CN115957319B

CN115957319B - Injection preparation of anti-NKG 2A monoclonal antibody

Info

Publication number: CN115957319B
Application number: CN202211256384.4A
Authority: CN
Inventors: 白义; 贾蒙蒙; 肖超
Original assignee: BEIJING JINGYI TAIXIANG TECHNOLOGY DEVELOPMENT CO LTD; Beijing Dongfang Baitai Biotechnology Co ltd
Current assignee: BEIJING JINGYI TAIXIANG TECHNOLOGY DEVELOPMENT CO LTD; Beijing Dongfang Baitai Biotechnology Co ltd
Priority date: 2022-10-14
Filing date: 2022-10-14
Publication date: 2023-06-30
Anticipated expiration: 2042-10-14
Also published as: CN115957319A

Abstract

The invention relates to the field of biological medicine, and in particular provides an injection preparation of an anti-NKG 2A monoclonal antibody, which comprises 120-200mg/ml of the anti-NKG 2A monoclonal antibody, 10-40mM of buffer salt, 50-200mM of protein protectant and 0.005-0.04% (w/v) of surfactant, wherein the pH value of the injection preparation is 5.5-6.5. The anti-NKG 2A monoclonal antibody provided by the invention can relieve the inhibition effect of NKG2A signal channels on immune cells through acting on NKG2A inhibitory receptors on the immune cells, and enhance the cell activities of NK cells, T cells and the like, thereby activating the killing of the immune cells of the NK cells and the like on tumors; the anti-NKG 2A monoclonal antibody preparation provided by the invention has the advantages of simple preparation process, low cost, higher concentration, better stability and the like.

Description

Injection preparation of anti-NKG 2A monoclonal antibody

Technical Field

The invention relates to the technical field of biological medicines, in particular to an injection preparation of an anti-NKG 2A monoclonal antibody.

Background

Tumors become the first factor threatening the life and health of people, and the problem of tumor intervention faces serious tests. With the continuous application and development of biomedical technology, immunotherapy has made a major breakthrough in the field of cancer treatment, and the focus of immunotherapy is to utilize the adaptive immune system in patients, and most importantly, to kill tumor cells by using cd8+ T, but more and more research data indicate that many cancers can develop various strategies to evade the recognition of cd8+ T cells, so that new immunotherapeutic approaches against cancers need to be developed.

NKG2A is a member of the C lectin superfamily, which forms an inhibitory receptor with CD94 in the form of a heterodimer, expressed on about half of peripheral blood NK cells, part of cd8+ αβt cells, NKT cells and γδ T cells. Non-classical HLA-class I molecules (human HLA-E or mouse Qa-1 molecules) are the primary ligands for NKG2A-CD94, which are often abnormally high expressed on tumor cells. Binding of the NKG2A-CD94 receptor to its ligand can inhibit the tumor inhibiting effect of NK cells and T cells, the intracellular part of NKG2A contains tyrosine inhibition motif (ITIM), after the NKG2A-CD94 receptor is bound to the ligand, phosphorylated ITIM can accumulate tyrosine phosphatase (SHP-1), and SHP-1 can transmit inhibitory signals to NK cells and T cells. High expression of NKG2A on tumor infiltrating NK cells and T cells and high expression of HLA-E on tumor cells are both positively correlated with poor prognosis of cancer, and currently, no drug is marketed against this target of NKG2A, so NKG2A/HLA-E has become an immune checkpoint for new tumor treatment and has been attracting attention.

In the process of technological research and development, the anti-NKG 2A monoclonal antibody is different from other antibodies in stability, is easy to generate conformational changes such as denaturation, aggregation, precipitation and other chemical and physical degradation, and can greatly influence the safety of the anti-NKG 2A monoclonal antibody, therefore, the invention provides an injection preparation of the anti-NKG 2A monoclonal antibody, which can ensure higher affinity, stronger drug effect and more stability of the anti-NKG 2A monoclonal antibody, in the process of technological research and development of the anti-NKG 2A monoclonal antibody.

Disclosure of Invention

In order to ensure the stability of the anti-NKG 2A monoclonal antibody in the long-term storage, transportation or use process, the invention provides an injection preparation of the anti-NKG 2A monoclonal antibody through a great deal of experimental conditions.

The specific technical scheme of the invention is as follows:

the invention provides an injection preparation of an anti-NKG 2A monoclonal antibody, which comprises the following components in percentage by weight:

wherein the pH value of the injection preparation is 5.5-6.5.

The injection preparation provided by the invention has higher antibody protein concentration, and provides a proper storage environment for the anti-NKG 2A monoclonal antibody through the synergistic cooperation of the buffer salt, the protein protectant and the surfactant, so that the drug effect and long-term stability of the anti-NKG 2A monoclonal antibody can be ensured.

Further, the anti-NKG 2A monoclonal antibody comprises a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is shown as SEQ ID No. 14, and the amino acid sequence of the light chain variable region is shown as SEQ ID No. 15.

Further, the anti-NKG 2A monoclonal antibody further comprises a heavy chain constant region and a light chain constant region, wherein the amino acid sequence of the heavy chain constant region is shown as SEQ ID No. 18, and the amino acid sequence of the light chain constant region is shown as SEQ ID No. 20.

Further, the protein content of the anti-NKG 2A monoclonal antibody is 120-150mg/ml.

Further, the buffer salt is citrate buffer or phosphate buffer.

Further, the buffer salt is contained in an amount of 20 to 30mM.

Further, the pH value of the injection preparation is 6.0-6.5.

Further, the protein protectant comprises one or more of sorbitol, mannitol, trehalose, sucrose, arginine, glycine and proline.

Further, the surfactant comprises one or more of polysorbate 20, polysorbate 80 and poloxamer.

Preferably, the surfactant is present in an amount of 0.01% to 0.02% (w/v).

