CN102492038B - Anti-human Tim-3 neutralized monoclonal antibody L3D and application thereof - Google Patents

Abstract

The invention discloses an anti-human Tim-3 neutralizing monoclonal antibody L3D. The present invention clones the antibody light and heavy chain variable region genes from the prepared anti-human Tim-3 neutralizing monoclonal antibody L3D hybridoma cells, and the obtained light chain and heavy chain variable region genes can encode the variable region of the monoclonal antibody district. Based on the light and heavy chain variable region genes of the above-mentioned monoclonal antibodies, a variety of small molecule genetically engineered antibodies can be constructed and expressed. Based on the polypeptides or proteins encoded by the above-mentioned genes, a variety of biologically active molecules can be cross-linked to prepare Tim- 3 Expression level detection reagents, diagnosis and treatment of diseases caused by abnormal expression of Tim-3.

Description

Neutralizing monoclonal antibody L3D against human Tim-3 and use thereof

technical field

The present invention relates to a monoclonal antibody, in particular to an anti-human Tim-3 neutralizing monoclonal antibody L3D, and also relates to the use of the monoclonal antibody in preparing Tim-3 detection reagents and diagnosing and treating Tim-3 high expression applications that lead to disease.

Background technique

Tim-3 is structurally a member of the T cell Immunoglobulin domain and Mucin domain protein (T cell Immunoglobulin domain and Mucin domain protein, Tim) family. Because the Tim family is widely involved in the immune regulation process of the body, its function is being paid more and more attention by people. Tim-3 is specifically expressed on the surface of activated Th1 and Th17 effector cells, but not on Th2 cells. It is now known that galactose-binding protein-9 (Galactin-9, Gal-9) is the natural ligand of Tim-3, and this molecule is widely expressed in the peripheral immune system. Gal-9 and Tim-3 molecules on Th1 and Th17 cells Specific binding can trigger the latter's apoptosis, downregulate the immune response, and induce immune tolerance. At present, studies have confirmed that Tim-3 molecules are widely involved in immune response processes such as autoimmune diseases, transplant rejection, and anti-infection mainly by regulating the functions of different CD4+T cell subsets [Anderson DE. TIM-3 as a therapeutic target in human inflammatory diseases .Expert Opin The Targets.2007;11(8):1005-9.Anderson AC, Anderson DE.TIM-3 inautoimmunity.Curr Opin Immunol.2006;18(6):665-9.Degauque N, Mariat C, Kenny J , et al.Regulation of T-cell immunity by T-cell immunoglobulin and mucindomain proteins.Transplantation 2007; 84:S12-16.Zhu C, Anderson AC, Schubart A, et al.The Tim-3ligand galectin-9 negatively regulates T helper type 1 immunity. Nat Immunol 2005;6:1245-1252.].

Current studies have confirmed that the abnormal expression of Tim-3 is closely related to many diseases. For example, it has been found that the expression of Tim-3 on T cells in HIV, HCV-infected patients and some tumor patients increases, because Tim-3 can mediate The apoptosis of T effector cells transmits negative regulatory signals, which can lead to paralysis of the body's immune function. Any factor that blocks the combination of Tim-3/Gal-9 can make Th1 and Th17 cells avoid death, enhance the activity of T effector cells, and restore immune function [Kassu A, Marcus RA, D'Souza MB, et al.Regulation ofVirus-Specific CD4+T Cell Function by Multiple Costimulatory Receptors duringChronic HIV Infection.Immunol.2010；185(5)：3007-18.Huang X，et al.Lymphomaendothelium preferentially expresses Tim-3 and facilitates the progression oflymphoma by mediating immune evasion .Exp Med.2010;207(3):505.N.Castelblanco,V.Kuchroo,D.R.Gretch,and H.R.Rosen.2009.Negative immuneregulator Tim-3 is overexpressed on T cells in hepatitis C virus infection and its blockade rescues dysCD + and CD8+ T cells. Virol. 83:9122-9130.].

