CN106632675A

CN106632675A - Anti-human Tim-3 monoclonal antibody 8E11 and preparation method thereof

Info

Publication number: CN106632675A
Application number: CN201611160547.3A
Authority: CN
Inventors: 夏瑞; 王信洁
Original assignee: Changzhou Health Biotechnology Co Ltd
Current assignee: Changzhou Health Biotechnology Co Ltd
Priority date: 2016-12-15
Filing date: 2016-12-15
Publication date: 2017-05-10

Abstract

The invention relates to the technical field of biology, and particularly relates to an anti-human Tim-3 monoclonal antibody 8E11 and a preparation method thereof. The antibody contains a light chain variable region and a heavy chain variable region, wherein the light chain variable region contains an amino acid sequence shown in SEQ ID NO:1, and the heavy chain variable region contains an amino acid sequence shown in SEQ ID NO:2. The monoclonal antibody has high specificity and high potency, thereby providing a brand-new means for Tim-3 detection.

Description

A kind of anti-human Tim-3 monoclonal antibodies 8E11 and preparation method thereof

Technical field

The present invention relates to biological technical field, and in particular to a kind of anti-human Tim-3 monoclonal antibodies 8E11 and its preparation side Method.

Background technology

Tim-3 genes and Tim-3 structural proteins：Mouse Tim-3 genes are located on o.11 chromosome, and transcript total length is 1015 bases；And mankind Tim-3 genes are located on No. 5 chromosome, transcript total length is 1116 bases.Tim families are extremely Rare four members, respectively Tim-1, Tim-2, Tim-3, Tim-4.Wherein Tim-1 for hepatitis A virus acceptor, Tim- 2 can with specific expressed in Th2 cells, Tim-3 albumen be activation Th1 cell surface markers, Tim-4 matching somebody with somebody as Tim-1 Body molecule, is expressed in the DC of maturation.

Current as shown by data, mouse Tim-3 molecules are made up of 281 amino acid, and the reaction and disease related to Th1 has Close；The I type memebrane proteins that mankind Tim-3 molecules are made up of 301 amino acid, its extracellular portion includes that is rich in a cysteine Ig samples region and a mucin region, Ig samples area include 4 conservative cysteines, mucin area be rich in threonine, silk Propylhomoserin and proline.Containing about 42～77 amino acid, this is the conservative area of topnotch in mouse and human homology's thing to its intracellular region Domain.

The contact of Tim-3 genes and immune system relevant disease and function aspects：Mankind Tim families are located at 5q33.2, this Area is the area of liability of asthma.Some atopies, such as asthma and allergic rhinitis, its sensitiveness and Tim-3 genes- The contact of 574T/G pleomorphism sites is the closest.In Allergic Rhinitis, Tim-3 genes in -1516G/T, -574T/G, There is significant difference in the genotype and gene frequency in 4259G/T sites and normal person.To two lists of Tim-3 promoter regions Relation between nucleotide polymorphism site and allergic asthma is studied, it was demonstrated that Tim-3 promoter region polymorphic positions There is close association between point and anaphylactia.

Autoimmunity disease is a kind of disease related to inflammatory reaction.If inflammatory reaction can be lowered, it is possible to reduce, very To the generation for being prevention autoimmunity disease.Some acceptor molecules (such as CTLA-4), cell factor (such as TGF-β), also adjust Section property cell (such as CD4⁺CD25⁺T cell) the common immunologic balance for keeping body.Tim family proteins are one kind of inflammatory reaction Main Molecular regulator, there is provided inhibition signal, lowers inflammatory reaction, reduces autoimmunity disease and anaphylactoid generation.Fri- The expression that sancho-Kiss etc. passes through the viral infected mice Tim-3 signals of reduction, can be in macrophage and mast cell induction association Reduce expressing with stimulation molecule CD80, CD4 can also be reduced⁺T cell CTLA-4 level, has ultimately resulted in subtracting for regulatory T-cell The increase of few and cardiac inflammatory reaction, illustrates that Tim-3 signals play critically important effect in terms of regulation and control adaptive immune response.

Immune factor is one of key factor that impact tumour cell is survived in vivo and expanded.Long-Time Service immunosupress The transplant recipient of agent, due to activating a large amount of functional T cells, usually there is the danger of concurrent some malignant tumours.Typically Think, T cell immunity is the main contents of tumour immunity, if can produce in vivo thin for the specificity of tumour cell enough Cytotoxic T cells (CTL), tumour cell is difficult survival.Tim-3 is the specific surfaces mark of Th1 type cells, is not expressed in just Beginning T cell, B cell, DC and hematopoietic cell.Speculate that Tim-3 by adjusting Th1 type cell effects, and then can adjust tumour Immunity.