The beneficial effects of the invention are as follows: the anti-NKG 2A monoclonal antibody provided by the invention can relieve the inhibition effect of NKG2A signal channels on immune cells through acting on NKG2A inhibitory receptors on the immune cells, and strengthen the cell activities of NK cells, T cells and the like, so as to activate the killing of the immune cells of the NK cells and the like on tumors, and the monoclonal antibody is a novel check point inhibitor for promoting anti-tumor immunity and is mainly used for preparing medicines for treating cancers or autoimmune diseases; in addition, anti-NKG 2A monoclonal antibodies may also be used in combination with immunomodulatory or anti-inflammatory agents to treat cancer or autoimmune diseases, especially when tumor cells escape killing of cd8+ T cells by low MHC-I expression, activated immune cells may kill tumor cells without MHC-I coordination, cancers including, but not limited to, colon, head and neck, non-small cell lung, pancreatic, renal, gastric, liver, ovarian, breast or melanoma; autoimmune diseases include, but are not limited to, systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, dermatomyositis, polymyositis, vasculitis, or xerosis; secondly, the preparation components suitable for long-term storage of the anti-NKG 2A monoclonal antibody are screened out through physicochemical properties brought by the anti-NKG 2A monoclonal antibody, and good storage environment is provided for the anti-NKG 2A monoclonal antibody through the interaction of buffer salt, protein protectant and surfactant and cooperative coordination, so that the generation rate of aggregates and degradation products of the antibody in the preparation can be effectively reduced in the storage and transportation processes, the physical stability of the antibody is improved, and the potential safety risk is reduced while the activity is ensured; the anti-NKG 2A monoclonal antibody preparation provided by the invention has the advantages of simple preparation process, low cost, higher concentration and better stability.

Drawings

FIG. 1 is a plasmid map of pScFv-Disb-HS vector in the method for screening an antibody organism provided in example 3 of the present invention;

FIG. 2 is a graph showing comparison of affinity of gradient dilution ELISA anti-NKG 2A phage monoclonal antibody in example 4 of the invention;

FIG. 3 is a map of the expression plasmid pTSE of the anti-NKG 2A whole-antibody provided in example 6 of the invention;

FIG. 4 is a graph showing the comparison of the binding ability of murine antibodies to NKG2A in example 7 of the present invention;

FIG. 5 is a graph showing the binding of the anti-NKG 2A monoclonal antibody MA-1 of example 8 of the invention to NKG2A, NKG2C, NKG E;

FIG. 6 is a graph showing the comparison of the binding ability of the humanized antibody molecule of example 13 of the present invention to NKG 2A;

FIG. 7 is a graph showing the effect of the anti-NKG 2A-monoclonal antibody of example 14 of the invention on activating human PBMC to kill HL-60;

FIG. 8 is a schematic diagram of an in vivo tumor suppression model of a monoclonal antibody of example 15 of the present invention.

Detailed Description

The invention will be described in further detail with reference to the following examples.

Example 1

The embodiment 1 of the invention provides an injection preparation of an anti-NKG 2A monoclonal antibody, which comprises the following components in percentage by weight:

wherein the pH value of the injection preparation is 5.5-6.5.

The anti-NKG 2A monoclonal antibody comprises a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is shown as SEQ ID No. 14, and the amino acid sequence of the light chain variable region is shown as SEQ ID No. 15.

SEQ ID No:14

EVQLVESGGGLVKPGGSLRLSCAASGFTFSDYDMSWVRQAPGKGLEWVSTISSGGSYTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCTSPRQVGLRKAFDYWGQGTTVTVSS；

SEQ ID No:15

DVVMTQSPAFLSVTPGEKVTITCSASSSVSYMYWYQQKPDQAPKLLIKLTSNLASGVPSRFSGSGSGTDFTFTISSLEAEDAATYYCQQWSSNPFTFGQGTKLEIK。

Example 2

The embodiment 2 of the invention provides an injection preparation of an anti-NKG 2A monoclonal antibody, which comprises the following components in percentage by weight:

wherein the pH value of the injection preparation is 6.0-6.5.

The buffer salt is citrate buffer or phosphate buffer; the protein protectant comprises one or more of sorbitol, mannitol, trehalose, sucrose, arginine, glycine and proline; the surfactant comprises one or more of polysorbate 20, polysorbate 80 and poloxamer.

Example 3 preparation of antigen and control antibody and construction and screening of phage antibody library

According to the invention, a mouse is immunized by using an NKG2A antigen (NKG 2A protein extracellular segment), an immunization method is optimized, a phage display library is created, an antigen site screening method is established, and the construction, screening and identification of the phage display library are as follows:

1. NKG2A antigen immunized mice:

experimental animals:

species strain: BALB/c, female, mouse;

weight of: 18-20g;

experimental animal provider: beijing Fukang biotechnology Co., ltd.

2. Immunization: mice were immunized with human NKG2A (a synthetic gene from the company, inc. Biotechnology, gold-sri, south kyo) as the immunizing antigen, and the company constructed the vector and expressed and purified.

3. Construction and screening of phage antibody library

The method comprises the steps of taking mouse spleen cells with higher titer, extracting total RNA in the mouse spleen cells by using Trizol reagent (purchased from Ambion, cat# 15596026), obtaining cDNA by RT-PCR, carrying out PCR amplification by using the cDNA as a template and degenerate primers (used in degenerate primer reference: journal of Immunological Methods (2000) 167-177) so as to obtain an immune mouse antibody heavy chain variable region gene library (VH) and a light chain variable region gene library (VL), respectively carrying out double enzyme digestion on the light chain and heavy chain, connecting the heavy chain and the light chain variable region gene library and the VL to a vector subjected to the same step-by-step enzyme digestion, and constructing a pScFv-Disb-VL gene library, wherein the PscFv-Disb-HS vector is obtained by modifying a vector pComb3 vector (purchased from a Chinese plasmid vector strain gene collection center) by adopting a series of gene cloning method, so that the vector pComb3 vector is used for constructing and expressing a phage single chain antibody library. The transformed vector is named pScFv-Disb-HS vector, the plasmid map of which is shown in figure 1 is obtained, and a mouse immune phage antibody library is constructed based on the vector.

