A method for purifying anti-nkg2a monoclonal antibodies

Abstract

The application relates to the field of biological medicine, and specifically provides a purification method of an anti-NKG2A monoclonal antibody, which comprises the following steps: using an affinity chromatography column to preliminarily purify and concentrate cell liquid containing the anti-NKG2A monoclonal antibody, and collecting a protein solution; after the collected protein solution is refined through cation exchange chromatography, the protein solution is directly purified through anion exchange chromatography to obtain the purified protein solution. The application provides the purification method of the anti-NKG2A monoclonal antibody, the target protein is captured through affinity chromatography, the aggregates are controlled through cation exchange chromatography in ion exchange chromatography, the residual impurities are removed through directly connected anion exchange chromatography, the anion and the cation are perfectly connected, no sample treatment or solution replacement is needed in the middle, the process connection is smooth, the treatment capacity is high, the antibody purity is high, the impurity content is low, the recovery rate is high, the production cost is reduced, the process cycle is shortened, and the pilot scale production is easy.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to a purification method of anti-NKG2A monoclonal antibody. BACKGROUND

[0002] Cancer has become an important killer threatening human life and health, and treating cancer by activating the immune system has become a research direction in the field of tumor treatment. Antibody drugs have become a hot spot for domestic and foreign pharmaceutical companies to layout due to their unique efficacy advantages and low side effects. For example, the research and development of anti-PD-1 / PD-L1 antibodies and CAR-T technology in immunotherapy. The focus of this immunotherapy is to use the adaptive immune system in the patient's body, and the most important thing is to use CD8+T to kill tumor cells. However, through a large number of studies, it is found that anti-PD-1 / PD-L1 antibodies are only effective for 20-30% of cancer patients, so it is necessary to develop new immunotherapy methods for cancer.

[0003] NKG2A is an "inhibitory" member of the NKG2 family, mainly expressed in CD56hi NK cells, NKT cells and CD8+αβ T cell subsets. Non-classical MHC class I molecule HLA-E is the main ligand of NKG2A-CD94, and the expression level is about 25 times lower than that of classical MHC class I molecule. It is expressed in most normal tissues, and the interaction between NKG2A and HLA-E can inhibit the activation of NK cells and T cells. Therefore, NKG2A on tumor-infiltrating NK cells and T cells and HLA-E on tumor cells have become new immune checkpoints for tumor treatment and have attracted much attention. At present, there is still no drug on the market targeting NKG2A. In order to meet the drug needs of cancer patients, it is urgent to develop an anti-NKG2A monoclonal antibody.

[0004] At present, in the development process of antibodies, the conventional antibody drug purification process generally includes three-step chromatography, but after each step of chromatography, the liquid needs to be changed or the buffer needs to be adjusted to adapt to the purification conditions of the next step. In batch production, too many intermediate operation steps not only waste time, but also affect the recovery rate, increase costs, and may face the risk of introducing new impurities. At the same time, the existence of market monopoly of imported chromatography media increases the production cost invisibly. Therefore, it is urgent to develop a purification method specially applicable to the anti-NKG2A monoclonal antibody provided by the present application. SUMMARY

[0005] In order to meet the market demand, realize the batch production of anti-NKG2A monoclonal antibody, improve the purification efficiency and recovery rate of anti-NKG2A monoclonal antibody, and ensure the stability of the biological activity of anti-NKG2A monoclonal antibody, the present application provides a purification method of anti-NKG2A monoclonal antibody.

[0006] The specific technical scheme of the present application is as follows:

[0007] The present application provides a purification method of anti-NKG2A monoclonal antibody, which comprises the following steps:

[0008] S1, affinity chromatography: using an affinity chromatography column to preliminarily purify and concentrate the cell liquid containing anti-NKG2A monoclonal antibody, and collecting the protein solution;

[0009] S2, ion exchange chromatography: composed of cation exchange chromatography and anion exchange chromatography, after the protein solution collected in step S1 is refined by the cation exchange chromatography, it is directly purified by the anion exchange chromatography to obtain the purified protein solution.

[0010] Further, the anti-NKG2A monoclonal antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises three heavy chain complementarity determining regions represented by HCDR1, HCDR2 and HCDR3 respectively, the light chain variable region comprises three light chain complementarity determining regions represented by LCDR1, LCDR2 and LCDR3 respectively, the amino acid sequence of the heavy chain complementarity determining region HCDR1 is shown as SEQ ID No: 1; the amino acid sequence of the heavy chain complementarity determining region HCDR2 is shown as SEQ ID No: 2; the amino acid sequence of the heavy chain complementarity determining region HCDR3 is shown as SEQ ID No: 3; the amino acid sequence of the light chain complementarity determining region LCDR1 is shown as SEQ ID No: 4; the amino acid sequence of the light chain complementarity determining region LCDR2 is shown as SEQ ID No: 5; the amino acid sequence of the light chain complementarity determining region LCDR3 is shown as SEQ ID No: 6.

[0011] Further, the anti-NKG2A monoclonal antibody is a murine antibody molecule, the amino acid sequence of the heavy chain variable region of the murine antibody molecule is shown as SEQ ID No: 7, and the amino acid sequence of the light chain variable region of the murine antibody molecule is shown as SEQ ID No: 8.

[0012] Further, the murine antibody molecule further comprises a heavy chain constant region selected from IgG1, IgG2a, IgG2b or IgG3 of mouse and a mouse C ka heavy chain constant region of an IgG1 type, a heavy chain constant region of an IgG2a type, a heavy chain constant region of an IgG2b type, or a heavy chain constant region of an IgG3 type; wherein the amino acid sequence of the heavy chain constant region of the IgG1 type is shown as SEQ ID No: 10, the amino acid sequence of the heavy chain constant region of the IgG2a type is shown as SEQ ID No: 11, the amino acid sequence of the heavy chain constant region of the IgG2b type is shown as SEQ ID No: 12, and the amino acid sequence of the heavy chain constant region of the IgG3 type is shown as SEQ ID No: 13; and the amino acid sequence of the light chain constant region of the mouse C k the amino acid sequence of the light chain constant region of the IgG1 type is shown as SEQ ID No: 9.

[0013] Further, the anti-NKG2A monoclonal antibody is a humanized antibody molecule, the amino acid sequence of the heavy chain variable region of the humanized antibody molecule is shown as SEQ ID No: 14, and the amino acid sequence of the light chain variable region of the humanized antibody molecule is shown as SEQ ID No: 15.

[0014] Further, the humanized antibody molecule further comprises a human antibody constant region, the human antibody constant region comprises a heavy chain constant region selected from an IgG1 type or an IgG4 type of human and a light chain constant region of a human C k the amino acid sequence of the heavy chain constant region of the IgG1 type is shown as SEQ ID No: 18, the amino acid sequence of the heavy chain constant region of the IgG4 type is shown as SEQ ID No: 19, and the amino acid sequence of the light chain constant region of the human C k the amino acid sequence of the light chain constant region of the IgG1 type is shown as SEQ ID No: 9.