Tim-3/Tim-3L and Th immune responses：Part (Tim-3L) expression of Tim-3 is more much broader than Tim-3.Zhu Deng research be found that the high expression of Tim-3L in a kind of TK-1 (CD8⁺Mouse lymphoma cell) on, and be accredited as half curdling - 9 (Galectin-9) of element.Galectin-9 is a soluble sugar egg A being attached on cell membrane, can induce thymocyte and outer The CD4 in week⁺、CD8⁺T cell proliferation, plays an important role in the dynamic equilibrium and inflammatory reaction for adjusting immunocyte.Separately have Research discovery, blocks Tim-3/Galectin-9 paths, CD4⁺CD25⁺What regulatory T cells were mediated can extend alloskin The survival event resolves of skin transplanting, it was demonstrated that Tim-3L is not only expressed in CD4⁺CD25⁺On regulatory T cells, and Tim- 3/Galectin-9 paths have impact on CD4⁺CD25⁺The performance of regulatory T cells function.Galectin-9 exists in few in spleen Number CD11⁺BMDC and CDllb⁺Monocytic surface, Tim-3L may be also expressed on macrophage and APC.

Tim-3 and Tim-3L (Galectin-9) is combined so as to lower Thl type immune responses, and can adjust CD4⁺ CD25⁺Regulatory T cells.In order to understand Tim-3 and its ligand binding so as to suppress the mechanism of Th1 cytological effect functions, Kuchroo etc. in Thl, Th2 cell of differentiation and maturation plus people Galectin-9, the flow of calcium ions for noting abnormalities and quickly Thl cell deaths, and Th2 cells and Tim-3^-/-Thl cells without this effect, so as to confirm the effect of Galectin-9 according to The combination of Lai Yu and Tim-3.The exception of Tim-3/Tim-3L signal paths is there is in the disease of many Thl abnormal reactions. Autoimmune Encephalomyelitis (EAE) belong to typical Thl types immune response, Monney etc. in mouse EAE model, apply Tim-3 antibody can accelerate the outbreak of disease, increase the seriousness and the death rate of disease.WangF etc. _ research shows in allogeneic Dermatoplasty mouse gives restructuring Galectin-9, can selectively removing Tim3⁺Th1 cells, decline IFN-γ secretion, significantly Inhibition nf allograft thing rejection, increases the survival rate of allograft skin.

Tim-3/Tim-3L and tumour immunity：It is generally acknowledged that with T lymphocytes as core in tumour immunity, CD8⁺T cell Cancer cell can be specifically killed directly as effector cell, be referred to as cytotoxic T lymphocyte (CTL).It is also another kind of It is NK cells to kill oncocyte, and such cell just can spontaneously kill target cell without the need for antigen presensitization, and with cell toxic effect Should.CD4⁺T cell is less subject to concern, but they also play an important role in the tumour immunity for adjusting body.CD4⁺T Cell Thl cells secretion IL-2, IFN-γ and TNF-α etc., they can be thin directly or indirectly through activation CTL cells, NK The cell toxicant and phagocytosis of born of the same parents and macrophage is promoting cellular immunity, therefore their anti tumor immune responses to body are non- It is often important.It is mainly Thl cells in advanced tumors patient to be suppressed, thus immunotherapy of tumors will try to make its Thl/ Th2 to Thl drifts about.Tim-3 for Thl type cells specific surfaces mark, be not expressed in T cells, B cell, DC and Hematopoietic cell.And then speculate that Tim-3 can be by adjusting Thl cell effects, so as to adjust tumour immunity.

Negativity regulation and control of the Tim-3/Tim-3L in tumour immunity

The research of Klibi etc. finds：There is substantial amounts of Galectin-9 to express in the exosome of nasopharyngeal carcinoma cell secretion, Galectin-9 inserts people and expresses in exosome, then be prevented from being cut by protease.The outer core physical efficiency of nasopharyngeal carcinoma cell secretion Nasopharyngeal carcinoma CD4 of enough inducing specifics⁺The a large amount of apoptosis of T cell, this effect can be by anti-Tim-3 antibody or anti-Galectin-9 Antibody is suppressed, and so as to demonstrate this path of blocking Tim-3/Galectin-9, can mitigate nasopharyngeal carcinoma exosome and Thl is exempted from The suppression reaction of epidemic disease response, so as to maintain the GVT of T cell, improves the effect of clinical immunotherapy nasopharyngeal carcinoma. The research of Zoltan etc. finds：Tim-3 has high expression on the mast cell and MC of people, what is interesting is Galectin-9 is also expressed on mast cell, and is not expressed on tumour cell；They also have found the fertilizer that TGF-β 1 stimulated Maxicell, the rising of the expression of its Tim-3 can suppress the anti-tumor capacity of body, and this rush knurl effect possibly to rely on The combination of Galectin-9 is expressed on the Tim-3 expressed on mast cell and itself mast cell, so they think that Tim-3 is Promote the development of this tumour cell of melanoma.Geng etc. constructs the eukaryon expression plasmid psTim-3 of sTim-3, transfection The animal of the expression plasmid, its internal MC tachyauxesis, this may can suppress in vivo cell with sTim-3 Factor IL-2, IFN-γ are relevant with the generation of TNF-β；And the activity decrease of antitumor CTL cell, the infiltration pouring in tumor tissues Bar cell quantity is reduced, and illustrates that sTim-3 can significantly reduce the antineoplastic immune of T cell；It is micro- in real-time quantitative PCR monitoring tumour Also find the down regulation of gene expression of Thl cell factors in the expression conditions of environment, and the gene related to regulatory T cells Foxp-3, IL-10, TGF-β expression are then basically unchanged.It is indicated above that sTim-3 may take part in T cell mediated immunity answering The negativity regulation and control answered.