4. Enrichment screening of phage libraries

The immune tube was coated with huFc-NKG2A/CD94 as antigen in an amount of 4. Mu.g/500. Mu.l/tube, coated overnight at 4℃and the immune tube and immune phage antibody library were blocked with 4% skim milk powder/PBST, respectively, at room temperature for 1h. Adding the blocked immune phage antibody library into immune tube to combine antigen and antibody, and adding phage with input of about 10 ⁹ ～10 ¹² After 1 hour of reaction at room temperature, unbound phage was washed off with PBST-and eluted with 0.1M Glycine-HCl at pH2.2, and finally the eluted phage antibody solution was neutralized with 1.5M Tris-HCl at pH 8.8 to about pH 7.0.

10ml of TG1 bacterial liquid growing to logarithmic phase after the neutralization of phage infection is placed in an incubator at 37 ℃ for 30min, part of bacterial liquid is taken out for gradient dilution and coated on a 2YTAG plate, and the phage yield is calculated. The remaining bacterial liquid was centrifuged to discard the supernatant, the bacterial pellet was resuspended in a small amount of medium, aspirated and spread on a 2YTAG large plate, ready for the next round of screening.

Scraping the infected thallus coated with the plate from a large plate, inoculating the thallus to a 2YTAG liquid culture medium, shaking the thallus to a logarithmic phase, adding M13KO7 auxiliary phage for superinfection, culturing the bacteriophage at 220rpm overnight at 28 ℃ to prepare phage, and settling and purifying the phage by PEG/NaCl for the next round of screening. Three rounds of phage library enrichment screening were performed together.

5. Screening of phage Single-chain antibody Positive clones

After three rounds of screening, selecting well-separated monoclonal colonies, inoculating to a 96-well deep-hole plate with 2YTAG liquid culture medium, culturing at 37deg.C and 220rpm to logarithmic phase, and adding about 10 per well ¹⁰ Is statically infected with helper phage M13KO7 at 37℃for 30min.4000rpm, centrifuging for 15min, discarding the supernatant, re-suspending the pellet with 2YTAK, and culturing overnight at 28deg.C and 220 rpm. After centrifugation at 4000rpm at 4℃for 15min, the amplified phage supernatant was aspirated for ELISA identification:

antigen huFc-NKG2A/CD94 was coated at a concentration of 0.5. Mu.g/ml, primary antibody was 100. Mu.l 1:3 diluted phage supernatant, and blocking solution was used as negative control. After incubation of HRP-anti-M13 secondary antibody, TMB substrate is used for color development, OD450nm is detected in an enzyme labeling instrument after 20min of color development, and the reading value is more than 0.3, and positive clone is considered.

Antibody-specific primary screening was performed using positive clone supernatants: binding of each phage supernatant to the different antigens huFc-NKG2A/CD94, huFc-NKG2C/CD94 or huFc-NKG2C/CD94 was detected by ELISA as described above, and subsequent screening was performed by selecting a monoclonal molecule having an OD450nm reading higher than three times the reading when the supernatant bound to NKG2A than when the supernatant bound to NKG2C/E, at which time 1 monoclonal molecule was co-screened and designated as anti-NKG 2A monoclonal antibody MA-1.

An anti-NKG 2A monoclonal antibody MA-1 comprising a heavy chain variable region comprising 3 heavy chain complementarity determining regions represented by HCDR1, HCDR2 and HCDR3, respectively, and a light chain variable region comprising 3 light chain complementarity determining regions represented by LCDR1, LCDR2 and LCDR3, respectively, the amino acid sequence of heavy chain complementarity determining region HCDR1 being shown in SEQ ID No. 1; the amino acid sequence of the heavy chain complementarity determining region HCDR2 is shown in SEQ ID No. 2; the amino acid sequence of the heavy chain complementarity determining region HCDR3 is shown in SEQ ID No. 3; the amino acid sequence of the light chain complementary determining region LCDR1 is shown as SEQ ID No. 4; the amino acid sequence of the light chain complementary determining region LCDR2 is shown as SEQ ID No. 5; the amino acid sequence of the light chain complementarity determining region LCDR3 is shown in SEQ ID No. 6, and the specific sequence is as follows.

The heavy chain variable region sequence and the light chain variable region sequence of the anti-NKG 2A monoclonal antibody MA-1 are as follows:

specifically, SEQ ID No. 7 (amino acid sequence of the heavy chain variable region of MA-1):

EVKLEESGGGLVKPGGSLKLSCAASGFAFSDYDMSWVRQTPEKRLEWVATISSGGSYTYYPDSVKGRFTISRDNARNTLYLQMSSLRSEDTALYYCTSPRQVGLRKAFDYWGQGTTLTVSS；

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

DIVVTQSPALMSASPGEKVTMTCSASSSVSYMYWYQQKPRSSPKSWIYLTSNLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPFTFGSGTKLEIKRAD。

example 4 gradient dilution ELISA comparison of affinity of anti-NKG 2A phage monoclonal antibody

The murine antibody molecule (MA-1) obtained in example 3 was subjected to monoclonal phage display and purification, followed by phage gradient dilution ELISA experiments to identify affinity, as follows:

coating NKG2A antigen with carbonate buffer solution of pH9.6, coating 100 ng/well/100. Mu.L, overnight at 4deg.C, washing three times with PBST, and subjecting phage monoclonal antibody MA-1 selected in example 2 to five-fold gradient with PBST, respectivelyDiluted, 100. Mu.l of diluted sample was added to each well, and the mixture was allowed to stand at room temperature for 1 hour. The ELISA plate was washed with PBST, and the HRP-anti-M13 (purchased from Bio-view stone, cat# GE 27-9421-01) monoclonal antibody diluted with PBST was added to the ELISA plate and left at room temperature for 1h. TMB chromogenic kit developed, developed at room temperature for 10 min, with 2M H ₂ SO ₄ After termination, the microplate reader reads at 450nm and calculates the corresponding EC50 values as follows:

through the data and as shown in FIG. 2, the murine antibody molecule MA-1 screened in example 2 was able to bind to NKG2A, and both the murine antibody molecule MA-1 provided by the invention and NKG2A have higher affinity.