On the one hand, in some autoimmune diseases such as systemic lupus erythematosus (SLE), asthma and other diseases, due to the increase of Gal-9 or Tim-3 expression, the function of Th1 cells is inhibited, thereby breaking the immune balance in the body, The activity of pathological Th2 cells is enhanced, leading to the pathogenesis of the disease. In this case, any factor that blocks the combination of Tim-3/Gal-9 can help restore the immune balance in the body and alleviate the disease process. For example, injecting anti-Tim-3 antibody or recombinant Tim-3 fusion protein into asthma model animals can block Tim-3/Gal-9 binding, enhance Th1 cell activity, correct asthma symptoms mediated by Th2 cells, and restore Th1 in vivo /Th2 cell balance [Hu WK, Lu XX, Yang S et al.Expression of theTh1-specific cell-surface protein Tim-3 increases in a murine model of atopic asthma.Asthma.2009;46(9):872.Pan HF , Zhang N, Li WX, Tao JH, Ye DQ. TIM-3 as a new therapeutic target in systemic lupus erythematosus. Mol Biol Rep.2010; 37(1): 395-8. Wang Y, Meng J, Wang X, Expression of human TIM-1 and TIM-3 on lymphocytes from systemic lupus erythematosus patients. Scand Immunol. 2008; 67(1): 63-70. Kearley J, McMillan SJ, Lloyd CM. Th2-driven, allergen-induced airway inflammation is reduced after treatment with anti-Tim-3 antibody in vivo. Exp Med2007;204:1289; Fukushima A, Sumi T, Fukuda K, Kumagai N, et al.Antibodies to T-cell Ig and mucin domain-containing proteins(Tim)-1 and -3 suppress the induction and progression of murine allergic conjunctivitis. Biochem Biophys Res Commun. 2007; 353(1): 211.]. On the other hand, in some autoimmune diseases such as inflammatory bowel disease and type I diabetes models, studies have found that blocking the activity of Tim-3 enhances the function of Th1 effector cells in vivo, further aggravating autoimmune damage, which proves again Tim-3 plays an important role in the maintenance of immune balance in the body [Sanchez-Fueyo A, Tian J, Picarella D, et al. Tim-3 inhibits T helpertype 1-mediated auto-and alloimmune responses and promotes immunological tolerance. Nat Immunol 2003; 4 : 1093-1101. Li X, et al. Clinical Immunol. 2010, 134: 169-177.].

The above research data show that the Tim-3 pathway has an important immune regulatory function, and its abnormal expression is closely related to the occurrence and development of various diseases. Although studies have shown that neutralizing antibodies to Tim-3 also play a good role in intervening in some disease models, there are currently no Tim-3 antibodies on the market at home and abroad, so the establishment and development of antibody-based antibodies with independent intellectual property rights It has important practical significance for the intervention of various related diseases.

Contents of the invention

The invention discloses an anti-human Tim-3 monoclonal antibody L3D. The monoclonal antibody includes a light chain and a heavy chain, and its amino acid variable region sequences are respectively as SEQ ID NO: 1 and SEQ ID NO: in the sequence table. 2, its coding genes are respectively shown in SEQ ID NO: 3 and SEQ ID NO: 4 in the sequence listing.

The invention also discloses the application of anti-human Tim-3 neutralizing monoclonal antibody L3D, including the application in preparation of Tim-3 detection reagent, diagnosis and treatment of diseases caused by high expression of Tim-3.

The diseases caused by the high expression of Tim-3 in the present invention include HIV and HCV infection, tumors, autoimmune diseases such as systemic lupus erythematosus (SLE), asthma and the like.

The amino acid sequences of the complementary determining regions CDR1, CDR2, and CDR3 of the variable region of the light chain protein molecule of the monoclonal antibody of the present invention are respectively shown in the sequence listing as SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7. The amino acid sequences of the complementary determining regions CDR4, CDR5, and CDR6 of the variable region of the heavy chain protein molecule of the monoclonal antibody are respectively shown in SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10 in the sequence listing.