The content of the invention

In view of the situation of prior art, it is an object of the invention to provide a kind of anti-human Tim-3 monoclonal antibodies 8E11 and Its preparation method.

To achieve these goals and other related purposes, the present invention is adopted the following technical scheme that：

A first aspect of the present invention provides a kind of anti-human Tim-3 monoclonal antibodies, and the antibody contains light chain variable district And weight chain variable district.

Preferably, light chain variable district contains such as SEQ ID NO：Amino acid sequence shown in 1.

Preferably, weight chain variable district contains such as SEQ ID NO：Amino acid sequence shown in 2.

Second aspect present invention provides a kind of polynucleotides, its described anti-human Tim-3 monoclonal antibody of coding.

The polynucleotides of the present invention can be DNA form or rna form.DNA form includes cDNA, genomic DNA or people The DNA of work synthesis.DNA can be single-stranded or double-strand.

The polynucleotide sequence for encoding the monoclonal antibody can be by well known to those skilled in the art any appropriate Technology prepare.The technology sees the general description of this area, such as《Molecular Cloning:A Laboratory guide》(J. Pehanorm Brookers etc., Science Press, 1995).The including but not limited to method such as recombinant DNA technology, chemical synthesis.

In the preferred embodiment of this case, the polynucleotides for encoding said monoclonal antibody are DNA molecular, and the DNA divides Son is containing the coding monoclonal antibody light chain variable region nucleotide sequence and encodes the monoclonal antibody heavy variable region Nucleotide sequence.

Encode the monoclonal antibody light chain variable region nucleotide sequence such as SEQ ID NO：Shown in 3.Encode the Dan Ke The nucleotide sequence of grand heavy chain of antibody variable region such as SEQ ID NO：Shown in 4.

Third aspect present invention provide a kind of expression vector, its contain the polynucleotides and with the series of operations Connected expression regulation sequence.

Method well-known to those having ordinary skill in the art can be used to build the carrier.These methods include recombinant DNA technology, DNA synthetic technologys etc..The DNA for encoding the monoclonal antibody can be effectively connected on the MCS in carrier, to refer to MRNA synthesis and then expressing protein are led, or for homologous recombination.

Preferably, the carrier is prokaryotic vector or shuttle plasmid etc..

Fourth aspect present invention provides a kind of host cell, and it is converted by the carrier.

Host cell can be prokaryotic, such as bacterial cell；Or low eukaryotic, such as yeast cells；Or it is high Deng eukaryotic, such as mammalian cell.Representative example has:Escherichia coli, streptomyces；Salmonella typhimurtum, Liszt Bacterium；Fungal cell's such as yeast；Plant cell；The insect cell of fruit bat S2 or Sf9；CHO, COS.293 cell or Bowes are black Zooblast of plain oncocyte etc..

Fifth aspect present invention provides a kind of method for preparing said monoclonal antibody, and the method specifically includes following step Suddenly：

A) genes of interest fragment is obtained；

B) genes of interest is merged in fragment inserting expressioning carrier；

C) by the expression vector conversion eukaryotic host cell for obtaining；

D) eukaryotic host cell under conditions of the monoclonal antibody expression is adapted to obtained by incubation step c)；

E) the acquisition recombinant monoclonal antibodies are isolated and purified.

Preferably, in step a), the genes of interest contains SEQ ID NO：The coding monoclonal antibody shown in 3 is light Chain variable region nucleotide sequence, and SEQ ID NO：The nucleotides of the coding monoclonal antibody heavy variable region shown in 4 Sequence.

Preferably, in step b), the expression vector can be conventional prokaryotic system expression vector, preferred pNP expression Carrier.

Preferably, in step c), can be by the expression vector conversion Escherichia coli for obtaining, preferred escherichia coli DH5a；Host Cell, preferable Chinese hamster ovary celI.

Preferably, in step e), isolation and purification method is：It is straight from cells and supernatant with ProteinG affinity columns Connect and isolate and purify out said monoclonal antibody.

Sixth aspect present invention, there is provided the monoclonal antibody answering in Tim-3 detections or diagnostic kit is prepared With.

Seventh aspect present invention, there is provided a kind of Tim-3 detections or diagnostic kit, containing aforementioned monoclonal antibody.

