is shown as SEQ ID NO.5, and specifically comprises the following steps:

IleArgAsnLysAlaAsnGlyTyrThrThr。

the amino acid sequence of the complementarity determining region 2(CDR2) of the heavy chain variable region is shown in SEQ ID NO.6, and specifically comprises the following components:

AlaArgAsp LeuAspTyr。

the amino acid sequence of the light chain variable region of the human Tim-3 monoclonal antibody CDT1 is shown in SEQ ID NO.7, and specifically comprises the following steps:

the amino acid sequence of the heavy chain variable region of the human Tim-3 monoclonal antibody CDT1 is shown in SEQ ID NO.8, and specifically comprises the following steps:

the encoding nucleotide sequence of the light chain variable region of the human Tim-3 monoclonal antibody CDT1 is shown as SEQ ID NO.9, and specifically comprises the following steps:

the encoding nucleotide sequence of the heavy chain variable region of the human Tim-3 monoclonal antibody CDT1 is shown in SEQ ID NO.10, and specifically comprises the following steps:

example 4: anti-human Tim-3 monoclonal antibody CDT1 promotes CD8⁺T cell proliferation assay

1. Isolation of human Whole blood mononuclear cells (PBMC)

(1) Blood was collected using an anticoagulation tube, shaken up and diluted with an equal volume (1:1) of Hank's solution or PBS. 2ml of the lymphocyte separation medium was taken and put into a 15ml centrifuge tube.

(2) The centrifuge tube is inclined at an angle of 45 degrees, diluted blood is sucked by a suction tube, and is slowly added along the wall of the test tube at a position 1cm above the liquid level of the separation layer, so that the diluted blood is superposed on the separation layer liquid, and the interface between the diluted blood and the separation layer liquid is kept clear (the volume ratio of the diluted blood to the separation layer liquid is about 2: 1).

(3) The horizontal centrifuge is centrifuged at room temperature at 300^ g/min for 20min, the centrifuged content is divided into four layers, the upper layer is plasma (containing platelets), the middle layer is a layering liquid, the bottom layer is red blood cells and granulocytes, and a milky turbid mononuclear cell layer can be seen at the interface of the upper layer liquid and the middle layer liquid.

(4) Gently inserting the leukocyte membrane layer by using a capillary tube (or a 1ml syringe), sucking boundary layer mononuclear cells along the periphery of the tube wall, transferring the boundary layer mononuclear cells into another centrifuge tube, and sucking out PBMC as completely as possible to avoid sucking excessive stratified fluid and plasma.

(5) Adding 5ml Hank's solution or RPMI1640, washing for 2 times, mixing, centrifuging at 300 × g/min for 10min, and removing supernatant.

(6) After the final centrifugation, the supernatant was discarded, and 2ml of RPMI1640 medium containing 10% fetal bovine serum was used to resuspend the cells for use.

CFSE-labeled PBMC

(1) The isolated PBMCs were adjusted to a cell count of 0.5X 10⁶-10×10⁷Between each ml, put into a 15ml centrifuge tube.

(2) The tube was placed flat, 110. mu.L of PBS was added to the tube, 1.1. mu.L of 5mM CFSE stock solution was mixed with PBS, quickly covered with a cap and mixed by inversion.

(3) Cells were incubated for 5min at room temperature in the dark.

(4) 10 volumes of PBS (containing 5% inactivated fetal bovine serum) were added, centrifuged at 300 × g for 5min, the supernatant carefully discarded, and the cells washed twice.

(5) The cells were suspended by adding the culture medium, and 100. mu.L of each of the 96-well plates was used, and the volume of the plate was 1X 10⁵Individual cells/well.

3. Antibody-facilitated T cell proliferation assay

(1) After the CFSE-treated PBMC cells were seeded in a 96-well plate, CD3CD28 antibody-coated magnetic beads and IL-2 cytokine-activated T cells were added, and the mixture was placed in a cell culture incubator at 37 ℃ and 5% CO₂Culturing for 3 days under the condition, and detecting the expression of Tim-3 on the cell surface by flow, wherein the result shows that the expression of Tim-3 is obviously up-regulated.

(2) Activated cells are divided into three groups: PBS was added to the blank control group, Isotype control group was added to the Isotype control group having the same subtype as CDT1 (name: Purified Mouse IgG2a,. kappa.Isotype Ctrl Antibody brand: Biolegend cat # 401501), Antibody group was added to CDT1 Antibody at a final concentration of 10ug/ml, and the Antibody group was placed in a cell culture incubator at 37 ℃ with 5% CO₂Cultured under the conditions for 3 days.

(3) The cells were transferred to 1.5ml ep tubes, centrifuged at 2000rpm for 5min, washed with 1ml PBS and resuspended in 100. mu.L PBS.

(4) Adding flow-type antibody fluorescently labeled by CD3-APC and CD8-PE, incubating for 30min at 4 ℃, washing with PBS, and detecting CD8 by an up-flow machine⁺T cell proliferation, as shown in FIG. 2, indicates that CDT1 antibody is directed against CD8⁺T cells have a pronounced effect on promoting proliferation. As shown in FIG. 3, CDT1 antibody was compared with a commercial Tim-3 functional antibody, and the results showed that CDT1 antibody was directed against CD8⁺T cells have a proliferative effect that is stronger than commercial functional antibodies.