The present invention also discloses a preparation method of the above-mentioned monoclonal antibody L3D, which mainly includes the following:

1. Construction of anti-human Tim-3 monoclonal antibody hybridoma cell line

First, prokaryotic expression of human Tim-3 protein, Balb/c mice were immunized, and cell fusion was carried out by conventional methods. Positive cell clones were screened by indirect ELISA and subcloned repeatedly until all hybridoma cell culture supernatants were detected as 100% positive. The chromosome karyotype of hybridoma L3D was analyzed, and the average number of chromosomes of hybridoma L3D was 106. The biphasic agar diffusion test proved that the immunoglobulin subtype secreted by L3D hybridoma was IgG2a. Figure 1 shows that monoclonal antibody L3D can specifically bind to human Tim-3 molecule.

2. Screening and identification of anti-human Tim-3 monoclonal antibody hybridoma cell lines

Using FACS method, the binding experiment of human Tim-3 antibody to Tim-3 molecules on human U937 cells was carried out. The results showed that L3D can bind the Tim-3 molecule on U937, and exhibited cross-reactivity with mouse Tim-3 (Fig. 2). At the same time, the neutralizing activity of Tim-3 antibody was verified by lymphocyte apoptosis experiment. The results showed that L3D significantly inhibited Gal-9-induced apoptosis of human THP1 cells (Figure 3)

3. In vivo intervention effect of monoclonal antibody L3D on autoimmune injury disease model

The ascites of Balb/c mouse hybridoma cells was purified by an affinity column, measured by an ultraviolet spectrophotometer, and the protein content was calculated. C57BL/6 mice were used to establish a sepsis model and an animal model of autoimmune inflammatory bowel disease. After injecting mice with L3D antibody or isotype control antibody, observe the changes in mouse body weight or survival rate, and analyze the biological activity of monoclonal antibody L3D in vivo. The results showed that L3D antibody significantly changed the survival rate of sepsis mice (Fig. 4), and affected the degree of inflammation in inflammatory bowel disease mice (Fig. 5). The above results suggested that anti-human Tim-3 monoclonal antibody L3D in Has good neutralizing activity in vivo.

4. Fishing for L3D light and heavy chain genes of hybridoma cells

The neutralizing monoclonal antibody L3D cell RNA was extracted, and the light and heavy chain genes of the antibody were captured by RT-PCR with two pairs of specific primers. Connect into the vector by conventional method, transform competent bacteria, pick a single colony after culture, extract the plasmid for PCR identification, and then conduct DNA sequencing analysis.

Through the above steps, a vector containing human Tim-3 neutralizing antibody L3D light and heavy chain genes was constructed. After sequence analysis and comparison, the coding sequences were mouse immunoglobulin light and heavy chain genes, and their amino acid sequences were SEQ ID NO: 1 and SEQ ID NO: 2.

5. Determination of the gene sequence and amino acid sequence of the light and heavy chain variable regions of the monoclonal antibody L3D

Use www.expasy.org online software to translate the nucleotide sequence of the light and heavy chain variable regions of the human Tim-3 neutralizing monoclonal antibody L3D into its encoded amino acid sequence, and the light and heavy chains of the monoclonal antibody L3D The amino acid sequence of the variable region is shown in SEQ ID NO: 1 and SEQ ID NO: 2 in the sequence listing. According to the Kabat database, the amino acid sequences of the complementarity determining regions CDR1, CDR2 and CDR3 in the light chain variable region sequence are respectively shown in SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7 in the sequence listing. The amino acid sequences of the complementarity determining regions CDR4, CDR5 and CDR6 in the heavy chain variable region sequence are respectively shown in SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10 in the sequence listing.

The present invention uses a set of designed primers to successfully clone antibody light and heavy chain variable region genes from cultured human Tim-3 neutralizing monoclonal antibody L3D tumor cells. The resulting light and heavy chain variable region genes encode the correct mouse antibody variable regions. Figure 6 shows the molecular weights of anti-human Tim-3 antibodies. The monoclonal antibody of the present invention is based on the human Tim-3 neutralizing monoclonal antibody L3D light and heavy chain variable region genes that have been cloned above, and can construct and express a variety of small molecule genetically engineered antibodies, such as single chain antibodies, monoclonal antibodies, Domain antibodies, chimeric antibodies, Fab antibodies, antibody fusion proteins, etc.; based on the polypeptides or proteins encoded by the above genes, various biologically active molecules can be cross-linked to prepare for the detection, diagnosis and treatment of Tim-3 expression levels. 3 Drugs for the diagnosis or treatment of diseases caused by high expression.