[0015] Further, in step S1, affinity chromatography: the cell liquid containing the anti-NKG2A monoclonal antibody is subjected to preliminary purification and concentration by using an affinity chromatography column, and a protein solution is collected, which specifically comprises the following steps:

[0016] S101, using a Tris-hydrochloric acid buffer with a pH of 7.0-7.4 and a concentration of 50mM to equilibrate the affinity chromatography column, the medium of the affinity chromatography column is NMab Pro, the bed height of the affinity chromatography column is 15-21cm, and the Tris-hydrochloric acid buffer with a concentration of 50mM contains 150mM of sodium chloride;

[0017] S102, loading the cell liquid containing the anti-NKG2A monoclonal antibody onto the affinity chromatography column at a flow rate of 60-180cm / h;

[0018] S103, after the sample is loaded, the acetate buffer solution with pH 5.0-5.6, 50mM is used for rebalancing, and after the ultraviolet signal is stable, the acetate buffer solution with pH 5.0-5.6, 50mM is used for at least one intermediate elution, until the curve of the ultraviolet absorption value is stable, the flushing is stopped, and the acetate buffer solution is sodium acetate buffer solution or potassium acetate buffer solution;

[0019] S104, the acetate buffer solution with pH 3.4-3.6 is used for elution of the affinity chromatography column, when the ultraviolet absorption value rises to 100mAU, the protein solution is collected, and when the ultraviolet absorption value falls to 100mAU, the collection is ended, and the protein solution is reserved.

[0020] Further, in step S2, the protein solution collected in step S1 is purified by the cation exchange chromatography, and the method comprises the following steps:

[0021] (1) the cation exchange chromatography column is balanced by using the acetate buffer solution with pH 5.0-5.2, 30-50mM, the medium of the cation exchange chromatography column is NanoGel-50SP, the bed height of the cation exchange chromatography column is 15-21cm, and the acetate buffer solution is sodium acetate buffer solution or potassium acetate buffer solution;

[0022] (2) the protein solution collected in step S1 is loaded onto the cation exchange chromatography column at a flow rate of 60-180cm / h;

[0023] (3) the cation exchange chromatography column is washed by using the acetate buffer solution with pH 5.5-5.7, 60-80mM;

[0024] (4) the cation exchange chromatography column is eluted by using the phosphate buffer solution with pH 7.4-7.6, 30-50mM, when the ultraviolet absorption value rises to 100mAU, the eluate is collected, and when the ultraviolet absorption value falls to 100mAU, the collection is ended, and the phosphate buffer solution is sodium phosphate buffer solution or potassium phosphate buffer solution.

[0025] Further, in step S2, the protein solution is purified by the anion exchange chromatography, and the method comprises the following steps:

[0026] The eluate collected by the cation exchange chromatography is loaded onto the anion exchange chromatography column at a flow rate of 60-180cm / h, the medium of the anion exchange chromatography column is NanoGel-50Q, and the bed height of the anion exchange chromatography column is 10-20cm;

[0027] After the sample is loaded, the anion exchange chromatography column is flushed with the phosphate buffer with pH 7.4-7.6 and 30-50 mM, and when the ultraviolet absorption value rises to 100 mAU, the purified protein solution is collected, and when the ultraviolet absorption value drops to 200 mAU, the collection is ended.

[0028] Preferably, in step S1, after the affinity chromatography, the collected protein solution is further subjected to pH adjustment, specifically including:

[0029] The collected protein solution in step S1 is incubated at room temperature for 60-90 min;

[0030] The pH value of the protein solution is adjusted to 3.5-3.7 by using 1M acetic acid solution;

[0031] The pH value of the protein solution is adjusted back to 5.0-5.2 by using 1M Tris buffer, and the filtrate is collected by filtering through a 0.22 μm filter and is ready for ion exchange chromatography.

[0032] The present application has the following advantages: the present application provides a purification method specifically for anti-NKG2A monoclonal antibodies, and the anti-NKG2A monoclonal antibodies disclosed in the present application can act on the NKG2A inhibitory receptor on immune cells, release the inhibition of the NKG2A signaling pathway on immune cells, enhance the activity of cells such as NK cells and T cells, thereby activating the killing of immune cells such as NK cells on tumors, and the monoclonal antibodies are new checkpoint inhibitors for promoting anti-tumor immunity, and are mainly used for preparing drugs for treating cancer or autoimmune diseases; the method provided by the present application captures the target protein through affinity chromatography, realizes control of aggregates through cation exchange chromatography in ion exchange chromatography, removes residual impurities through directly connected anion exchange chromatography, and the anion and cation are perfectly connected without sample treatment or solution replacement in the middle, the process connection is smooth, the treatment capacity is high, the antibody purity is high, the impurity content is low, the recovery rate is high, the work intensity is reduced, the production cost is reduced, the process cycle is shortened, and the production is easy to scale up. BRIEF DESCRIPTION OF DRAWINGS

[0033] Figure 1 The plasmid map of the pScFv-Disb-HS vector in the antibody biological screening method provided for Example 3 of the present application;

[0034] Figure 2 The comparison chart of the affinities of the gradient dilution ELISA anti-NKG2A phage monoclonal antibodies in Example 4 of the present application;

[0035] Figure 3 The map of the expression plasmid pTSE of the anti-NKG2A whole antibody provided for Example 6 of the present application;

[0036] Figure 4 Figure for comparing the binding ability of the mouse-derived antibody in the embodiment 7 of the present application to NKG2A;

[0037] Figure 5 Figure for the binding curve of the anti-NKG2A monoclonal antibody MA-1 to NKG2A, NKG2C and NKG2E in the embodiment 8 of the present application;

[0038] Figure 6 Figure for comparing the binding ability of the humanized antibody molecule in the embodiment 13 of the present application to NKG2A;

[0039] Figure 7 Figure for the effect of the anti-NKG2A monoclonal antibody in the embodiment 14 of the present application on activating human PBMC to kill HL-60;

[0040] Figure 8 Figure for the in vivo tumor inhibition model of the monoclonal antibody in the embodiment 15 of the present application. DETAILED DESCRIPTION

[0041] The present application will be further described in detail below in combination with the following embodiments.

[0042] Embodiment 1

[0043] The present application provides a purification method of the anti-NKG2A monoclonal antibody, which comprises the following steps:

[0044] S1, affinity chromatography: the cell liquid containing the anti-NKG2A monoclonal antibody is subjected to preliminary purification and concentration by using an affinity chromatography column, and the protein solution is collected;

[0045] S2, ion exchange chromatography: it is composed of cation exchange chromatography and anion exchange chromatography, and the protein solution collected in step S1 is subjected to purification by the cation exchange chromatography and then directly subjected to purification by the anion exchange chromatography to obtain the purified protein solution.

[0046] The anti-NKG2A monoclonal antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises three heavy chain complementarity determining regions represented by HCDR1, HCDR2 and HCDR3 respectively, the light chain variable region comprises three light chain complementarity determining regions represented by LCDR1, LCDR2 and LCDR3 respectively, the amino acid sequence of the heavy chain complementarity determining region HCDR1 is shown as SEQ ID No: 1; the amino acid sequence of the heavy chain complementarity determining region HCDR2 is shown as SEQ ID No: 2; the amino acid sequence of the heavy chain complementarity determining region HCDR3 is shown as SEQ ID No: 3; the amino acid sequence of the light chain complementarity determining region LCDR1 is shown as SEQ ID No: 4; the amino acid sequence of the light chain complementarity determining region LCDR2 is shown as SEQ ID No: 5; and the amino acid sequence of the light chain complementarity determining region LCDR3 is shown as SEQ ID No: 6.

[0047]

[0048] Example 2 Preparation of antigen and construction and screening of phage antibody library

[0049] The present application optimizes the immunization method by immunizing mice with NKG2A antigen (extracellular segment of NKG2A protein), creates a phage display library and establishes an antigen site screening method. The construction and screening of the phage display library are specifically as follows:

[0050] 1. Immunization of mice with NKG2A antigen

[0051] Experimental animals:

[0052] Species strain: BALB / c, female, mouse;

[0053] Body weight: 18-20g;

[0054] Supplier of experimental animals: Beijing Huafukang Biotechnology Co., Ltd.