Compared with prior art, beneficial effects of the present invention are：

The anti-human Tim-3 monoclonal antibody specificities of the present invention are high, and potency is high, relative to R＆D conventional in the market The anti-human Tim-3 antibody binding efficiencies of company and eBioscience companies are higher, and the detection for Tim-3 provides brand-new hand Section.

Description of the drawings

Fig. 1：The anti-human Tim-3 monoclonal antibodies 8E11 antibody labeling L929-Tim3 cells of the present invention, then mark sheep anti mouse PE bis- resists (eBioscience), flow cytomery result, while arranging commercial antibody and Isotype control.

Specific embodiment

The present invention relates to a kind of anti-human Tim-3 monoclonal antibodies, the antibody includes weight chain variable district and light chain variable district.Gently Contain such as SEQ ID NO chain variable region：Amino acid sequence shown in 1.Weight chain variable district contains such as SEQ ID NO：Ammonia shown in 2 Base acid sequence.

Term " monoclonal antibody (monoclonal antibody) " used herein refers to the antibody obtained from the substantially uniform colony of a class, i.e., should The single antibody included in colony is identical, in addition to the mutation of minority natural generation that may be present.Monoclonal antibody Gao Te Single antigen point is directed to different in naturely.And, (generally there is the difference for different determinants to resist from conventional polyclonal antibody preparation Body) it is different, each monoclonal antibody is for the single determinant on antigen.In addition to their specificity, monoclonal antibody Benefit also resides in them by hybridoma culture to synthesize, and will not be polluted by other immunoglobulin (Ig)s.Modifier " Dan Ke It is grand " characteristic of antibody is illustrated, it is to obtain from substantially uniform antibody population, this is not construed as needing with any special Method is producing antibody.

Term " antibody " used herein and " immunoglobulin (Ig) " are about 150000 dalton for having identical architectural feature Different four glycan albumen, it is made up of two identical light chains (L) and two identical heavy chains (H).Every light chain is common by one Valency disulfide bond is connected with heavy chain, and the disulfide bond number between the heavy chain of different Immunoglobulin Isotypes is different.Every heavy chain and The intrachain disulfide bond at light chain also regular interval.There is variable region (VH) one end of every heavy chain, is followed by multiple constant regions.Per bar By variable region (VL), the other end has constant region for one end of light chain：The constant region of light chain is relative with the first of heavy chain constant region, gently The variable region of chain is relative with the variable region of heavy chain.Special amino acid forms interface between light chain and the variable region of heavy chain.

" variable " some parts for representing variable region in antibody of term used herein are different in sequence, and it is formed Combination and specificity of the various specific antibodies to its specific antigen.However, changeability and be unevenly distributed over whole antibody can In becoming area.It is concentrated in three fragments in being referred to as complementary determining region (CDR) or hypervariable region in light chain and weight chain variable district.Can The part for becoming more conservative in area is referred to as framework area (FR).Each self-contained four FR areas in the variable region of native heavy and light chain, it Generally be in the folding configurations of p mono-, be connected by three CDR for forming ring in succession, in some cases can forming part p fold knot Structure.CDR in every chain is by FR areas firmly against the antigen binding that together form antibody together and with the CDR of another chain I, 647-669 page (1991) (referring to Kabat etc., NIH PuB, No.91-3242, are rolled up) in position, and constant region does not directly participate in antibody And the combination of antigen, but they show different effector functions, such as participate in the cytotoxicity for depending on antibody of antibody.

Monoclonal antibody can be obtained with various methods well known to those skilled in the art.For example, monoclonal antibody can use Hybridoma method is (by Kohler etc., Nature, 256:495 (1975) propose first) it is obtained, or with the recombinant DNA method (U.S. Patent No.4,816,567) it is obtained.Monoclonal antibody also can use such as Clackson etc., Nature, 352：624-628(1991) With Marks etc., J.Mol.Biol., 222：Technology described in 581-597 (1991) is separated from phage antibody library and obtained.

Present invention also offers the DNA molecular of coding anti-human Tim-3 monoclonal antibodies of the invention.In a preferably example In, the DNA molecular contains SEQ ID NO：The nucleotide sequence of the coding monoclonal antibody light chain variable district shown in 3, and SEQ ID NO：The nucleotide sequence of the coding monoclonal antibody weight chain variable district shown in 4.

In the nucleotide sequence for obtaining coding anti-human Tim-3 monoclonal antibody heavies variable region of the invention and light chain variable district Afterwards, the monoclonal antibody of the present invention generally can by the following method be prepared.

First, there is provided be connected containing the nucleotide sequence for encoding monoclonal antibody of the invention and with the series of operations Expression regulation sequence expression cut out body.