Description of drawings

Figure 1 The ELISA method to screen and identify the specific binding activity of human Tim-3 neutralizing monoclonal antibody L3D to antigen. The control antigen was thioephrin (Trx).

Figure 2 The binding activity of monoclonal antibody L3D to Tim-3 on human U937 cells and the cross-binding activity to Tim-3 on mouse RAW264.7 cells were detected by flow cytometry (FACS). A: the binding activity of monoclonal antibody L3D to Tim-3 on human U937 cells; B: the cross-binding activity of monoclonal antibody L3D to Tim-3 on mouse RAW264.7 cells.

Fig. 3 In vitro neutralizing activity analysis of monoclonal antibody L3D. Gal-9 protein can induce the apoptosis of THP1 through Tim-3 on the cells. In this figure, PI and Annexcin-V were used for apoptotic cell staining experiments to observe the neutralization resistance of Tim-3 antibody to Gal-9-induced apoptosis. break effect.

Fig. 4 Effect of monoclonal antibody L3D on the course of sepsis. Establish a sepsis model in C57BL/6 mice, give L3D or isotype control antibody at the same time, then observe the survival rate of the animals (A), and analyze the inflammatory mediator IL-6 after antibody intervention by ELISA method (B), Expression changes of IL-1beta (C).

Figure 5 Effect of monoclonal antibody L3D on the course of inflammatory bowel disease in mice. The inflammatory bowel disease model was established in C57BL/6 mice, and L3D or isotype control antibody was given at the same time, and then the body weight changes of the mice and the changes of cytokines IL-17 and IFN-g were observed. A: Changes in body weight of mice over time; B: Changes in cytokine IL-17A; C: Changes in cytokine IFN-g.

Fig. 6 Protein electrophoresis pattern of monoclonal antibody L3D. Each lane is A: non-reducing electrophoresis, B: reducing electrophoresis (showing heavy chain and light chain respectively), and C: molecular weight marker.

Detailed ways

The content of the present invention can be understood more easily by referring to the following examples, which are only for further illustrating the present invention, and are not meant to limit the scope of the present invention.

Embodiment 1 Construction of anti-human Tim-3 monoclonal antibody hybridoma cell line

1. Materials

Freund's complete adjuvant and Freund's incomplete adjuvant, chromogenic reagent TMB: product of Sigma; 20% fetal bovine serum: product of Beijing Yuanheng Shengma Biotechnology Institute; serum-free RPMI 1640: product of Gibco; SP2/ 0 cells: imported from ATCC and preserved by the Institute of Basic Medical Sciences of the Academy of Military Medical Sciences; Balb/c and C57BL/6 mice: provided by the Experimental Animal Center of the Academy of Military Medical Sciences; other reagents were purchased from the market.

2. Methods and Results

(1) Immunization of Balb/c mice. Six 4- to 6-week-old female Balb/c mice were selected and immunized subcutaneously with 100 μg ricin in the groin. The first injection was added with Freund's complete adjuvant, and the second injection was added with Freund's incomplete adjuvant. The immunization was once every 3 weeks. A total of 3 times of immunization. Blood was collected from the tail vein after the third immunization, and the antibody production was detected by indirect ELISA. Three days before the fusion, 100 μg ricin was administered intraperitoneally for a booster immunization, and fusion was performed on the third day.