[0055] 2. Immunization: The mice were immunized with human NKG2A (synthetic gene by Nanjing Kingsriver Biotechnology Co., Ltd., and the vector was constructed and expressed and purified by the company).

[0056] 3. Construction and screening of phage antibody library

[0057] The mouse spleen cells with higher titer were taken, and the total RNA in the mouse spleen cells was extracted by using Trizol reagent (purchased from Ambion, item number: 15596026), and the cDNA was obtained by RT-PCR, and the degenerate primer (the degenerate primer used is referred to the reference: Journal of Immunological Methods 233 (2000) 167-177) was used for PCR amplification, so as to obtain the immune mouse antibody heavy chain variable region gene library (VH) and light chain variable region gene library (VL), and the light and heavy chains were double-digested respectively, and were connected to the vector which was also treated by enzyme in the same step, so as to construct the pScFv-Disb-HS-VH-VL gene library. The pScFv-DisB-HS vector is obtained by using a series of gene cloning methods to modify the vector pComb3 vector (purchased from the Chinese plasmid vector strain cell strain gene preservation center), so as to be used for constructing and expressing the phage single-chain antibody library. The modified vector is named as pScFv-Disb-HS vector, and the plasmid map thereof is shown in Fig. 1, and the mouse immune phage antibody library is constructed based on the vector. Figure 1 The phage library is constructed based on the vector.

[0058] 4. Enrichment and screening of the phage library

[0059] The immunotubes were coated with huFc-NKG2A / CD94 as an antigen, and the antigen coating amount was 4 μg / 500 μl / tube, and the immunotubes were coated overnight at 4°C, and then 4% skimmed milk / PBST was used to block the immunotubes and the immune phage antibody library, and the blocking was performed at room temperature for 1 h. After the blocking, the immune phage antibody library was added into the immunotubes for antigen-antibody combination, and the phage input amount was about 10 9 ~ 10 12 After the reaction at room temperature for 1 h, the unbound phage was washed away by using PBST, and was eluted by using 0.1M pH 2.2 Glycine-HCl, and finally the eluted phage antibody solution was neutralized to about pH 7.0 by using 1.5M pH 8.8 Tris-HCl.

[0060] The phage after the neutralization was used to infect 10 ml of TG1 bacteria liquid which was grown to the logarithmic phase, and was placed in a 37°C incubator for 30 min, and part of the bacteria liquid was taken out for gradient dilution, and was coated on 2YTAG plates, and was used for calculating the phage output. The remaining bacteria liquid was centrifuged, and the supernatant was discarded, and the bacteria precipitate was resuspended in a small amount of culture medium, and was sucked out and coated on a 2YTAG large plate, so as to prepare for the next round of screening.

[0061] The bacteria from the above-mentioned infected and plated bacteria were scraped from the large plate and inoculated into 2YTAG liquid medium, and after being shaken to the logarithmic phase, M13KO7 helper phage was superinfected, and the phage was prepared by culturing overnight at 28°C and 220 rpm. The phage was purified by PEG / NaCl precipitation for the next round of screening. Three rounds of phage library enrichment screening were performed.

[0062] 5. Screening of phage single-chain antibody positive clones

[0063] After three rounds of screening, well-separated monoclonal colonies were picked and inoculated into 2YTAG liquid medium added to a 96-well deep well plate, and cultured at 37°C and 220 rpm until the logarithmic growth phase. About 10 10 μl of helper phage M13KO7 was added to each well, and the temperature was set to 37°C for stationary infection for 30 min. Centrifugation was performed at 4000 rpm for 15 min, and the supernatant was discarded. The bacteria were resuspended and precipitated with 2YTAK, and cultured overnight at 28°C and 220 rpm. After centrifugation at 4000 rpm and 4°C for 15 min, the amplified phage supernatant was aspirated and subjected to ELISA identification:

[0064] The antigen huFc-NKG2A / CD94 was coated at a concentration of 0.5 μg / ml, and the primary antibody was 100 μl of 1:3 diluted phage supernatant, with blocking solution as a negative control. After incubation with HRP-anti-M13 secondary antibody, TMB substrate was used for color development, and after 20 min of color development, the OD 450 nm was detected in a microplate reader, and a reading value >0.3 was considered as a positive clone.

[0065] Antibody specificity screening was performed using the supernatant of the positive clone: the binding of each phage supernatant to different antigens huFc-NKG2A / CD94, huFc-NKG2C / CD94, or huFc-NKG2C / CD94 was detected by the above-mentioned ELISA method, and the monoclonal molecules with OD450nm reading values more than three times higher when the supernatant bound to NKG2A than when it bound to NKG2C / E were selected for subsequent screening. At this time, a total of one monoclonal molecule was screened, which was named MA-1.

[0066] Murine antibody molecules Heavy chain variable region sequences Light chain variable region sequences MA-1 SEQ ID No: 7 SEQ ID No: 8

[0067] Specifically, SEQ ID No: 7 (amino acid sequence of the heavy chain variable region of MA-1):

[0068] EVKLEESGGGLVKPGGSLKLSCAASGFAFSDYDMSWVRQTPEKRLEWVATISSGGSYTYYPDSVKGRFTISRDNARNTLYLQMSSLRSEDTALYYCTSPRQVGLRKAFDYWGQGTTLTVSS;

[0069] SEQ ID No: 8 (amino acid sequence of the light chain variable region of MA-1):

[0070] DIVVTQSPALMSASPGEKVTMTCSASSSVSYMYWYQQKPRSSPKSWIYLTSNLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPFTFGSGTKLEIKRAD.

[0071] Example 3 Gradient dilution ELISA to compare the affinity of anti-NKG2A phage monoclonal antibodies

[0072] The murine antibody molecule (MA-1) obtained in Example 2 was subjected to monoclonal phage display and purification, and then subjected to phage gradient dilution ELISA to identify the affinity, and the specific method is as follows:

[0073] NKG2A antigen was coated with carbonate buffer at pH 9.6, 100 ng / well / 100 μL, coated overnight at 4°C, washed three times with PBST, and the phage monoclonal antibody MA-1 screened in Example 2 was diluted with PBST at a gradient of 5 times, 100 μl of the diluted sample was added to each well, and incubated at room temperature for 1 hour. Wash the ELISA plate with PBST, add HRP-anti-M13 (purchased from Bio-viewshine, item number: GE27-9421-01) monoclonal antibody diluted with PBST to the ELISA plate, and incubate at room temperature for 1 h. Color development with TMB color development kit, color development at room temperature for 10 minutes, and after termination with 2M H2SO4, read at 450nm with a microplate reader, and calculate the corresponding EC50 value, and the specific data are as follows:

[0074] From the above data and as shown in Figure 2 , the murine antibody molecule MA-1 screened in Example 2 can bind to NKG2A, and the murine antibody molecule MA-1 provided by the present application has high affinity with NKG2A.