Term " expression regulation sequence " used herein is often referred to participate in the sequence of control nucleotide sequence expression.Expression is adjusted Control sequence includes promoter and the termination signal being operatively connected with Target Nucleotide Sequence.They generally contain and include nucleotides sequence Sequence needed for the appropriate translation of row." being operatively connected " refers to that some parts of linear DNA molecule can affect same linear DNA The activity of sequence other parts.For example, if promoter or enhancer increased the transcription of coded sequence, it and coded sequence It is operatively connected.

The DNA sequence dna of coding monoclonal antibody of the present invention can be obtained with conventional meanses well known to those skilled in the art. For example, can according to sequence disclosed by the invention it is artificial synthesized or with PCR methods amplification obtain encode the monoclonal antibody heavy it is variable Area and the nucleotide sequence of light chain variable district.Then, by selecting suitable digestion it is a little with various methods well known in the art These suitable expression of nucleotide sequence insertion are cut out in body, the CH for making them entrained in body is cut out in expression respectively Before coded sequence and constant region of light chain coded sequence, and make them in same frame.

It is that body is cut out in various commercially available expression well known by persons skilled in the art that body is cut out in expression used by the present invention, for example, be purchased from The expression sanction body of Qiagen and Promega companies, and commercially available expression sanction body pMG18 (《Entered according to INCP-9 plasmid sequences The instrument moral exploitation of row environmental monitoring》(DEVELOPMENT OFTOOLS FORENVIROMNENTAL MONITORING BASED ON INCP-0PLASMIDS SEQUENCES), A.Created, R.Krasowiak, M.Titok, C.M.ThomasSchool of Biological Sciences,University of Birmingham,Edgbaston,Birmingham B152TT, UK and Faculty of Biology,Dept of MicroBiology,Belarus State UniversityScoring Av.4,Minsk 220080Belarus).

Subsequently, cut out body with the expression of above-mentioned acquisition and convert suitable host cell.It is thin that " host cell " generally comprises protokaryon Born of the same parents and eukaryotic.The example of conventional prokaryotic host cell includes Escherichia coli, hay bacillus etc..Conventional eucaryon host is thin Born of the same parents include yeast cells, insect cell core mammalian cell.In the present invention, preferably host cell is Chinese hamster ovary celI.Use table There are many kinds up to the method for cutting out body transformed host cell, Transformation Program used depends on host to be transformed.By heterologous multinuclear The method that thuja acid is imported in mammalian cell is known in the art, it include the transfection that glucan circle leads, calcium phosphate precipitation, Polybrene (methylene of 1,5 one one 1,5 one phenodiazine of dimethyl 11 gathers smelly compound) mediated transfection, protoplast fusion, electricity are worn Hole, liposome mediated transfection and by the direct microinjections of DNA in karyon.In the present invention, preferably method is electroporation Method or liposome mediated-method etc..For example can be using the liposome method kit of Invitrogen companies come transfection CHO cell.

Then, under conditions of monoclonal antibody expression of the present invention is adapted to, the host cell obtained by culture conversion.Then use Conventional immunoglobulin purification step, such as albumin A-Sepharose hydroxyapatite chromatographies, gel electrophoresis, dialysis, ion are handed over Changing the routine well known to those skilled in the art such as chromatography, hydrophobic chromatography, sieve chromatography or affinity chromatography, to isolate and purify means pure Change obtains the restructuring anti-bisphenol A monoclonal antibody of the present invention.

Gained monoclonal antibody can be identified with conventional meanses.The binding specificity of monoclonal antibody can with immunoprecipitation or External tuberculation (such as radioimmunoassay Munson, Anal.Biochem., 107:220 (1980) Scatchard) Analyze to determine.

Embodiments of the present invention are illustrated below by way of specific instantiation, those skilled in the art can be by this specification Disclosed content understands easily other advantages and effect of the present invention.The present invention can also pass through concrete realities different in addition The mode of applying is carried out or applies, the every details in this specification can also based on different viewpoints with application, without departing from Various modifications and changes are carried out under the spirit of the present invention.

Before the specific embodiment of the invention is further described, it should be appreciated that protection scope of the present invention is not limited to down State specific specific embodiment；It is also understood that the term used in the embodiment of the present invention is specific concrete in order to describe Embodiment, rather than in order to limit the scope of the invention；In description of the invention and claims, unless in text Explicitly point out in addition, singulative " one ", " one " and " this " include plural form.

When embodiment provides number range, it should be appreciated that except non-invention is otherwise noted, two ends of each number range Any one numerical value can select between point and two end points.Unless otherwise defined, the present invention used in all technologies and Scientific terminology is identical with the meaning that those skilled in the art of the present technique are generally understood that.Except the concrete grammar used in embodiment, equipment, Outside material, according to those skilled in the art to the grasp of prior art and the record of the present invention, can also use and this Any method of the similar or equivalent prior art of method, equipment described in inventive embodiments, material, equipment and material come real The existing present invention.