(2) Cell fusion. The immunized mice were sacrificed by decapitating the eyeballs, and the splenocytes of the mice were aseptically removed, and cell fusion was carried out according to conventional methods. The specific method is as follows: ①The immunized mice were sacrificed by taking off the eyeballs and bleeding, soaked in 75% alcohol for 3 minutes, aseptically removed the spleen, ground the single cell suspension with a 200-mesh steel mesh, washed twice with serum-free RPMI 1640 and counted ; ② Collect SP2/0 cells in the logarithmic growth phase, wash twice with serum-free RPMI1640 and count; ③ Mix the two kinds of cells according to the ratio of SP2/0 cells: splenocytes = 1:5, and wash once with RPMI 1640 , discard the supernatant, and gently break up the cells; ④ slowly add 1ml of 50% PEG ( _MW : 1500) solution within 1min, and place in a 37°C water bath for 1min; , 5ml, 10ml, 10ml; ⑥Centrifuge at 800r/min for 7min, discard the supernatant, and suspend the cells as lightly as possible; ⑦Add HAT(Sigma)-RPMI 1640 culture medium containing 20% FCS, and adjust the cell concentration to 2×10 ⁶ /ml, mixed evenly and added dropwise to a 96-well culture plate (Gibco) covered with trophoblast cells (1×10 ⁴ cells/well), 100 μl/well, and cultured in a 5% CO ₂ incubator at 37°C.

(3) Anti-human Tim-3 monoclonal antibody hybridoma cells were screened by indirect ELISA. The ELISA plate was coated with 10 μg/ml recombinant human Tim-3 (rhTim-3), overnight at 4° C. and blocked. Add the culture supernatant of the cells to be tested (37°C for 1h, wash the plate 4 times with PBST), and 50 μl of HRP-GAM diluted 1:500 (37°C for 45min, wash the plate 4 times with PBST). After color development with TMB substrate, the OD value was measured at a wavelength of 450 nm.

(4) Cloning of hybridoma cells. Positive cell clones were screened by indirect ELISA and subcloned repeatedly until all hybridoma cell culture supernatants were detected to be 100% positive. Cloning of hybridoma cells using limiting dilution method: ①Prepare trophoblasts on the day of cloning or one day before: kill Kunming mice by neck dislocation, sterilize the skin by soaking in 75% alcohol, peel off the abdominal skin aseptically, and extract 5ml of 1640 culture with a syringe The liquid was injected into the peritoneal cavity of the mouse, and after repeated washing, the peritoneal washing liquid was sucked out, diluted with 1640 culture medium with 20% fetal bovine serum, and dropped into a 96-well plate, about 0.1ml per well. ② Take a small amount of hybridoma cells to be cloned and transfer to another sterile test tube, and count them accurately. ③Limited dilution method for subcloning. ④ Place the culture plate in a 5% CO ₂ incubator at 37°C for culture, and cell clones can be observed under a microscope after about 5 days. Change the medium at the right time, test, take the positive monoclonal cell line for expansion culture, and freeze the cell line in time.

(5) Determination of immunoglobulin subtypes of hybridoma cells. Using goat anti-mouse IgG1, IgG2a, IgG2b and IgG3, the concentrated culture supernatant of hybridoma cells was used for biphasic agar diffusion experiments. The results showed that the culture supernatant of hybridoma cells could only form binding bands with goat anti-mouse IgG2a antibodies. It was proved that the immunoglobulin subtype secreted by L3D hybridoma cells was IgG2a.

Through the above steps, human Tim-3 monoclonal antibody L3D was screened.

Example 2 Screening and Identification of Hybridoma Cell Lines with Human Tim-3 Neutralizing Monoclonal Antibody

1. Materials

Same as embodiment one.

2. Methods and Results

(1) Use ELISA method to screen and identify the specific binding activity of human Tim-3 neutralizing monoclonal antibody L3D to antigen: respectively coat recombinant human Tim-3 protein and thioepyn (Trx) control protein, add different Diluted anti-human Tim-3 antibody and HRP-labeled anti-mouse IgG antibody were used to detect the antigen-binding activity and specificity of L3D by indirect ELISA. The results showed that L3D can specifically bind to human Tim-3 molecule with high titer.

(2) Flow cytometry (FACS) was used to detect the binding of monoclonal antibody L3D to Tim-3 on human U937 cells and the cross-binding activity to Tim-3 on mouse RAW264.7 cells. Experiments were carried out by conventional FACS method. The results showed that L3D can specifically bind to human Tim-3 molecules, and also has a certain cross-binding activity with mouse Tim-3 molecules.