[0075] Example 4

[0076] The mouse antibody molecule of the embodiment 4 is further limited based on the embodiment 2, and further comprises a heavy chain constant region selected from IgG1, IgG2a, IgG2b or IgG3 of mouse and a light chain constant region of C k mouse; wherein the amino acid sequence of the heavy chain constant region of IgG1 is shown as SEQ ID No: 10, the amino acid sequence of the heavy chain constant region of IgG2a is shown as SEQ ID No: 11, the amino acid sequence of the heavy chain constant region of IgG2b is shown as SEQ ID No: 12, and the amino acid sequence of the heavy chain constant region of IgG3 is shown as SEQ ID No: 13; the amino acid sequence of the light chain constant region of C k mouse is shown as SEQ ID No: 9; and the specific sequence is as follows:

[0077] SEQ ID No: 9 (the amino acid sequence of the light chain constant region of C k mouse) :

[0078] ADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC; SEQ ID No: 10 (the amino acid sequence of the heavy chain constant region of IgG1 of mouse) :

[0079] AKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPG;

[0080] SEQ ID No: 11 (the amino acid sequence of the heavy chain constant region of IgG2a of mouse) :

[0081] AKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK;

[0082] SEQ ID No: 12 (amino acid sequence of heavy chain constant region of mouse IgG2b type):

[0083] AKTTPPSVYPLAPGCGDTTGSSVTLGCLVKGYFPESVTVTWNSGSLSSSVHTFPALLQSGLYTMSSSVTVPSSTWPSQTVTCSVAHPASSTTVDKKLEPSGPISTINPCPPCKECHKCPAPNLEGGPSVFIFPPNIKDVLMISLTPKVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTIRVVSTLPIQHQDWMSGKEFKCKVNNKDLPSPIERTISKIKGLVRAPQVYILPPPAEQLSRKDVSLTCLVVGFNPGDISVEWTSNGHTEENYKDTAPVLDSDGSYFIYSKLNMKTSKWEKTDSFSCNVRHEGLKNYYLKKTISRSPGK;

[0084] SEQ ID No: 13 (amino acid sequence of heavy chain constant region of mouse IgG3 type):

[0085] ATTTAPSVYPLVPGCSDTSGSSVTLGCLVKGYFPEPVTVKWNYGALSSGVRTVSSVLQSGFYSLSSLVTVPSSTWPSQTVICNVAHPASKTELIKRIEPRIPKPSTPPGSSCPPGNILGGPSVFIFPPKPKDALMISLTPKVTCVVVDVSEDDPDVHVSWFVDNKEVHTAWTQPREAQYNSTFRVVSALPIQHQDWMRGKEFKCKVNNKALPAPIERTISKPKGRAQTPQVYTIPPPREQMSKKKVSLTCLVTNFFSEAISVEWERNGELEQDYKNTPPILDSDGTYFLYSKLTVDTDSWLQGEIFTCSVVHEALHNHHTQKNLSRSPELELNETCAEAQDGELDGLWTTITIFISLFLLSVCYSASVTLFKVKWIFSSVVQVKQTAIPDYRNMIGQGA.

[0086] Example 5 Preparation of murine full-length antibody molecule

[0087] Example 5 of the present application is based on Example 2, the plasmid extracted from the monoclonal antibody MA-1 identified in Example 2 is cloned into the vector pTSE containing the gene of mouse IgG2a heavy chain (the amino acid sequence is shown as SEQ ID No: 11) and the gene of mouse C k light chain constant region (the amino acid sequence is shown as SEQ ID No: 9) respectively, and the pTSE vector structure is shown as Figure 3 (pTSE vector preparation process, see CN103525868A specification page 3

[0019] paragraph).

[0088] The HEK293E cells (purchased from the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, item number GNHu43) are transiently transfected for antibody expression, and the complete monoclonal antibody molecule is obtained by protein A affinity column purification using AKTA instrument.

[0089] Example 6 Binding ability of murine antibody to NKG2A

[0090] NKG2A was coated with carbonate buffer at pH 9.6, 100 ng / well / 100 μl, coated overnight at 4°C. Washed five times with 300 μl / well PBST, then added 1% BSA-PBST to block at 37°C for 1 h, added MA-1 mouse antibody at different dilution concentrations, the highest initial concentration of whole antibody was 5 μg / ml, each antibody was diluted by 3 times, and 12 gradients were made for each antibody, incubated at 37°C for 1 h. Washed five times with 300 μl / well PBST, then added Anti-Mouse Fc-HRP diluted with 1% BSA-PBST at 1:10000, incubated at 37°C for 1 h. Color development with TMB color development kit, 100 μl / well, color development at room temperature for 8 min, then color development was terminated with 2M H2SO4. Read at 450 nm, and calculate the corresponding EC50 value, the data are shown in Figure 4 .

[0091] It can be known from Figure 4 that the screened mouse antibody MA-1 can bind to NKG2A and has high affinity.

[0092] Example 7 Binding specificity experiment of mouse anti-NKG2A monoclonal antibody to NKG2A, NKG2C and NKG2E

[0093] The EC50 value of the monoclonal antibody MA-1 obtained in Example 5 for binding to huFc-NKG2A / CD94, huFc-NKG2C / CD94 or huFc-NKG2E / CD94 was detected by ELISA, wherein NKG2C (extracellular segment of NKG2C protein) and NKG2E (extracellular segment of NKG2E protein) have high sequence identity with NKG2A and are NK cell activating type receptors:

[0094] The anti-NKG2A monoclonal antibody MA-1 was gradient diluted as a primary antibody, and anti-mouse Ig-HRP was used as a secondary antibody, which were respectively combined with NKG2A, NKG2C and NKG2E, and then the dose-read value curve was drawn and nonlinear regression curve was made after ELISA detection, and the EC50 value of each antibody for binding to the three antigens was calculated. The binding curve of the anti-NKG2A monoclonal antibody MA-1 to NKG2 protein is shown in Figure 5 .

[0095] It can be known from Figure 5 that the anti-NKG2A monoclonal antibody MA-1 screened in the application can specifically bind to NKG2A and does not bind to NKG2C and NKG2E and other NKG2 family proteins.

[0096] Example 8

[0097] The monoclonal antibody or the antigen binding fragment thereof is a chimeric antibody molecule, the chimeric antibody molecule comprises a heavy chain variable region of a murine antibody molecule, a light chain variable region of the murine antibody molecule and a human antibody constant region. The human antibody constant region comprises a heavy chain constant region selected from human IgG1 or IgG4 and a human C k The amino acid sequence of the heavy chain constant region of the IgG1 is shown as SEQ ID No: 18, the amino acid sequence of the heavy chain constant region of the IgG4 is shown as SEQ ID No: 19, and the amino acid sequence of the light chain constant region of the human C k The amino acid sequence of the heavy chain constant region of the IgG1 is shown as SEQ ID No: 18, the amino acid sequence of the heavy chain constant region of the IgG4 is shown as SEQ ID No: 19, and the amino acid sequence of the light chain constant region of the human C

[0098] SEQ ID No: 18 (the amino acid sequence of the heavy chain constant region of the human IgG1) :

[0099] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0100] SEQ ID No: 19 (the amino acid sequence of the heavy chain constant region of the human IgG4) :

[0101] ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0102] SEQ ID No:20 (human C k chain constant region amino acid sequence):

[0103] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGE C.

[0104] Preparation of chimeric antibody molecules

[0105] The preferred human antibody constant region of Example 9 of the present application is further based on the human IgGl type heavy chain constant region (the amino acid sequence of which is shown as SEQ ID No: 18) and the human C k type light chain constant region (the amino acid sequence of which is shown as SEQ ID No: 20).

[0106] Specific preparation method:

[0107] The heavy chain variable region VH (SEQ ID No: 7) and the light chain variable region VL gene (SEQ ID No: 8) of the ideal anti-NKG2A monoclonal antibody molecule MA-1 screened out are kept as the murine sequence, and are respectively cloned into the vector pTSE (as shown in Figure 3 SEQ ID No: 19) carrying the heavy chain constant region and the light chain constant region gene, the heavy chain constant region is the human IgGl type (the amino acid sequence is shown as SEQ ID No: 18), and the light chain constant region is the human C kChimeric antibody CA-1 was obtained by transient transfection of HEK293E cells (purchased from: Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, item number: GNHu43) for antibody expression.