Example 5

Example 5 of the present invention further defines on the basis of example 3 that the murine antibody molecule further comprises a heavy chain constant region selected from the group consisting of murine IgG1, igG2a, igG2b, or IgG3 and murine C _k A light chain constant region of the 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; mouse C _k The amino acid sequence of the constant region of the light chain is shown as SEQ ID No. 9; the specific sequence is as follows:

SEQ ID No. 9 (mouse C) _k Light chain constant region amino acid sequence):

ADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC；

SEQ ID No. 10 (murine heavy chain constant region amino acid sequence of IgG1 type):

AKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPG；

SEQ ID No. 11 (murine heavy chain constant region amino acid sequence of IgG2a type):

AKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK；

SEQ ID No. 12 (murine heavy chain constant region amino acid sequence of IgG2b type):

AKTTPPSVYPLAPGCGDTTGSSVTLGCLVKGYFPESVTVTWNSGSLSSSVHTFPALLQSGLYTMSSSVTVPSSTWPSQTVTCSVAHPASSTTVDKKLEPSGPISTINPCPPCKECHKCPAPNLEGGPSVFIFPPNIKDVLMISLTPKVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTIRVVSTLPIQHQDWMSGKEFKCKVNNKDLPSPIERTISKIKGLVRAPQVYILPPPAEQLSRKDVSLTCLVVGFNPGDISVEWTSNGHTEENYKDTAPVLDSDGSYFIYSKLNMKTSKWEKTDSFSCNVRHEGLKNYYLKKTISRSPGK；

SEQ ID No. 13 (heavy chain constant region amino acid sequence of murine IgG3 type):

ATTTAPSVYPLVPGCSDTSGSSVTLGCLVKGYFPEPVTVKWNYGALSSGVRTVSSVLQSGFYSLSSLVTVPSSTWPSQTVICNVAHPASKTELIKRIEPRIPKPSTPPGSSCPPGNILGGPSVFIFPPKPKDALMISLTPKVTCVVVDVSEDDPDVHVSWFVDNKEVHTAWTQPREAQYNSTFRVVSALPIQHQDWMRGKEFKCKVNNKALPAPIERTISKPKGRAQTPQVYTIPPPREQMSKKKVSLTCLVTNFFSEAISVEWERNGELEQDYKNTPPILDSDGTYFLYSKLTVDTDSWLQGEIFTCSVVHEALHNHHTQKNLSRSPELELNETCAEAQDGELDGLWTTITIFISLFLLSVCYSASVTLFKVKWIFSSVVQVKQTAIPDYRNMIGQGA。

EXAMPLE 6 preparation of murine Total anti molecules

Example 6 of the present invention on the basis of example 3, plasmids were extracted from the monoclonal antibody MA-1 screened and identified in example 3, and the genes encoding heavy chain VH and light chain VL were cloned into a mouse-equipped IgG2a heavy chain (amino acid sequence shown as SEQ ID No: 11) and mouse C, respectively _k Vector pTSE of light chain constant region (amino acid sequence shown as SEQ ID No. 9) gene, pTSE vector structure is shown in FIG. 3 (see page 3 [0019 ] of CN103525868A description for pTSE vector preparation process)]Segments).

HEK293E cells (purchased from the institute of basic medicine, national academy of sciences, under the trade designation GNHu 43) were transiently transfected for antibody expression and purified by protein A affinity column using an AKTA instrument to obtain complete monoclonal antibody molecules.

EXAMPLE 7 binding Capacity of murine antibody to NKG2A

NKG2A,100 ng/well/100. Mu.l, was coated with carbonate buffer at pH9.6 at a temperature of 4℃overnight. Washing with 300 μl/well PBST for five times, adding 1% BSA-PBST, blocking at 37deg.C for 1 hr, adding MA-1 murine antibody with different dilution concentration, initial maximum concentration of whole antibody is 5 μg/ml, respectively diluting 3 times, making 12 gradients of each antibody, and incubating at 37deg.C for 1 hr. Five washes with 300. Mu.l/well PBST were performed, and Anti-Mouse Fc-HRP diluted with 1% BSA-PBST 1:10000 was added and incubated for 1h at 37 ℃. TMB chromogenic kit, 100. Mu.l/well, room temperature for 8min, then 2MH ₂ SO ₄ The color development was terminated. Read at 450nm and calculate the corresponding EC50 values, data shown in figure 4.

As can be seen from FIG. 4, the screened murine antibody MA-1 was able to bind to NKG2A with higher affinity.

EXAMPLE 8 experiments on the binding specificity of murine anti-NKG 2A monoclonal antibody to NKG2A, NKG2C, NKG2E

The monoclonal antibody MA-1 obtained in example 6 was tested for its EC50 value binding to huFc-NKG2A/CD94, huFc-NKG2C/CD94 or huFc-NKG2E/CD94 by ELISA, wherein both NKG2C (extracellular portion of the NKG2C protein) and NKG2E (extracellular portion of the NKG2E protein) were identical to the sequence of NKG2A and were NK-cell-activated receptors:

the anti-NKG 2A monoclonal antibody MA-1 is used as a primary antibody by gradient dilution, the anti-mouse Ig-HRP is used as a secondary antibody, the primary antibody is respectively combined with NKG2A, NKG C and NKG2E, a dose-reading curve is performed after ELISA detection, a nonlinear regression curve is performed, the EC50 value of each antibody respectively combined with three antigens is calculated, and the combination curve of the anti-NKG 2A monoclonal antibody MA-1 and the NKG2 protein is shown in figure 5.

As shown in FIG. 5, the anti-NKG 2A monoclonal antibody MA-1 selected according to the invention can specifically bind to NKG2A but not to other NKG2 family proteins such as NKG2C and NKG 2E.