(3) is the neutralizing activity analysis of the monoclonal antibody L3D. Add recombinant Gal-9 to human THP1 cell culture to induce apoptosis of the latter, add different concentrations of L3D antibody to the above system respectively, and after culturing for 24-48 hours, use flow cytometry to detect the apoptosis ratio of THP1 cells . The results showed that Gal-9 could induce the apoptosis of human THP1 cells (Annexcin-V ⁺ PI ^- cells), and the apoptosis rate induced by Gal-9 decreased significantly after adding L3D to the above system, suggesting that the monoclonal antibody L3D had a better effect in vitro. Good neutralizing activity.

Through the above steps, a human Tim-3 galactose binding site neutralizing monoclonal antibody named L3D was obtained. In vitro experiments, L3D can neutralize the apoptosis induced by GAL-9/Tim-3 , the neutralizing monoclonal antibody L3D also has a certain cross-reactivity with mouse Tim-3.

Example 3 Fishing of Human Tim-3 Neutralizing Monoclonal Antibody Hybridoma L3D Light and Heavy Chain Genes

1. Materials

Primers: PHs1, see SEQ ID NO: 11 in the sequence listing; PHs2, see SEQ ID NO: 12 in the sequence listing; PHa1, see SEQ ID NO: 13 in the sequence listing; PLs1, see SEQ ID NO: 14 in the sequence listing, PLs2 See SEQ ID NO: 15 in the sequence listing; PLA1, see SEQ ID NO: 16 in the sequence listing; DNA fragment purification kit: product of OMEGA Biotechnology Company; T4DNA ligase: product of New England Biolabs; vector PGEM Teasy: product of Promega Company; competent bacteria JM109: purchased from Promega Company. All the other are with embodiment two.

2. Method results

(1) Take 5×(10 ⁶ -10 ⁷ ) neutralizing monoclonal antibody L3D cells in the logarithmic growth phase, centrifuge to remove the supernatant, and pop the cells evenly. Add 1ml TRIzol (Invitrogen) and pipette repeatedly to fully lyse the cells. After shaking for 5 minutes, add 0.2ml chloroform, shake for 15 seconds, place at room temperature for 2-3 minutes, centrifuge at 12000r/min at 2°C-8°C for 15 minutes, and take the supernatant in In another new tube, add 500 μl of isopropanol and mix well, then place at room temperature for 10 minutes, and centrifuge at 12000 r/min for 10 minutes at 2°C to 8°C. The precipitate was washed with 75% ethanol, and after drying, the precipitate was dissolved with 20 μl RNase-free deionized water.

(2) Take a solution containing 1 μg of total RNA, add 4 μl of AMV5× buffer, 0.5 μl of Oligo (dT) (500ng/μl), 2 μl of 2.5mmol/L dNTP, 0.5 μl of Rnasin (50U/μl), and deionize Water to 20μl, reverse transcriptase 2~5U, extend at 42°C for 1 hour. Denature at 95°C for 5 minutes, place in an ice bath, and the product is the first strand of cDNA. Using two pairs of specific primers PHs1, PHa1 and PLs1, PLA1, in a 20μl PCR reaction system, add 2μl of reverse transcription product, 2μl of Taq enzyme 10×buffer, 1μl of upstream and downstream primers, 1μl of 2.5mmol/L dNTP, and add Taq enzyme 1 ~ 2U, add deionized water to 20μl. Denaturation at 95°C for 2 minutes, cycle parameters: 94°C for 1min, 55°C for 1min, 72°C for 1min, a total of 30 cycles, and 72°C for 10min extension.

(3) Using the DNA fragments to be recovered, cut off the gel blocks containing the target DNA fragments under long-wave ultraviolet light, put them into a centrifuge tube, add three times the gel volume of the gel solution, and dissolve the gel blocks completely in a 55°C water bath. Use the DNA fragment purification kit to recover the DNA fragments and put the purified DNA fragments in the aqueous solution, mix the recovered PCR products in the T4 DNA ligase buffer at a ratio (molar ratio) of 2:1 with the carrier PGEMTeasy, and then add 0.5 U The T4 DNA ligase was ligated overnight at 16°C, and the total volume of the ligation reaction was 10 μL.