[0108] Humanization of murine antibody molecule MA-1

[0109] First, the sequence of murine antibody molecule MA-1 in Example 2 and the human antibody germline database (v-base) were compared to find higher homologous human antibody light and heavy chain germlines as candidate sequences, and then the sequences of the CDRs of murine antibody molecule MA-1 were transplanted onto the human candidate sequences for homology modeling. Then, through three-dimensional structure simulation calculation, the key framework amino acid residues that may play an important role in maintaining the CDR loop structure were designed for the back mutation of the humanized antibody. The designed light and heavy chain variable regions of the humanized antibody containing the back mutation were synthesized by Nanjing Kingsriver Biotechnology Co., Ltd., and then connected to the transient expression vector. The light and heavy chains obtained by humanization were combined and analyzed, and the following humanized antibody molecules were obtained: HA-1, HA-2, and the sequences of the two monoclonal antibodies screened above are as follows:

[0110]

[0111]

[0112] Specifically, SEQ ID No: 14 (amino acid sequence of the heavy chain variable region of HA-1):

[0113] EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYDMSWVRQAPGKGLEWVSTISSGGSYTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCTSPRQVGLRKAFDYWGQGTTVTVSS;

[0114] SEQ ID No: 16 (amino acid sequence of the heavy chain variable region of HA-2):

[0115] EVQLVESGGGLVKPGGSLRLSCAASGFAFSDYDMSWVRQAPGKGLEWVATISSGGSYTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCTSPRQVGLRKAFDYWGQGTTVTVSS;

[0116] SEQ ID No: 15 (amino acid sequence of the light chain variable region of HA-1):

[0117] DVVMTQSPAFLSVTPGEKVTITCSASSSVSYMYWYQQKPDQAPKLLIKLTSNLASGVPSRFSGSGSGTDFTFTISSLEAEDAATYYCQQWSSNPFTFGQGTKLEIK;

[0118] SEQ ID No:17 (Amino acid sequence of the light chain variable region of HA-2):

[0119] DIVVTQSPAFLSVTPGEKVTITCSASSSVSYMYWYQQKPDQAPKLLIKLTSNLASGVPSRFSGSGSGTDFTFTISSLEAEDAATYYCQQWSSNPFTFGQGTKLEIK.

[0120] Example 11 Preparation of humanized all-antibody molecules

[0121] Example 11 of the present invention further specifies, based on Example 10, that the humanized antibody molecule also includes a human antibody constant region, which includes a heavy chain constant region selected from human IgG1 or IgG4 and human C k The amino acid sequences of the light chain constant region of type IgG1 and the heavy chain constant region of type IgG4 are shown in SEQ ID No:18 and SEQ ID No:19, respectively. k The amino acid sequence of the light chain constant region of the type is shown in SEQ ID No:20.

[0122] The specific sequence of the constant region of the human antibody described above is the same as that in Example 8.

[0123] The coding genes for the heavy chain VH and light chain VL of the two humanized antibody molecules obtained in Example 10 above were cloned into the vector pTSE (e.g., ...) containing the heavy chain constant region and light chain constant region genes, respectively. Figure 3 As shown), each humanized antibody molecule selects the heavy chain constant region of IgG1 type (amino acid sequence as shown in SEQ ID No:19), and selects human C k All antibody molecules were prepared from the light chain constant region of type (amino acid sequence as shown in SEQ ID No:20), transiently transfected into HEK293E cells (purchased from the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, catalog number GNHu43) for antibody expression, and purified using an AKTA instrument via a protein A affinity column to obtain one monoclonal antibody, as detailed below.

[0124] Humanized antibody molecules Humanized whole antibody molecules HA-1 HA-1 -IgG1 HA-2 HA-2 -IgG1

[0125] Example 12

[0126] Embodiment 12 of the present application further limits the following scheme on the basis of the above-mentioned embodiments:

[0127] The present application also provides a polynucleotide molecule encoding the anti-NKG2A monoclonal antibody.

[0128] The present application also provides a recombinant DNA expression vector comprising the polynucleotide molecule.

[0129] The present application also provides a host cell transfected with the above-defined recombinant DNA expression vector, wherein the host cell is a prokaryotic cell, a yeast cell, an insect cell or a mammalian cell.

[0130] Preferably, the host cell is a mammalian cell, and the mammalian cell is a HEK293 cell, a CHO cell or an NS0 cell.

[0131] The present application also provides a medicament comprising the anti-NKG2A monoclonal antibody.

[0132] The present application also provides the use of the anti-NKG2A monoclonal antibody for specifically binding to NKG2A, relieving the inhibition of NK cells by the NKG2A signaling pathway, activating the killing of tumors by NK cells, and preparing a medicament for treating cancer or autoimmune diseases.

[0133] The cancer includes, but is not limited to, colon cancer, head and neck cancer, non-small cell lung cancer, pancreatic cancer, kidney cancer, gastric cancer, liver cancer, ovarian cancer, breast cancer or melanoma.

[0134] The autoimmune disease includes, but is not limited to, systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, dermatomyositis, polymyositis, vasculitis or Sjogren's syndrome.

[0135] The present application also provides the use of the anti-NKG2A monoclonal antibody in combination with an immunomodulatory drug or an anti-inflammatory factor drug for preparing a medicament for treating cancer or autoimmune diseases.

[0136] The immunomodulatory drug includes, but is not limited to, an anti-PD-1 monoclonal antibody, an anti-PD-L1 monoclonal antibody, an anti-CTLA4 monoclonal antibody, an anti-4-1BB monoclonal antibody, an anti-OX-40 monoclonal antibody, an anti-PD-L2 monoclonal antibody, an anti-LAG-3 monoclonal antibody, an anti-TIGIT monoclonal antibody, an anti-GITR monoclonal antibody, an anti-ICOS monoclonal antibody, an anti-PVR monoclonal antibody, an anti-PVRIG monoclonal antibody, an anti-VISTA monoclonal antibody or an anti-TIMS monoclonal antibody.

[0137] Anti-inflammatory drugs include, but are not limited to: anti-TNFα monoclonal antibodies, anti-IL-1β monoclonal antibodies or IL-1 receptor antagonists, anti-IL-6R monoclonal antibodies, anti-IL-8 monoclonal antibodies, recombinant IL-15 or IL-15 agonists;

[0138] Cancers include, but are not limited to, colon cancer, head and neck cancer, non-small cell lung cancer, pancreatic cancer, kidney cancer, stomach cancer, liver cancer, ovarian cancer, breast cancer, or melanoma;

[0139] Autoimmune diseases include, but are not limited to, systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, dermatomyositis, polymyositis, vasculitis, or Sjögren's syndrome.

[0140] Example 13: Binding experiment of humanized all-antibody molecule to NKG2A protein

[0141] NKG2A antigen was coated with carbonate buffer (pH 9.6) at a rate of 200 ng / well / 100 μL overnight at 4°C. The cells were washed five times with 300 μL / well PBST, then blocked with 1% BSA-PBST at 37°C for 1 h. Humanized antibodies HA-1, HA-2, and the chimeric antibody CA-1 prepared in Example 9 were added at different dilutions. The initial maximum concentration of all three antibodies was 50 μg / mL. Each antibody was diluted 3-fold, and ten gradients were prepared for each antibody. The cells were incubated at 37°C for 1 h. The cells were washed five times with 300 μL / well PBST, then Goat Anti Human IgG-HRP (purchased from Beijing Zhongshan Jinqiao Biotechnology Co., Ltd., catalog number: ZB-2304) diluted 1:5000 with 1% BSA-PBST was added, and the cells were incubated at 37°C for 1 h. The TMB chromogenic kit was used for color development, with 100 μL / well, incubated at room temperature for 5 min, followed by termination with 2M H2SO4. Readings were taken at 450 nm using a microplate reader, and the corresponding EC50 values ​​were calculated. Experimental results are shown below. Figure 6 As shown.