Unless otherwise indicated, disclosed in this invention experimental technique, detection method, preparation method using this technology lead The conventional molecular biology in domain, biochemistry, chromatin Structure and analysis, analytical chemistry, cell culture, recombinant DNA technology and The routine techniques of association area.The perfect explanation in existing document of these technologies, specifically can be found in Sambrook etc. MOLECULAR CLONING：A LABORATORY MANUAL, Second edition, Cold Spring Harbor Laboratory Press, 1989and Third edition, 2001；Ausubel etc., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley＆Sons, New York, 1987and periodic updates；the Series METHODS IN ENZYMOLOGY, Academic Press, San Diego；Wolffe, CHROMATIN STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998；METHODS IN ENZYMOLOGY, Vol.304, Chromatin (P.M.Wassarman and A.P.Wolffe, eds.), Academic Press, San Diego, 1999；With METHODS IN MOLECULAR BIOLOGY, Vol.119, Chromatin Protocols (P.B.Becker, ed.) Humana Press, Totowa, 1999 etc..

The preparation of anti-human Tim-3 monoclonal antibodies 8E11 of embodiment 1 and acquisition

First, animal immune

With the recombined human Tim-3 albumen (R＆DSystems, 2365-TM) of purifying for immunogene, immune 6～8 week old BALB/c mouse, immunity 1 time per 2 weeks, altogether immunity 3 times are first 4 days in merging, again with booster immunization.

2nd, the preparation of myeloma cell

The front murine myeloma cell Ag8 of recovery in 10 days of fusion, using the DMEM medium cultures containing 10% hyclone, treats Cell growth is vigorous, form good (perfectly round, bright, size is homogeneous), and cell viability can more than 95% (trypan blu e dyeing) To merge.Cell concentration is adjusted to 3 × 10 by the front 24～36h fresh cultures of fusion⁵/mL。

3rd, the foundation of hybridoma cell strain

DMEM basal mediums, PEG solution are placed in pre-temperature in 37 DEG C of water-baths by experiment before starting.Collect 2 × 10⁷Individual growth Good Ag8 cells, with 1 × 10⁸Individual spleen cell is mixed in 50ml centrifuge tubes, and with plasma-free DMEM medium two are washed Time, most supernatant is abandoned after 1000rpm centrifugation 5min, with finger attack ttom of pipe, make two kinds of cells fully be mixed into pasty state.By plastic tube In being placed in 37 DEG C of thermos cups, the 50% of 1ml 37 DEG C of pre-temperatures of Jing PEG solution is drawn, tip straw is gently inserted cell suspension In, at the uniform velocity add in 1min, and side edged is gently mixed, and then stands 90s in 37 DEG C of water-baths.Then in 3 1min The serum-free DMEM of 37 DEG C of pre-temperatures of 1ml, 2ml, 3ml is equally at the uniform velocity separately added into, the serum-free DMEM of 37 DEG C of pre-temperatures is eventually adding Make liquid in pipe final volume be 40ml, the static 5min of room temperature, 800rpm centrifugation 10min, abandon supernatant, sedimentation cell is gently placed in 100ml is containing in 2%HAT, the DMEM culture mediums of 10% hyclone.It is added dropwise after mixing in 96 well culture plates containing feeder cells In, 100 μ l/ holes, 37 DEG C, 5%CO₂Culture, the later half amounts of 3～4d are changed liquid, use HT culture mediums after 10d instead, and conversion contains 10% after 2 weeks The DMEM culture mediums of hyclone.

4th, the screening of positive hybridoma cell

When hybridoma cell clone is covered with 1/3-1/4 culture holes (culture medium color is in yellow), you can draw supernatant and use Indirect immunofluorescence labeling and flow cytometry are screened.It is thin as positive antibody screening with L929-Tim-3 gene transfecting cells Born of the same parents, L929 is negative antibody screening cell, while the anti-human TIM-3 monoclonal antibodies (R＆DClone#344823) marked with commercialization PE For positive control.Draw hybridoma culture supernatant to be screened with indirect immunofluorescence labeling method and flow cytometer showed, obtain positive Change liquid after clone in good time and carry out secondary screening as stated above in time.

5th, the colonized culture of positive hybridoma cell

After using cell accurate counting of the limiting dilution assay by positive hole, with HAT Selective agar mediums gradient dilution be 10/ Averagely contain 1 cell, 37 DEG C, 5%CO in 96 well culture plates, per hole in ml cells, the μ l/ holes of cell suspension 100 after dilution₂ Culture.Culture medium is changed in good time according to the growth conditions of cell and is carried out by the screening technique of the positive colony set up in time multiple Sieve.The hybridoma that single clonal growth, form are good, antibody titer is high is selected to continue to be subcloned, until antibody-secreting is positive Property rate be more than 98%；Amplification Culture and in time liquid nitrogen cryopreservation.

Mouse-anti-human T im-3 monoclonal antibodies 8E11 of acquisition are carried out into variable region sequencing.

Total serum IgE is separated from hybridoma cell strain, cDNA storehouses are transcribed into.PCR amplification codings heavy chain of antibody and light chain variable Area DNA.PCR primer to be run and send sequencing after recovery purifying after glue.