Example 9

Example 9 of the present invention further defines the monoclonal antibody or antigen binding fragment thereof as a chimeric antibody molecule comprising a heavy chain variable region of a murine antibody molecule, a light chain variable region of a murine antibody molecule, and a human antibody constant region. The human antibody constant region includes a heavy chain constant region selected from the group consisting of human IgG1 or IgG4 type and human C _k The amino acid sequence of the light chain constant region of the type IgG1 and the heavy chain constant region of the type IgG4 is shown as SEQ ID No. 18, the amino acid sequence of the heavy chain constant region of the type IgG4 is shown as SEQ ID No. 19, and the human C is _k The amino acid sequence of the constant region of the light chain is shown as SEQ ID No. 20;

SEQ ID No. 18 (heavy chain constant region amino acid sequence of human IgG1 type):

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK；

SEQ ID No. 19 (heavy chain constant region amino acid sequence of human IgG4 type):

ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK；

SEQ ID No. 20 (human C _k Light chain constant region amino acid sequence of chain):

RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC。

EXAMPLE 10 preparation of chimeric antibody molecules

Example 10 of the present invention further preferred human antibody constant region based on example 9 comprises a heavy chain constant region of human IgG1 type (ammonia thereofThe base acid sequence is shown as SEQ ID No. 18) and human C _k A light chain constant region of the type (the amino acid sequence of which is shown as SEQ ID No. 20).

The specific preparation method comprises the following steps:

the heavy chain variable region VH (SEQ ID No: 7) and the light chain variable region VL gene (SEQ ID No: 8) of the selected ideal anti-NKG 2A monoclonal antibody molecule MA-1 are kept unchanged in murine sequences and cloned into the vector pTSE (shown in FIG. 3) harboring the heavy chain constant region and the light chain constant region gene, respectively, the heavy chain constant region being of human IgG1 type (amino acid sequence shown in SEQ ID NO: 18) and the light chain constant region being of human C _k Type (amino acid sequence shown as SEQ ID NO: 20). HEK293E cells (purchased from the institute of basic medicine of the national academy of sciences of medicine, accession number GNHu 43) were transiently transfected and antibody expression was performed to obtain chimeric antibody CA-1.

EXAMPLE 11 humanization of the murine antibody molecule MA-1

Firstly, comparing the sequence of the murine antibody molecule MA-1 in the example 3 with a human antibody germline database (v-base), searching human antibody light and heavy chain germline with higher homology as candidate sequences, and then transplanting the sequence of the CDR of the murine antibody molecule MA-1 to the human candidate sequences for homologous modeling. The back mutations of the humanized antibodies were then designed by three-dimensional structure modeling to calculate key framework amino acid residues that might play an important role in maintaining the CDR loop structure. The light chain variable region and the heavy chain variable region of the designed humanized antibody containing the back mutation are respectively synthesized by the Nanjing Jinsri biotechnology Co., ltd, then are connected to a transient expression vector, and the light chain and heavy chain combination analysis obtained by humanization is carried out to obtain the following humanized antibody molecules: HA-1, HA-2, the sequences of the 2 monoclonal antibodies selected above were as follows: :

specifically, SEQ ID No. 14 (amino acid sequence of the heavy chain variable region of HA-1):

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

EVQLVESGGGLVKPGGSLRLSCAASGFAFSDYDMSWVRQAPGKGLEWVATISSGGSYTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCTSPRQVGLRKAFDYWGQGTTVTVSS；

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

DVVMTQSPAFLSVTPGEKVTITCSASSSVSYMYWYQQKPDQAPKLLIKLTSNLASGVPSRFSGSGSGTDFTFTISSLEAEDAATYYCQQWSSNPFTFGQGTKLEIK；

SEQ ID No. 17 (amino acid sequence of the light chain variable region of HA-2):

DIVVTQSPAFLSVTPGEKVTITCSASSSVSYMYWYQQKPDQAPKLLIKLTSNLASGVPSRFSGSGSGTDFTFTISSLEAEDAATYYCQQWSSNPFTFGQGTKLEIK。

EXAMPLE 12 preparation of humanized Total anti molecules

Example 12 of the present invention further defines on the basis of example 11 that the humanized antibody molecule further comprises a human antibody constant region comprising a heavy chain constant region selected from the group consisting of human IgG1 or IgG4 and human C _k The amino acid sequence of the light chain constant region of the type IgG1 and the heavy chain constant region of the type IgG4 is shown as SEQ ID No. 18 and SEQ ID No. 19 respectively, and human C _k The amino acid sequence of the light chain constant region of the type is shown as SEQ ID No. 20.

The specific sequence of the constant region of the human antibody is the same as that of example 9.

The genes encoding the heavy chain VH and the light chain VL of the 2 humanized antibody molecules obtained in example 11 above were cloned into vector pTSE (shown in FIG. 3) harboring the heavy chain constant region and the light chain constant region genes, respectively, and each humanized antibody molecule selected from the heavy chain constant region of the IgG1 type (amino acid sequence shown in SEQ ID No: 19), and human C was selected _k The light chain constant region with the amino acid sequence shown as SEQ ID No. 20 is used for preparing full antibody molecules, HEK293E cells (purchased from the national institute of medicine and the basic science, code number GNHu 43) are transiently transfected, antibody expression is carried out, and 1 single antibody is obtained by purifying protein A affinity column by using an AKTA instrumentThe cloned antibodies are specifically as follows.

EXAMPLE 13 binding experiments of humanized Total antibody molecules to NKG2A protein

The NKG2A antigen was coated with 200 ng/well/100. Mu.L in carbonate buffer pH9.6 at a temperature of 4℃overnight. Five washes with 300. Mu.L/well PBST, 1% BSA-PBST was added and blocked at 37℃for 1 hour, humanized antibodies HA-1, HA-2 of different dilution concentrations and chimeric antibodies CA-1,3 antibodies prepared in example 10 were added, each having an initial maximum concentration of 50. Mu.g/mL, each antibody was subjected to 10 gradients after 3-fold dilution, and incubated at 37℃for 1 hour. Five washes with 300. Mu.L/well PBST were performed, and Goat Anti Human IgG-HRP (purchased from Abies media Biotechnology Co., ltd., product number ZB-2304) diluted with 1% BSA-PBST 1:5000 was added thereto and incubated at 37℃for 1 hour. TMB chromogenic kit, 100. Mu.L/well, 5min at room temperature, then 2M H ₂ SO ₄ The color development was terminated. The microplate reader was read at 450nm and the corresponding EC50 values were calculated, and the experimental results are shown in fig. 6.