[0142] pass Figure 6 It can be seen that both different humanized antibody molecules can bind to NKG2A, and the EC50 values ​​of the two humanized antibody molecules are close to those of the chimeric antibody CA-1, indicating that the humanized antibody molecules retain the high binding ability of the mouse parent antibody MA-1 to NKG2A.

[0143] Example 14: In vitro killing effect detection of anti-NKG2A monoclonal antibody

[0144] HL-60 tumor cells with high expression of HLA-E were selected as target cells, and human PBMC cells were selected as effector cells. The density of the target cells and the effector cells was adjusted, and the effector and target ratio was 50:1 after mixing in the same volume. The sample was diluted with different antibodies and added to the corresponding position of the 96-well plate. After incubation at 37°C in a CO2 incubator for 18 hours, the supernatant was detected for LDH release, and the cell killing rate was calculated. The results are shown in Figure 7 .

[0145] By Figure 7 It can be concluded that the anti-NKG2A monoclonal antibodies HA-1 and HA-2 provided by the present application can activate and promote the killing effect of human PBMC cells on HL-60 tumor cells.

[0146] Example 15 Inhibition experiment of anti-NKG2A monoclonal antibody HA-1 on MC38-NKG2A colorectal cancer in mice

[0147] 1. Experimental animals:

[0148] Species and strain: C57BL / 6JGpt mice;

[0149] Weeks of age: 6-8 weeks;

[0150] Supplier of experimental animals: Baoxietu (Beijing) Pharmaceutical Technology Co., Ltd.

[0151] 2. Cell culture:

[0152] MC38 tumor cells (YK-CL-256-02) (purchased from: Biovector NTCC Inc., Beijing) are original cells, and MC38-NKG2A tumor cell lines are constructed.

[0153] Each mouse was subcutaneously inoculated with MC38 tumor cells stably transfected on the right side of the back, and after tumor formation, 18 mice with similar tumor sizes were randomly divided into three groups, 6 mice in each group. Intraperitoneal injection of anti-NKG2A monoclonal antibody HA-1 (10 mg / kg) provided in the present application, while setting up a control group injected with the same amount of Z270 control antibody Z270 and its humanized antibody huZ270 VH and VL sequences (from patent US8993319B2), and the negative control group was injected with the same amount of isotype IgG. Drug administration was twice a week, and tumor volume and body weight were measured twice a week. The tumor growth was observed and recorded, and when the tumor volume exceeded 3000mm 3 , the mice were sacrificed by cervical dislocation, and each tumor was carefully separated with surgical tweezers and weighed. The results are shown in Figure 8 .

[0154] AsFigure 8 As shown in the figure, the tumor volume of the group injected with the anti-NKG2A monoclonal antibody HA-1 is significantly smaller than that of the negative control group, and the tumor volume of the group injected with the anti-NKG2A monoclonal antibody HA-1 is closer to that of the control antibody Z270. It can be seen that the anti-NKG2A monoclonal antibody HA-1 in the present application can significantly inhibit tumor growth.

[0155] Example 16

[0156] The embodiment 16 of the present application provides a purification method of the anti-NKG2A monoclonal antibody based on the above-mentioned embodiments 1-15, and the purification method comprises:

[0157] S1, affinity chromatography: using an affinity chromatography column to preliminarily purify and concentrate the cell liquid containing the anti-NKG2A monoclonal antibody, and collecting the protein solution, wherein the anti-NKG2A monoclonal antibody is the anti-NKG2A monoclonal antibody HA-1 screened in the embodiments 2-15;

[0158] S101, using a Tris-hydrochloric acid buffer solution with pH 7.0 and 50mM to equilibrate the affinity chromatography column, wherein the medium of the affinity chromatography column is NMab Pro, the bed height of the affinity chromatography column is 15cm, and the Tris-hydrochloric acid buffer solution with 50mM contains 150mM of sodium chloride;

[0159] S102, loading the cell liquid containing the anti-NKG2A monoclonal antibody to the affinity chromatography column at a flow rate of 60cm / h;

[0160] S103, after the loading is completed, using an acetate buffer solution with pH 5.0 and 50mM to re-equilibrate, and after the ultraviolet signal is stable, using the acetate buffer solution with pH 5.0 and 50mM to perform at least one intermediate washing elution, and stopping the washing until the curve of the ultraviolet absorption value is reduced to be stable, wherein the acetate buffer solution is a sodium acetate buffer solution;

[0161] S104, using an acetate buffer solution with pH 3.4 to elute the affinity chromatography column, starting to collect the protein solution when the ultraviolet absorption value rises to 100mAU, and ending the collection when the ultraviolet absorption value falls to 100mAU, for standby.

[0162] In step S1, after the affinity chromatography, the collected protein solution is further subjected to pH adjustment, specifically including: incubating the protein solution collected in step S1 at room temperature for 60min; using a 1M acetic acid solution to adjust the pH value of the protein solution to 3.5; using a 1M Tris buffer solution to adjust the pH value of the protein solution to 5.0, and filtering through a 0.22μm filter to collect the filtrate, for standby of ion exchange chromatography.

[0163] S2, ion exchange chromatography: it is composed of cation exchange chromatography and anion exchange chromatography connection, the protein solution collected in step S1 is purified by cation exchange chromatography, and then directly purified by anion exchange chromatography to obtain the purified protein solution, and the sample outlet of the cation exchange chromatography column is communicated with the sample inlet of the anion exchange chromatography column.

[0164] In step S2, the protein solution collected in step S1 is purified by cation exchange chromatography, which specifically includes the following methods:

[0165] (1) using pH5.0, 30mM acetate buffer to balance the cation exchange chromatography column, the medium of the cation exchange chromatography column is NanoGel-50SP, the bed height of the cation exchange chromatography column is 15cm, and the acetate buffer is sodium acetate buffer; (2) the protein solution collected in step S1 is loaded onto the cation exchange chromatography column at a flow rate of 60cm / h; (3) using pH5.5, 60mM acetate buffer to wash the cation exchange chromatography column; (4) using pH7.4, 30mM phosphate buffer to elute the cation exchange chromatography column, when the ultraviolet absorption value rises to 100mAU, start collecting the eluate, when the ultraviolet absorption value drops to 100mAU, end collecting, and the phosphate buffer is sodium phosphate buffer.

[0166] In step S2, the protein solution collected in step S1 is purified by cation exchange chromatography, which specifically includes the following methods:

[0167] The eluate collected by the cation exchange chromatography is automatically loaded into the anion exchange chromatography column at a flow rate of 60cm / h, the medium of the anion exchange chromatography column is NanoGel-50Q, and the bed height of the anion exchange chromatography column is 10cm; after the loading is completed, the anion exchange chromatography column is flushed with pH7.4, 30mM phosphate buffer, when the ultraviolet absorption value rises to 100mAU, the purified protein solution is collected, and when the ultraviolet absorption value drops to 200mAU, the collection is ended, and the purified protein solution is obtained.