Learn, anti-human Tim-3 monoclonal antibodies 8E11 of the present invention, the amino acid sequence that light chain variable district has such as SEQ ID NO：Shown in 1, specially：

LDIVLTQSPSYLAAPPGETITINCRASKSISKYLAWYQEKPGKTNKLLIYSGSTLQSGIPSRFSGSGSGTDFTLTIS NLEPEDFAMYYRQQHNEYPWTFGGGTKLEIKRI。

The amino acid sequence that weight chain variable district has such as SEQ ID NO：Shown in 2, specially：

HRVESGPGLVQPSQSLSITCTVSGFSLTTYGVHWVRQSPGKGLEWLGVIWSGGSTDYNAAFLSRLNITMDNSKSQVF FKMNSLQADDTAIYYCARDLYHYAMDYWGQGTSITVSSKSQSFPNVFPLISCDSPLSDKNLV。

Encode the monoclonal antibody light chain variable region nucleotide sequence such as SEQ ID NO：Shown in 3, specially：

CTCGATATTGTGTTGACACAGTCTCCATCTTATCTTGCTGCACCTCCTGGAGAAACCATTACTATTAATTGCAGGGC AAGTAAGAGCATTAGCAAATATTTAGCCTGGTATCAAGAGAAACCTGGGAAAACTAATAAACTTCTTATATACTCTG GATCCACTTTGCAATCTGGAATTCCATCAAGGTTCAGTGGCAGTGGATCTGGTACAGATTTCACTCTCACCATCAGT AACCTGGAGCCTGAAGATTTTGCAATGTATTACCGTCAACAGCATAATGAATACCCGTGGACGTTCGGTGGAGGCAC CAAGCTGGAAATAAAACGTATA。

Encode the nucleotide sequence such as SEQ ID NO of the monoclonal antibody heavy variable region：Shown in 4, specially：

GCTGCTCGAGCGGCCGCCAGTGTGATGGATATCTGCAGAATTCGGCTTCAGGTGCACCGGGTGGAGTCAGGACCTGG CCTAGTGCAGCCCTCACAGAGCCTGTCCATCACCTGCACAGTCTCTGGTTTCTCATTAACTAcCTATGGTGTACACT GGGTTCGCCAGTCTCCAGGAAAGGGTCTGGAGTGGCTGGGAGTGATATGGAGTGGTGGAAGCACAGACTATAATGCA GCTTTCCTATCCAGACTGAACATCACCATGGACAATTCCAAGAGCCAAGTTTTCTTTAAAATGAACAGCCTGCAAGC TGATGACACAGCCATATATTACTGTGCCAGAGACCTTTACCACTATGCTATGGACTACTGGGGTCAAGGAACCTCAA TCACCGTCTCCTCAAAGAGTCAaTCCTTCCCAAATGTCTTCCCCCTCaTCTCCTGCGACAGCCCCCTGTCTGATAAG AATCTGGTGGC。

The potency evaluation of the anti-human Tim-3 monoclonal antibodies of embodiment 2

Culture L929-Tim3 cells, are transferred to 96 orifice plates, per hole 1 × 10⁵Cell be suspended in 100 μ l containing 1%FBS's In PBS.The antibody of the μ g of equal amount 0.2, including anti-human Tim-3 monoclonal antibodies 8E11, homotype prepared by embodiment 1 are added per hole Control and the anti-human Tim-3 antibody of R＆D and eBioscience companies.It is incubated 20 minutes under the conditions of 4 DEG C of lucifuges, is then centrifuged for Supernatant discarded, in being resuspended in the PBS containing 1%FBS, Flow cytometry.

As a result as shown in figure 1, compared with R＆D companies and antibody known to eBioscience companies, the present invention's is anti-human Tim-3 monoclonal antibodies, can be with the Percentage bound of higher efficiency under same concentrations.

Embodiment above is, in order to illustrate embodiment disclosed by the invention, can not to be interpreted as the limit to the present invention System.Additionally, method, the change of composition in various modifications listed herein and invention, without departing from the scope of the present invention With for those skilled in the art be obvious on the premise of spirit.Although having combined the various concrete of the present invention Preferred embodiment has carried out specific description to the present invention, it is to be understood that, the present invention should not be limited only to these specific embodiments. In fact, various modifications obvious for those skilled in the art as above all should include obtaining invention Within the scope of the invention.

SEQUENCE LISTING

<110>Changzhou Ge Lukang biological medicines Science and Technology Ltd.