As can be seen from FIG. 6, 2 different humanized antibody molecules were able to bind to NKG2A, and the EC50 values of the 2 humanized antibody molecules were all closer to that of chimeric antibody CA-1, indicating that the humanized antibody molecules retained the high binding capacity of murine parent antibody MA-1 and NKG 2A.

Example 14 detection of in vitro killing Effect of anti-NKG 2A monoclonal antibody

HL-60 tumor cells with high HLA-E expression are selected as target cells, human PBMC cells are selected as effector cells, the densities of the target cells and the effector cells are adjusted, and the target ratio is 50 after uniform mixing in equal volume: 1, add to the corresponding position in 96 well plate. Samples of the different antibodies were diluted and added to the plate to give final concentrations of 50. Mu.g/ml. CO at 37 DEG C ₂ After incubation for 18h in the incubator, the supernatant was taken to detect LDH release, and the cell killing rate was calculated, and the result is shown in fig. 7.

As can be seen from FIG. 7, the anti-NKG 2A monoclonal antibodies HA-1 and HA-2 provided by the invention can activate and promote the killing effect of human PBMC cells on HL-60 tumor cells.

EXAMPLE 15 experiment of inhibition of MC38-NKG2A colorectal cancer by the anti-NKG 2A monoclonal antibody HA-1 in mice

1. Experimental animals:

species strain: c57BL/6JGpt, mice;

week-old: for 6-8 weeks;

experimental animal provider, baiosema (Beijing) pharmaceutical technologies Co., ltd.

2. Cell culture:

MC38 tumor cells (YK-CL-256-02) (purchased from general Biovector NTCC Inc. of Beijing Co., ltd., product number NTCC-MC 38) were used as primordial cells to construct MC38-NKG2A tumor cell lines.

The MC38 tumor cells are stably transformed by subcutaneous inoculation on the back of the right side of each mouse, and 18 mice with the approximate tumor size are randomly divided into three groups after the tumor formation, and 6 mice are selected from each group. The anti-NKG 2A monoclonal antibody HA-1 (10 mg/kg) provided in the present invention was intraperitoneally injected, while a control group was set up to inject an equivalent amount of Z270 control group, the control antibody Z270 and VH and VL sequences of its humanized antibody huZ (from patent US8993319B 2), a negative control group was injected with an equivalent amount of isotype IgG, administered twice a week, tumor volumes were measured twice a week and body weights were measured. Observing and recording tumor growth, and measuring tumor volume exceeding 3000mm ³ Mice were sacrificed by cervical scission, individual tumors were carefully isolated with surgical scissors forceps and weighed. The results are shown in FIG. 8.

As shown in fig. 8, the tumor volume of the anti-NKG 2A monoclonal antibody HA-1 injected was significantly smaller than that of the negative control group, and the tumor volume of the anti-NKG 2A monoclonal antibody HA-1 injected was closer to that of the control group antibody Z270. It can be seen that the anti-NKG 2A monoclonal antibody HA-1 of the invention can significantly inhibit the growth of tumor.

EXAMPLE 16 screening of formulation buffers

The preparation method of the antibody preparation comprises the following steps: the stock solution of the anti-NKG 2A monoclonal antibody HA-1 obtained above is changed into the target buffer salt through a ultrafiltration tube, the changed solution is concentrated, the sample is diluted to the required concentration, the sample is sterile filtered by a 0.22 mu m filter, and the sample is packaged into 2ml penicillin bottles with the concentration of 1ml per bottle. And after the split charging is finished, detecting the thermal stability of the protein, simultaneously carrying out a stability test at 40+/-2 ℃, and screening proper pH and type of the buffer solution by taking purity and charge isomers as key indexes.

The analysis and detection method comprises the following steps: thermal stability: detecting a thermal denaturation temperature (Tm) and an aggregation temperature (Tagg) by using a multifunctional protein stability analysis system (Uncle);

and (3) purity detection: analyzing by adopting a size exclusion chromatography high performance liquid chromatography (SEC-HPLC);

charge isomers: the charge isomer main peak content (CEX-HPLC) was determined by cation exchange chromatography.

Injection formulation pH range screening: screening was performed at the pH range commonly used for commercial protein formulations. Protein conformational stability (Tm) and colloidal stability (Tagg) were examined by a multifunctional protein stability analysis system (Uncle), protein purity (SEC-HPLC) and charge isomer (CEX-HPLC) main peak content changes were examined by accelerated stability at 40+ -2deg.C, and a relatively stable pH range was screened.

(1) The appropriate buffer pH was screened as follows:

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the thermal stability test results were as follows:

accelerated stability test results at 40+ -2deg.C

The thermal stability result shows that the buffer solution pH is 4.5, the Tm and Tagg of the protein are lower than those of other buffer solutions, and the protein has poor conformational stability and colloid stability under the condition of pH 4.5.

The results of the accelerated stability show that the aggregates are increased to different degrees when the aggregates are placed for 2 weeks at 40+/-2 ℃, the increase rate of the experimental example 1 is the greatest (increase of 6.1 percent), and the experimental example is 5 times (increase of 2.4 percent); the purity of the monomer is reduced to different degrees, the reduction ratio of the experimental example 5 is the most (8.4 percent), the experimental example is 1 time (7.0 percent), and the other prescriptions are reduced by about 2 percent; the drop Jie Feng is increased to different degrees, the increase proportion of experimental example 5 is the maximum (about 5% increase), and other prescriptions are increased by about 1%; the main peak of the charge isomer of each prescription sample is obviously reduced, the reduction ratio of the experimental example 5 is the most (64.2 percent), the reduction ratio of the experimental example 4 times is about 32 percent, the reduction ratio of the experimental example 2 and the experimental example 3 is the least, and the reduction ratio of the experimental example 2 and the experimental example 3 is about 14 percent; the pH, the particle size and the concentration are not obviously changed. Preliminary results showed that the anti-NKG 2A monoclonal antibody HA-1 was relatively stable at a pH ranging from 5.5 to 6.5.