[0168] Example 17

[0169] The embodiment 17 of the present application provides a purification method of anti-NKG2A monoclonal antibody based on the above-mentioned embodiments 1-15, which comprises:

[0170] S1, affinity chromatography: using an affinity chromatography column to preliminarily purify and concentrate the cell liquid containing anti-NKG2A monoclonal antibody, and collecting the protein solution, wherein the anti-NKG2A monoclonal antibody is the anti-NKG2A monoclonal antibody HA-1 screened in embodiments 2-15;

[0171] S101, using pH 7.4, 50mM Tris-hydrochloric acid buffer to balance the affinity chromatography column, the medium of the affinity chromatography column is NMab Pro, the bed height of the affinity chromatography column is 21cm, 50mM Tris-hydrochloric acid buffer contains 150mM sodium chloride;

[0172] S102, the cell liquid containing anti-NKG2A monoclonal antibody is loaded onto the affinity chromatography column at a flow rate of 180cm / h;

[0173] S103, after the loading is completed, pH 5.6, 50mM acetate buffer is used for rebalancing, and after the ultraviolet signal is stable, pH 5.6, 50mM acetate buffer is used for at least one intermediate washing elution until the curve of ultraviolet absorption value is reduced to stable, then stop washing, and the acetate buffer is potassium acetate buffer;

[0174] S104, using pH 3.6 acetate buffer to elute the affinity chromatography column, when the ultraviolet absorption value rises to 100mAU, start collecting the protein solution, when the ultraviolet absorption value drops to 100mAU, end the collection, and reserve.

[0175] In step S1, the collected protein solution is further subjected to pH adjustment after affinity chromatography, specifically including: the collected protein solution in step S1 is incubated at room temperature for 90min; the pH value of the protein solution is adjusted to 3.7 using 1M acetic acid solution; the pH value of the protein solution is adjusted back to 5.2 using 1M Tris buffer, and the filtrate is filtered through a 0.22μm filter and collected for ion exchange chromatography.

[0176] S2, ion exchange chromatography: it is composed of cation exchange chromatography and anion exchange chromatography, after the protein solution collected in step S1 is purified by cation exchange chromatography, it is directly purified by anion exchange chromatography to obtain the purified protein solution, and the outlet of the cation exchange chromatography column is communicated with the inlet of the anion exchange chromatography column.

[0177] In step S2, the protein solution collected in step S1 is purified by cation exchange chromatography, specifically including the following methods:

[0178] (1) using pH 5.2, 50mM acetate buffer to equilibrate the cation exchange chromatography column, the medium of the cation exchange chromatography column is NanoGel-50SP, the bed height of the cation exchange chromatography column is 21cm, and the acetate buffer is potassium acetate buffer; (2) the protein solution collected in step S1 is loaded onto the cation exchange chromatography column at a flow rate of 180cm / h; (3) using pH 5.7, 80mM acetate buffer to wash the cation exchange chromatography column; (4) using pH 7.6, 50mM phosphate buffer to elute the cation exchange chromatography column, when the ultraviolet absorption value rises to 100mAU, the eluate begins to be collected, and when the ultraviolet absorption value drops to 100mAU, the collection ends, and the phosphate buffer is potassium phosphate buffer.

[0179] In step S2, the purified protein solution is obtained by anion exchange chromatography, and the specific method comprises the following steps:

[0180] The eluate collected by the cation exchange chromatography is automatically loaded onto the anion exchange chromatography column at a flow rate of 180cm / h, the medium of the anion exchange chromatography column is NanoGel-50Q, and the bed height of the anion exchange chromatography column is 20cm; after the loading is completed, the anion exchange chromatography column is flushed with pH 7.6, 50mM phosphate buffer, when the ultraviolet absorption value rises to 100mAU, the purified protein solution begins to be collected, and when the ultraviolet absorption value drops to 200mAU, the collection ends, and the purified protein solution is obtained.

[0181] Comparative Example 1

[0182] The purification method of the anti-NKG2A monoclonal antibody provided in Comparative Example 1 of the application is the same as that in Examples 16 and 17, and the anti-NKG2A monoclonal antibody is the anti-NKG2A monoclonal antibody HA-1 screened in Examples 2-15.

[0183] Compared with Example 16, the cation exchange chromatography and the anion exchange chromatography are two-step chromatography without connection, the medium of the cation exchange chromatography column is Capto SP, the medium of the anion exchange chromatography column is Capto Q, and the other methods are all the same as those in Example 16.

[0184] Comparative Example 2

[0185] The purification method of the anti-NKG2A monoclonal antibody provided in Comparative Example 2 of the application is based on Example 16, and further limits the medium of the affinity chromatography column to be replaced by MabSelect SuRe LX of GE Company, and the other methods and parameters are all the same as those in Example 16.

[0186] Comparative Example 3

[0187] The application comparative example 3 provides a purification method of the anti-NKG2A monoclonal antibody, which is based on example 16, and the pH 5.5 and 60 mM acetate buffer used for washing the cation exchange column is deleted, and all other methods and parameters are the same as those in example 16.

[0188] Comparative example 4

[0189] The application comparative example 4 provides a purification method of the anti-NKG2A monoclonal antibody, which is based on example 16, and the pH 5.2 mixed buffer used for eluting the cation exchange column in the cation exchange chromatography is further limited, the mixed buffer comprises 40 mM sodium phosphate and 200 mM NaCl, and the sample needs to be subjected to liquid exchange treatment or dilution conductivity treatment before the anion exchange chromatography, and all other methods and parameters are the same as those in example 16.

[0190] Physical and chemical detection of the anti-NKG2A monoclonal antibody and related impurity detection

[0191] The anti-NKG2A monoclonal antibody purified by the purification method provided in examples 16 and 17 and comparative examples 1-4 of the application is detected by using gel chromatography technology means to detect the protein purity, analyze the content of aggregates, monomers and degradation products in the sample in the purification process; at the same time, ion chromatography technology means is used to analyze the content of charge isomer acid-base peaks.

[0192]

[0193]

[0194] The anti-NKG2A monoclonal antibody purified by the purification method provided in examples 16-17 and comparative examples 1-4 of the application is detected by using a special kit to detect the content of process-related impurities.

[0195]

[0196] The experimental results show that, compared with comparative example 1, examples 16 and 17 use integrated ion exchange chromatography, realize that the antibody recovery rate is greater than 97%, the final antibody purity is greater than 99.5%, and the process time is significantly shortened.

[0197] Compared with Comparative Example 2, Example 16 and 17 further limit the affinity chromatography medium, and the new generation of affinity chromatography medium NMab Pro of Nanjing Magway Bio-technology Co., Ltd. is used to capture the target protein in the cell culture solution in the purification process. Compared with the traditional affinity chromatography, the new generation of affinity chromatography medium NMab Pro has higher load, higher protein yield, excellent binding specificity, alkali resistance and pressure-flow rate characteristics, lower ligand shedding and host protein residue, and is equivalent to the imported MabSelect SuRe LX medium of GE Company in the control of protein purity and the ability of impurity removal. However, the production cost can be significantly reduced. The recovery rate of Example 16 and 17 is obviously higher than that of Comparative Example 2, and the impurity residue is obviously lower than that of Comparative Example 2.

[0198] Compared with Comparative Example 3, Example 16 further limits the pH 5.5, 60 mM acetate buffer used for washing the cation exchange chromatography column. The elution method can effectively control the HCP residue, remove the acidic isomer, improve the protein purity, and ensure that each residual impurity in the antibody protein product after the final purification meets the quality standard. Therefore, the elution step is essential.

[0199] Compared with Example 16, Comparative Example 4 changes the elution buffer of the cation exchange chromatography. The buffer can also effectively control the protein purity, but is easy to cause the collected protein solution to have a high conductivity value. The sample needs to be diluted or the solution needs to be replaced before the next purification, which greatly prolongs the process time. Therefore, the pH 7.6, 50 mM phosphate buffer provided by the purification process of Example 16 and 17 for eluting the cation exchange chromatography column can not only ensure the recovery rate and the protein purity, but also greatly shorten the purification process time.