<120>A kind of anti-human Tim-3 monoclonal antibodies 8E11 and preparation method thereof

<130> 164867

<160> 4

<170> PatentIn version 3.3

<210> 1

<211> 110

<212> PRT

<213> Artificial

<220>

<223>The amino acid sequence that light chain variable district has

<400> 1

Leu Asp Ile Val Leu Thr Gln Ser Pro Ser Tyr Leu Ala Ala Pro Pro

1 5 10 15

Gly Glu Thr Ile Thr Ile Asn Cys Arg Ala Ser Lys Ser Ile Ser Lys

20 25 30

Tyr Leu Ala Trp Tyr Gln Glu Lys Pro Gly Lys Thr Asn Lys Leu Leu

35 40 45

Ile Tyr Ser Gly Ser Thr Leu Gln Ser Gly Ile Pro Ser Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Leu Glu

65 70 75 80

Pro Glu Asp Phe Ala Met Tyr Tyr Arg Gln Gln His Asn Glu Tyr Pro

85 90 95

Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ile

100 105 110

<210> 2

<211> 139

<212> PRT

<213> Artificial

<220>

<223>The amino acid sequence that weight chain variable district has

<400> 2

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

1 5 10 15

Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Thr Tyr Gly Val

20 25 30

His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu Gly Val

35 40 45

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

50 55 60

Leu Asn Ile Thr Met Asp Asn Ser Lys Ser Gln Val Phe Phe Lys Met

65 70 75 80

Asn Ser Leu Gln Ala Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Arg Asp

85 90 95

Leu Tyr His Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Ile Thr

100 105 110

Val Ser Ser Lys Ser Gln Ser Phe Pro Asn Val Phe Pro Leu Ile Ser

115 120 125

Cys Asp Ser Pro Leu Ser Asp Lys Asn Leu Val

130 135

<210> 3

<211> 330

<212> DNA

<213> Artificial

<220>

<223>Encode the monoclonal antibody light chain variable region nucleotide sequence

<400> 3

ctcgatattg tgttgacaca gtctccatct tatcttgctg cacctcctgg agaaaccatt 60

actattaatt gcagggcaag taagagcatt agcaaatatt tagcctggta tcaagagaaa 120

cctgggaaaa ctaataaact tcttatatac tctggatcca ctttgcaatc tggaattcca 180

tcaaggttca gtggcagtgg atctggtaca gatttcactc tcaccatcag taacctggag 240

cctgaagatt ttgcaatgta ttaccgtcaa cagcataatg aatacccgtg gacgttcggt 300

ggaggcacca agctggaaat aaaacgtata 330

<210> 4

<211> 473

<212> DNA

<213> Artificial

<220>

<223>Encode the nucleotide sequence of the monoclonal antibody heavy variable region

<400> 4

gctgctcgag cggccgccag tgtgatggat atctgcagaa ttcggcttca ggtgcaccgg 60

gtggagtcag gacctggcct agtgcagccc tcacagagcc tgtccatcac ctgcacagtc 120

tctggtttct cattaactac ctatggtgta cactgggttc gccagtctcc aggaaagggt 180

ctggagtggc tgggagtgat atggagtggt ggaagcacag actataatgc agctttccta 240

tccagactga acatcaccat ggacaattcc aagagccaag ttttctttaa aatgaacagc 300

ctgcaagctg atgacacagc catatattac tgtgccagag acctttacca ctatgctatg 360

gactactggg gtcaaggaac ctcaatcacc gtctcctcaa agagtcaatc cttcccaaat 420

gtcttccccc tcatctcctg cgacagcccc ctgtctgata agaatctggt ggc 473

Claims

1. a kind of anti-human Tim-3 monoclonal antibodies, the antibody contains light chain variable district and weight chain variable district.

2. monoclonal antibody according to claim 1, it is characterised in that in the monoclonal antibody, light chain variable district contains Just like SEQ ID NO：Amino acid sequence shown in 1.

3. monoclonal antibody according to claim 1, it is characterised in that in the monoclonal antibody, weight chain variable district contains Just like SEQ ID NO：Amino acid sequence shown in 2.

4. a kind of polynucleotides, its coding monoclonal antibody as described in any one of claims 1 to 3.

5. a kind of expression vector, it contains polynucleotides as claimed in claim 4.

6. a kind of host cell, its carrier as described in requiring 5 such as power is converted.

7. a kind of preparation method of the monoclonal antibody as described in any one of claims 1 to 3, comprises the steps：

A) genes of interest fragment is obtained；

B) genes of interest is merged in fragment inserting expressioning carrier；

C) by the expression vector conversion eukaryotic host cell for obtaining；

E) the acquisition recombinant monoclonal antibodies are isolated and purified.

8. method according to claim 7, it is characterised in that in step a), the genes of interest contains such as SEQ ID NO：The coding monoclonal antibody light chain variable region nucleotide sequence shown in 3, and such as SEQ ID NO：Coding shown in 4 The nucleotide sequence of the monoclonal antibody heavy variable region.

9. according to any one of claims 1 to 3 monoclonal antibody prepare Tim-3 detection or diagnostic kit in should With.

10. in a kind of Tim-3 detection or diagnostic kit, it is characterised in that containing as described in any one of claims 1 to 3 singly Clonal antibody.