(2) Screening of buffer salt types and buffer salt molar concentrations of preparation

Selecting buffer salt with buffer capacity of about 5.5-6.5, developing buffer salt types and buffer solution molar concentration, inspecting protein conformation stability and colloid stability mainly through thermal stability (Tm, tagg) in this experiment, and screening a preparation buffer system favorable for stable storage of an anti-NKG 2A monoclonal antibody HA-1 by using protein purity (SEC-HPLC) and charge isomer (CEX-HPLC) as key indexes through an acceleration stability test at 40+/-2 ℃.

The buffer salt design composition was as follows:

the thermal stability test results were as follows:

accelerated stability test results at 40+ -2deg.C

The results of the thermostability show that in the pH range of 5.5-6.5, the Tm and Tagg of the protein are higher in acetate buffer, citrate buffer, histidine buffer and phosphate buffer.

The results of the accelerated stability show that the purity of each experimental example is reduced to different degrees when the experimental example is placed for 2 weeks at 40 ℃, the reduction ratio of the purity of the experimental example 9 and the purity of the experimental example 16 are the least (reduced by 1.6 percent), and the reduction ratio of the purity of the experimental example 13 is the most (reduced by 3.1 percent); the decrease in the monomer purity was gradually increased with increasing pH, and the decrease in the monomer purity was slightly smaller in examples 8 to 10 than in the other examples. After the sample was left at 40.+ -. 2 ℃ for 2 weeks, the main peak content of each sample was decreased, the decrease rate of the sample in the test example 9 was at least 12.0%, and the decrease rate of the sample in the test example 6 was at most 17.7%. Comprehensive investigation results show that under the condition of pH6.0-6.5, the stable characteristics of the protein in a citrate buffer system and a phosphate buffer system can be effectively maintained, and the citrate buffer system is superior to the phosphate buffer system; a buffer concentration of 20-30mM is better than 40 mM; the buffer at pH6.0 is preferred over pH6.5, for which reason the buffer salt is preferably citrate buffer or phosphate buffer, the buffer salt concentration preferably being 20-30mM, the pH preferably being 6.0.

Example 17 screening of protein protectants and surfactants.

The preparation method of the preparation provided in example 17 of the present invention is the same as that of example 16.

The analysis and detection method comprises the following steps: thermal stability: the multifunctional protein stability analysis system detects thermal denaturation temperature (Tm) and aggregation temperature (Tagg); and (3) purity detection: analyzing by adopting a size exclusion chromatography high performance liquid chromatography (SEC-HPLC); charge isomers: the charge isomer main peak content (CEX-HPLC) was determined by cation exchange chromatography. B22 (second dimension coefficient): the multifunctional protein stability analysis system detects the static scattering light intensity under different concentration conditions. Sub-visible particles: and detecting by adopting a microfluidic imaging method.

(1) Screening of protein protectants

The screening of the protein protectant and the surfactant is carried out on the basis of the previous round of buffer salt screening by an accelerated stability experiment at 40+/-2 ℃. The buffer is 20mM citrate buffer, pH6.0, protein concentration 150mg/ml, formulation prescription composition design is shown in the following table:

accelerated stability test results at 40+ -2deg.C

The results of the accelerated stability show that the sample aggregate of each experimental example grows to different degrees from the purity point of view when the sample aggregate is placed for 4 weeks at 40 ℃, wherein the experimental example 24 grows the most by about 3%, the experimental example 21 and the experimental example 22 grow the least by about 0.5%; the purity of the monomer is reduced to different degrees, wherein the reduction of the experimental example 21, the experimental example 22 and the experimental example 28 is least, the reduction is about 2%, the reduction of the purity of the experimental example 24 is most, and the reduction is 4.8%; from the charge isomer, the main peak content of the charge isomer is reduced by between 14.3% and 18.3%, wherein the main peak reduction ratio of experimental example 31 is the least, the reduction ratio is 14.3%, and the reduction ratio of experimental example 25 is the most, and the reduction ratio is 18.3%.

From the view of the sub-visible particles, the total particle number and the particle number larger than 25um in different experimental examples are increased to different degrees, the particle number larger than 25um in experimental examples 32-35 is obviously less than that in other experimental examples, and the surfactant plays a role in protecting proteins.

From a combination of examination results, it is shown that the protein protectant defined by the invention comprises one or more of sorbitol, mannitol, trehalose, sucrose, arginine, glycine and proline, which can protect against the NKG2A monoclonal antibody HA-1, and in addition, the surfactant-added experimental examples (experimental example 32, experimental example 33 and experimental example 34) show better sub-visible particles (the sample added with polysorbate 20 is relatively better), for which the surfactant is preferably polysorbate 20.

(2) Screening of complete Experimental examples

Through early preparation evaluation and screening, a citrate buffer solution is selected as a preparation buffer system, trehalose is used as a protein protecting agent, sorbitol is used as a candidate protein protecting agent, polysorbate 20 is used as a surfactant, and the content of the surfactant and a candidate experimental example are screened through an accelerated stability test at 40+/-2 ℃. The composition design of the experimental examples of the preparation is shown in the following table:

accelerated stability test results at 40+ -2deg.C

Can be obtained by Tagg test resultsThe anti-NKG 2A monoclonal antibody HA-1 HAs higher Tm value and Tagg in a citrate buffer system; the anti-NKG 2A monoclonal antibody HA-1 HAs better conformational stability and colloid stability in citrate buffer; through B ₂₂ As can be seen from the experimental results, sample B of each experimental example ₂₂ The values are positive, and the anti-NKG 2A monoclonal antibody HA-1 HAs weak repulsive force in the solution, and the weak repulsive force is equivalent.