(4) Take 10 μl of the connection solution, add it to 200 μl of competent bacteria JM109 and mix gently, put it in an ice bath for 30 minutes, heat shock in a 42°C water bath for 90 seconds, quickly transfer to an ice bath for 2 minutes, add 800 μl of LB medium, and transfer to 37°C Constant temperature shaker, shake at a speed of 150r/min for 45min, centrifuge at 4000r/min for 1min, discard 800μl supernatant, take the precipitate and spread it on a solid LB plate containing Amp (final concentration: 100μg/ml), and place the plate upside down at 37 ℃ incubator 12 ~ 18h.

(5) Pick a single clone from the above plate and inoculate it in LB medium containing ampicillin (100 μg/ml). 37 ℃ constant temperature shaker 170rpm, shaking culture overnight. Take 3ml of the bacterial solution and add it to a 1.5ml Eppendorf tube, centrifuge at 10000r/min for 1min, and discard the supernatant. Resuspend the precipitated bacteria in 100 μL of solution I, add 200 μL of freshly prepared solution II, and gently invert several times until the liquid becomes clear. Then, add 150 μL of solution III, and gently invert the solution several times to mix the liquid evenly, at this time a large amount of white flocculent precipitates appear. Centrifuge at 12000rpm for 5min at 4°C, add the supernatant to another Eppendorf tube, add an equal volume of Tris-HCl saturated phenol, shake vigorously, centrifuge at 12000rpm for 5min, and transfer the upper aqueous phase to a new tube. Then add 500 μL chloroform and extract again. Thereafter, carefully absorb the upper aqueous phase, transfer it to a new tube, add 2 times the volume of absolute ethanol to mix, and place it at -20°C for 3 hours. Centrifuge at 12,000 rpm for 10 min at 4°C, discard the supernatant, wash the precipitate twice with 70% ethanol, dry at room temperature for 20 min, dissolve in 40 μL sterile double distilled water, and perform PCR identification and DNA sequencing analysis.

The vectors containing the light and heavy chain genes of the human Tim-3 neutralizing antibody L3D were constructed through the above steps. After sequencing analysis and sequence alignment, they were mouse immunoglobulin light and heavy chain genes, and the corresponding amino acid sequences were SEQ ID NO: 1, SEQ ID NO: 2.

Example 4 Protein A Purification of Monoclonal Antibody L3D and Intervention Experiments on Animal Models of Sepsis and Inflammatory Bowel Disease

1. Materials

Protein A Sepharose CL 4B column protein column: product of Beijing Benyuan Zhengyang Biotechnology Co., Ltd.; the rest are the same as in Example 2.

2. Methods and Results

(1) The vector containing the light and heavy chain genes of human Tim-3 neutralizing antibody L3D is transformed into mammalian cells for expression. Collect the expression supernatant, add 1 mL pH8.0, 0.1moL/L phosphate buffer and adjust the pH to 9.0 with pH 9.0, 1moL/L TRIS-HCL. Add the antibody expression supernatant to the Protein A Sepharose CL 4B protein column that has been equilibrated with 0.1moL/L phosphate buffer pH 8.0, and wash the column with the above buffer until no foreign proteins are detected in the effluent. Elute with pH 3.0 citric acid buffer, collect the effluent, and immediately neutralize with 1moL/L pH 8.5 TRIS-HCL buffer, and dialyze with pH 7.2, 0.01M PBS for 72h. Samples were taken to measure OD ₂₆₀ and OD ₂₈₀ on an ultraviolet spectrophotometer, the protein content was calculated, and stored at -20°C after freeze-drying (Fig. 6 electrophoresis diagram of antibody protein).