Comparative example

In the research and development process, when the anti-human Tim-3 monoclonal antibody CDT1 hybridoma cell strain is constructed by adopting the experimental method, other two strains of antibodies capable of being combined with the Tim-3 extracellular region are constructed and screened: anti-human Tim-3 monoclonal antibody CPT1 hybridoma cell strain, anti-human Tim-3 monoclonal antibody CPT2 hybridoma cell strain. CD8 of its respective secreted monoclonal antibodies CPT1 and CPT2⁺Experimental study of T cell proliferation. The results are shown in FIG. 4, where CPT1 and CPT2 did not promote CD8⁺Function of T cell proliferation.

While the invention has been described with respect to a preferred embodiment, it will be understood by those skilled in the art that the foregoing and other changes, omissions and deviations in the form and detail thereof may be made without departing from the scope of this invention. Those skilled in the art can make various changes, modifications and equivalent arrangements, which are equivalent to the embodiments of the present invention, without departing from the spirit and scope of the present invention, and which may be made by utilizing the techniques disclosed above; meanwhile, any changes, modifications and variations of the above-described embodiments, which are equivalent to those of the technical spirit of the present invention, are within the scope of the technical solution of the present invention.

Sequence listing

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<213> Artificial Sequence (Artificial Sequence)

<400>7

Asn Ile Met Met Thr Gln Ser Pro Ser Ser Leu Ala Val Ser Ala Gly

1 510 15

Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Val Leu Tyr Ser

20 25 30

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

35 40 45

Ser Pro Glu Leu Leu Ile Tyr Trp Ala Ser Thr Arg Val 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 Ser Asn Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys His Gln

85 90 95

Tyr Leu Ser Leu Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile

100 105 110

<210>8

<211>114

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>8

Val Lys Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser

1 5 10 15

Leu Arg Leu Ser Cys Ala Thr Ser Gly Phe Thr Phe Thr Asp Tyr Tyr

20 25 30

Met Ser Trp Val Arg Gln Pro Pro Gly LysAla Leu Glu Trp Leu Gly

35 40 45

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

50 55 60

Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Tyr Ser Gln Ser Ile Leu

65 70 75 80

Tyr Leu Gln Met Asn Thr Leu Thr Ala Glu Asp Ser Ala Thr Tyr Phe

85 90 95

Cys Ala Arg Asp Leu Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val

100 105 110

Ser Ser

<210>9

<211>333

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>9

aacattatga tgacacagtc gccatcatct ctggctgtgt ctgcaggaga aaaggtcact 60

atgagctgta agtccagtca aagtgttttg tacagttcaa atcagaagaa cttcttggcc 120

tggtaccagc agaaaccagg gcagtctcct gaactgctga tctactgggc atccactagg 180

gtatctggtg tccctgatcg cttcacaggc agtggatctg ggacagattt tactcttacc 240

atcagcaatg tacaagctga agacctggca gtttattact gtcatcaata cctctccttg 300

cgcacgttcg gaggggggac caagctggag atc 333

<210>10

<211>342

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>10

gtgaaactgc aggagtctgg aggaggcttg gtacagcctg ggggttctct gagactctcc 60

tgtgcaactt ctgggttcac cttcactgat tactacatga gctgggtccg ccagcctcca 120

ggaaaggcac ttgagtggct gggttttatt agaaacaaag ctaatggtta cacaacagaa 180

tacagtgctt ctgtgaaggg tcggttcacc atctccagag attattccca aagcatcctc 240

tatcttcaaa tgaacaccct gacagctgag gacagtgcca cttatttctg tgcaagagat 300

ctggactact ggggccaagg gaccacggtc accgtctcct ca 342

Claims (11)

1. A monoclonal antibody directed against T cell immunoglobulin mucin molecule 3, said monoclonal antibody comprising a light chain and a heavy chain, said light chain comprising CDR1, CDR2 and CDR3, said CDR1 amino acid sequence is shown in SEQ ID No.1, said CDR2 amino acid sequence is shown in SEQ ID No.2, said CDR3 amino acid sequence is shown in SEQ ID No. 3; the heavy chain comprises a CDR1, a CDR2 and a CDR3, wherein the amino acid sequence of the CDR1 is shown in SEQ ID NO.4, the amino acid sequence of the CDR2 is shown in SEQ ID NO.5, and the amino acid sequence of the CDR3 is shown in SEQ ID NO. 6.

2. The monoclonal antibody of claim 1, wherein the amino acid sequence of the light chain variable region is SEQ ID No. 7and the amino acid sequence of the heavy chain variable region is SEQ ID No. 8.

3. The monoclonal antibody of claim 1, wherein the coding nucleotide sequence of the light chain variable region is SEQ ID No. 9and the coding nucleotide sequence of the heavy chain variable region is SEQ ID No. 10.

4. The monoclonal antibody of claim 1, wherein the monoclonal antibody is murine.

5. The monoclonal antibody of claim 1, wherein the monoclonal antibody is an immunoglobulin of the IgG2a heavy chain and Kappa type light chain subtypes.

6. An isolated DNA molecule encoding the variable region or full length amino acids of the heavy and/or light chain of a monoclonal antibody according to any one of claims 1-5.

7. A construct comprising the isolated DNA molecule of claim 6.

8. An expression system for monoclonal antibodies constructed by transfecting the construct of claim 7 into a host cell.

9. A method for producing a monoclonal antibody according to any one of claims 1 to 5, comprising the steps of: culturing said monoclonal antibody expression system under conditions suitable for expression of said antibody, thereby expressing said monoclonal antibody, and purifying and isolating said monoclonal antibody.

10. Use of a monoclonal antibody according to any one of claims 1-5 for the preparation of a medicament having the effect of promoting CD8⁺Use of drugs with T cell proliferation efficacy。

11. A pharmaceutical composition comprising a therapeutically effective amount of a monoclonal antibody or immunoconjugate thereof according to any one of claims 1-5.

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