[0200] The present application is not limited to the above best mode, and anyone can derive other various forms of products under the inspiration of the present application, but regardless of any changes in shape or structure, any technical solution with the same or similar technical solutions as the present application falls within the protection scope of the present application.

Claims

1. A method for purifying an anti-NKG2A monoclonal antibody, characterized in that, The purification method comprises: S1, affinity chromatography: using an affinity chromatography column to preliminarily purify and concentrate a cell liquid containing an anti-NKG2A monoclonal antibody, and collecting a protein solution; S2, ion exchange chromatography: composed of cation exchange chromatography and anion exchange chromatography, after the protein solution collected in step S1 is refined by the cation exchange chromatography, it is directly purified by the anion exchange chromatography to obtain the purified protein solution; The anti-NKG2A monoclonal antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises three heavy chain complementarity determining regions represented by HCDR1, HCDR2 and HCDR3 respectively, the light chain variable region comprises three light chain complementarity determining regions represented by LCDR1, LCDR2 and LCDR3 respectively, the amino acid sequence of the heavy chain complementarity determining region HCDR1 is shown as SEQ ID No: 1; the amino acid sequence of the heavy chain complementarity determining region HCDR2 is shown as SEQ ID No: 2; the amino acid sequence of the heavy chain complementarity determining region HCDR3 is shown as SEQ ID No: 3; the amino acid sequence of the light chain complementarity determining region LCDR1 is shown as SEQ ID No: 4; the amino acid sequence of the light chain complementarity determining region LCDR2 is shown as SEQ ID No: 5; and the amino acid sequence of the light chain complementarity determining region LCDR3 is shown as SEQ ID No:

6.

2. The method of purifying an anti-NKG2A monoclonal antibody according to claim 1, wherein, The anti-NKG2A monoclonal antibody is a murine antibody molecule, the amino acid sequence of the heavy chain variable region of the murine antibody molecule is shown as SEQ ID No: 7, and the amino acid sequence of the light chain variable region of the murine antibody molecule is shown as SEQ ID No:

8.

3. The method of purifying an anti-NKG2A monoclonal antibody according to claim 2, wherein the anti-NKG2A monoclonal antibody is an antibody according to any one of claims 1 to 2. The murine antibody molecule further comprises a heavy chain constant region selected from the group consisting of murine IgG1, IgG2a, IgG2b or IgG3, and a light chain constant region of murine C k type; wherein the amino acid sequence of the heavy chain constant region of IgG1 type is shown as SEQ ID No: 10, the amino acid sequence of the heavy chain constant region of IgG2a type is shown as SEQ ID No: 11, the amino acid sequence of the heavy chain constant region of IgG2b type is shown as SEQ ID No: 12, and the amino acid sequence of the heavy chain constant region of IgG3 type is shown as SEQ ID No: 13; and the amino acid sequence of the light chain constant region of murine C k type is shown as SEQ ID No:

9.

4. The method of purifying an anti-NKG2A monoclonal antibody according to claim 1, wherein, The anti-NKG2A monoclonal antibody is a humanized antibody molecule, the amino acid sequence of the heavy chain variable region of the humanized antibody molecule is shown as SEQ ID No: 14, and the amino acid sequence of the light chain variable region of the humanized antibody molecule is shown as SEQ ID No:

15.

5. The method for purifying anti-NKG2A monoclonal antibodies according to claim 4, characterized in that, The humanized antibody molecule further comprises a human antibody constant region, which comprises a heavy chain constant region selected from the group consisting of an IgGl type or an IgG4 type of human, and a light chain constant region of a human C k type, wherein the amino acid sequence of the heavy chain constant region of the IgGl type is as shown in SEQ ID No: 18, the amino acid sequence of the heavy chain constant region of the IgG4 type is as shown in SEQ ID No: 19, and the amino acid sequence of the light chain constant region of the human C k type is as shown in SEQ ID No:

20.

6. The method of purifying an anti-NKG2A monoclonal antibody according to claim 1, wherein, In step S1, affinity chromatography: using an affinity chromatography column to preliminarily purify and concentrate a cell liquid containing an anti-NKG2A monoclonal antibody, and collecting a protein solution, which specifically comprises the following steps: S101, using a Tris-hydrochloric acid buffer with a pH of 7.0-7.4 and a concentration of 50mM to equilibrate the affinity chromatography column, the medium of the affinity chromatography column is NMab Pro, the bed height of the affinity chromatography column is 15-21cm, and the Tris-hydrochloric acid buffer with a concentration of 50mM contains 150mM of sodium chloride; S102, loading the cell liquid containing the anti-NKG2A monoclonal antibody onto the affinity chromatography column at a flow rate of 60-180cm / h; S103, after the sample is loaded, the pH 5.0-5.6, 50mM acetate buffer is used for rebalancing, and after the ultraviolet signal is stable, the pH 5.0-5.6, 50mM acetate buffer is used for at least one intermediate elution, until the curve of the ultraviolet absorption value is reduced to stable, stop flushing, the acetate buffer is sodium acetate buffer or potassium acetate buffer; S104, using pH 3.4-3.6 acetate buffer to elute the affinity chromatography column, when the ultraviolet absorption value rises to 100mAU, start collecting the protein solution, when the ultraviolet absorption value drops to 100mAU, end collection, standby.

7. The method of purifying an anti-NKG2A monoclonal antibody according to claim 1, wherein the anti-NKG2A monoclonal antibody is an antibody according to any one of claims 2 to 6. In step S2, the protein solution collected in step S1 is purified by cation exchange chromatography, specifically including the following methods: (1) using pH 5.0-5.2, 30-50mM acetate buffer to balance the cation exchange chromatography column, the medium of the cation exchange chromatography column is NanoGel-50SP, the bed height of the cation exchange chromatography column is 15-21cm, and the acetate buffer is sodium acetate buffer or potassium acetate buffer; (2) the protein solution collected in step S1 is loaded onto the cation exchange chromatography column at a flow rate of 60-180cm / h; (3) using pH 5.5-5.7, 60-80mM acetate buffer to wash the cation exchange chromatography column; (4) using pH 7.4-7.6, 30-50mM phosphate buffer to elute the cation exchange chromatography column, when the ultraviolet absorption value rises to 100mAU, start collecting the eluate, when the ultraviolet absorption value drops to 100mAU, end collection, and the phosphate buffer is sodium phosphate buffer or potassium phosphate buffer.

8. The method for purifying anti-NKG2A monoclonal antibodies according to claim 7, characterized in that, In step S2, the purified protein solution is obtained by anion exchange chromatography, specifically including the following methods; The eluate collected by the cation exchange chromatography is loaded onto the anion exchange chromatography column at a flow rate of 60-180cm / h, the medium of the anion exchange chromatography column is NanoGel-50Q, and the bed height of the anion exchange chromatography column is 10-20cm; After the sample is loaded, the pH 7.4-7.6, 30-50mM phosphate buffer is used to flush the anion exchange chromatography column, and when the ultraviolet absorption value rises to 100mAU, the purified protein solution is collected, and when the ultraviolet absorption value drops to 200mAU, the collection is ended.

9. The method of purifying an anti-NKG2A monoclonal antibody according to claim 1, wherein, In step S1, the collected protein solution is further subjected to pH adjustment after affinity chromatography, specifically including: The protein solution collected in step S1 is incubated at room temperature for 60-90min; The pH value of the protein solution is adjusted to 3.5-3.7 using 1M acetic acid solution; The pH value of the protein solution is adjusted to 5.0-5.2 using 1M Tris buffer, and the filtrate is collected by filtering through a 0.22μm filter and entering the ion exchange chromatography standby.

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