(2) Reference methods (Xu.R, et al. Eur. Immunol. 2010.40: 1079; Li X, et al. Clinical Immunol. 2010, 134: 169). Establishment of sepsis and Inflammatory bowel disease model. Specific methods: 1) Sepsis model: first prepare an anesthetic, mix Sumianxin and ketamine 2:1.5 evenly, then dilute it twice with normal saline, and inject 60 μl intraperitoneally into each C57 mouse for about 1-2 minutes Can be anesthetized. After the mice were fully anesthetized, they were fixed on their backs on the operating table, and the abdomen was disinfected with cotton balls dipped in 75% alcohol. Cut a 2cm incision along the linea alba at the lower finger of the xiphoid process from top to bottom, carefully cut the peritoneum to expose the abdomen, carefully lift the peritoneum with tweezers, and find the cecum at the lower right of the abdominal incision. Its color is slightly lighter and swollen. Gently pull out the swollen blind end, and ligate at two-thirds of the distance from the blind end on the cecum. The ligation should not be too tight (to prevent intestinal necrosis from affecting the experimental results) about 2/3 of the width. Use a No. 8 needle to puncture the intestinal wall twice on the outside of the ligation, carefully squeeze out an appropriate amount of contents, and put the cecum and contents back into the abdominal cavity, keeping the physiological position of the cecum as much as possible. The peritoneum and skin were closed layer by layer and ligated with sutures with needles. An important indicator of successful model establishment is that the one-week survival rate of mice is 20-30%. ; 2) Inflammatory bowel disease model: C57 male mice aged 6-8 weeks were divided into 2 groups, 10 in each group. The control group drank water normally, and the experimental group drank 4% DSS solution prepared by DSS. The body weight changes of the mice were observed every day.

For the sepsis model, the mice were divided into experimental group and control group, 10 mice in each group, were given L3D antibody (200ug/mouse, intraperitoneal injection) or the same amount of isotype control antibody 12 hours before modeling, and then observed The condition of the animals includes the change of survival rate and body weight, and the spleen cells of the experimental group and the control group are collected on the 5th to 7th day, and the expression of cytokines IL-1beta and IL-6 is analyzed by quantitative PCR. The results showed that L3D significantly increased the mortality of sepsis model animals, decreased the body weight of model animals, and significantly increased the expression of cytokines, IL-1beta, and IL-6, suggesting that L3D has good neutralizing activity in vivo.

For the inflammatory bowel disease model, the mice were divided into an experimental group and a control group, 10 in each group, and the L3D antibody (200ug/mouse, intraperitoneal injection) or the same amount of isotype control antibody was given to the model while the model was established, and then the animals were observed. Changes in body weight, and the spleen cells of the experimental group and the control group were collected on the 5th to 7th day, and the expression of cytokines IL-17 and IFN-g was analyzed by quantitative PCR. The results showed that L3D significantly reduced the body weight of model animals, and the expressions of cytokines IL-17 and IFN-g were significantly increased, suggesting that L3D has good neutralizing activity in vivo.

Claims (7)

1. an anti-human Tim-3 neutralizing monoclonal antibody, comprises light chain CDR1-3 and heavy chain CDR4-6, it is characterized in that, the aminoacid sequence of described light chain CDR1-3 is:

CDR1: as shown in SEQ ID NO:5 in sequence table;

CDR2: as shown in SEQ ID NO:6 in sequence table;

CDR3: as shown in SEQ ID NO:7 in sequence table;

The aminoacid sequence of described heavy chain CDR4-6 is:

CDR4: as shown in SEQ ID NO:8 in sequence table;

CDR5: as shown in SEQ ID NO:9 in sequence table;

CDR6: as shown in SEQ ID NO:10 in sequence table.

2. monoclonal antibody as claimed in claim 1, is characterized in that, the aminoacid sequence of described variable region of light chain is as shown in SEQ ID NO:1 in sequence table, and the aminoacid sequence of described variable region of heavy chain is as shown in SEQ ID NO:2 in sequence table.

3. the monoclonal antibody as described in any one in claim 1-2, it is characterized in that, the encoding sequence of described variable region of light chain is as shown in the nucleotide sequence of SEQ ID NO:3 in sequence table, and the encoding sequence of described variable region of heavy chain is as shown in the nucleotide sequence of SEQ ID NO:4 in sequence table.

4. a Tim-3 detection kit that contains the monoclonal antibody described in any one in claim 1-3.

5. a pharmaceutical composition, it comprises the monoclonal antibody described in any one in claim 1-3.

6. the application of the monoclonal antibody described in any one in preparation Tim-3 diagnostic kit in claim 1-3.

7. the application of the monoclonal antibody described in any one in preparation treatment pyemia or inflammatory bowel medicine in claim 1-3.

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