The results of the accelerated stability show that the anti-NKG 2A monoclonal antibody preparation has good stability after being placed for 4 weeks at 40+/-2 ℃ and being added with the non-ionic surfactant polysorbate 20 with different concentrations. When the content of the nonionic surfactant is too low, the particle number of the nonionic surfactant is more than or equal to 25 microns, the antibody protein tends to aggregate, and when the content of the nonionic surfactant is too high, side effects can be generated, so that the infusion safety of the preparation is affected. The content of the surfactant defined in the invention is 0.01% -0.02% (w/v) so as to meet the stability requirement of the preparation, and further preferably, the content of the surfactant in the antibody preparation is 0.02% of polysorbate 20.

EXAMPLE 18 Experimental example confirmation of anti-NKG 2A monoclonal antibody

The preparation method of the preparation provided in example 18 of the present invention is the same as that of example 16.

We selected citrate buffer as buffer system, trehalose as protein protectant, and polysorbate 20 as surfactant at 0.02% to confirm the experimental examples.

The composition design of the experimental examples of the preparation is shown in the following table:

the analysis and detection method comprises the following steps:

thermal stability: the multifunctional protein stability analysis system detects thermal denaturation temperature (Tm) and aggregation temperature (Tagg); and (3) purity detection: analyzing by adopting a size exclusion chromatography high performance liquid chromatography (SEC-HPLC); charge isomers: the charge isomer main peak content (CEX-HPLC) was determined by cation exchange chromatography. Sub-visible particles: and detecting by adopting a microfluidic imaging method. The prescription confirmation test includes an acceleration test (40.+ -. 2 ℃), a freeze-thaw stability test (-20 ℃), a shaking stability test (25.+ -. 2 ℃ C., 120 rpm) and an illumination stability test (25.+ -. 2 ℃ C., RH60%, 4500.+ -. 500 lx).

Accelerated stability test results

The result of the accelerated stability shows that when the anti-NKG 2A monoclonal antibody HA-1 is placed for 4 weeks at the temperature of 40+/-2 ℃ and the concentration is 150mg/ml and 120mg/ml, no obvious difference exists in the aspects of purity, charge isomer, number of sub-visible particles which are more than or equal to 25um and the like; at a concentration of 200mg/ml the purity and charge isomer were slightly worse, no obvious difference was seen for sub-visible particles. For this purpose, the preferred concentration of the anti-NKG 2A monoclonal antibody HA-1 in the formulation is preferably 120mg/ml to 150mg/ml.

Freeze thawing stability results

As can be seen from the above table, after freeze thawing 5 times at-20 ℃, the purity, the charge isomer main peak content and the number of sub-visible particles which are more than or equal to 25um of each experimental example are not obviously changed.

Results of the shaking stability test:

as can be seen from the above table, the anti-NKG 2A monoclonal antibody HA-1 had no obvious changes in the purity, the major peak charge isomer content and the number of sub-visible particles of 25um or more in each experimental example after horizontal shaking for 7 days at 25.+ -. 2 ℃.

Results of the light stability test:

as can be seen from the above table, after the anti-NKG 2A monoclonal antibody HA-1 is placed for 10 days under the conditions of 25 ℃ and the illumination intensity of 4500+/-500 lx, the concentration of the anti-NKG 2A monoclonal antibody HA-1 is 150mg/ml and 120mg/ml, and the purity, the charge isomer, the number of sub-visible particles which are more than or equal to 25um and the like are not obviously different; at a concentration of 200mg/ml the purity and charge isomer were slightly worse, but not significantly, the sub-visible particles were not significantly different.

As proved by the experiment, the concentration of the anti-NKG 2A monoclonal antibody HA-1 is very stable within the range of 120-200mg/ml, and the antibody concentration is preferably 120-150mg/ml.

Through an acceleration test, a freeze thawing test, a shaking test and a light test at 40 ℃, the anti-NKG 2A monoclonal antibody HA-1 with the antibody protein concentration of 120mg/ml-150mg/ml is further verified to have good stability in a preparation of 20mM citrate buffer solution, 160mM trehalose and 0.02% (w/v) polysorbate 20, pH 6.0.

The present invention is not limited to the above-described preferred embodiments, and any person who can obtain other various products under the teaching of the present invention, however, any change in shape or structure of the product is within the scope of the present invention, and all the products having the same or similar technical solutions as the present application are included.

Claims

1. An injectable formulation of an anti-NKG 2A monoclonal antibody, comprising the following components in amounts:

wherein the pH value of the injection preparation is 5.5-6.5;

the anti-NKG 2A monoclonal antibody comprises a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is shown as SEQ ID No. 14, and the amino acid sequence of the light chain variable region is shown as SEQ ID No. 15;

the protein protecting agent comprises one or more of sorbitol, mannitol, trehalose, sucrose, glycine and proline;

the surfactant comprises one or more of polysorbate 20, polysorbate 80 and poloxamer.

2. The injectable formulation of the anti-NKG 2A monoclonal antibody of claim 1, wherein the anti-NKG 2A monoclonal antibody further comprises a heavy chain constant region and a light chain constant region, wherein the amino acid sequence of the heavy chain constant region is shown in SEQ ID No. 18 and the amino acid sequence of the light chain constant region is shown in SEQ ID No. 20.

3. The injectable formulation of anti-NKG 2A monoclonal antibody according to claim 1, wherein the protein content of the anti-NKG 2A monoclonal antibody is 120-150mg/ml.

4. The injectable formulation of anti-NKG 2A monoclonal antibody of claim 1, wherein the buffer salt is a citrate buffer or a phosphate buffer.

5. The injectable preparation of the anti-NKG 2A monoclonal antibody according to claim 4, wherein the buffer salt is present in an amount of 20-30mM.

6. The injectable formulation of anti-NKG 2A monoclonal antibody according to claim 5, wherein the pH of the injectable formulation is 6.0-6.5.

7. The injectable formulation of anti-NKG 2A monoclonal antibody according to claim 1, wherein the surfactant is present in an amount of 0.01% -0.02% (w/v).