CN109678963A

CN109678963A - A kind of preparation and its application of the bispecific antibody for targeting CD24 and activating NK cell

Info

Publication number: CN109678963A
Application number: CN201811548656.1A
Authority: CN
Inventors: 张娟; 王旻; 韩月; 孙福谋; 王阳; 王斐; 蔡佳玲
Original assignee: China Pharmaceutical University
Current assignee: China Pharmaceutical University
Priority date: 2018-12-18
Filing date: 2018-12-18
Publication date: 2019-04-26

Abstract

The invention belongs to genetic engineering antibody technical fields, and in particular to a kind of preparation and its application of the bispecific antibody for targeting CD24 and activating NK cell.Anti- CD24 humanized chimeric antibody cG7 is passed through flexible peptide (Gly with having the MICA molecule raised and activate NK cell activity using technique for gene engineering by the present invention₄Ser it) connects, steady to turn in CHO-s eukaryotic expression system, picking stablizes overexpression cell line and expands culture expression cG7-MICA；Bispecific antibody cG7-MICA is selectively shown MICA in CD24 using the function that the targeting of cG7 can remold MICA⁺Tumor cell surface, the immunosurveillance of induced NK cell, and NK cell is further activated by its Fc sections of effect, play the cell mediated cytotoxicity (ADCC) of antibody dependent；By experimental verification, all there is killing CD24 in vivo and in vitro⁺The immunocompetence of liver cancer cells.

Description

A kind of preparation and its application of the bispecific antibody for targeting CD24 and activating NK cell

Technical field

The invention belongs to bioengineering fields, and in particular to it is a kind of new can simultaneously with leukocyte differentiation antigen CD24 (cluster of differentiation 24) and natural killer cells (natural killer cells；NK cells) The anti-CD24 that Activating receptor NKG2D (NK cell receptor NK group 2, member D) is specifically bound on surface Humanized chimeric antibody merges the bispecific antibody cG7-MICA of source of people MICA molecule, can be with using the targeting of cG7 The function of remodeling MICA selectively shows MICA on liver cancer cells surface, raises and activates NK cells play antibody dependent Cell mediated cytotoxicity (Antibody-Dependent Cell-mediated Cytotoxicity；ADCC).? CD24⁺In the subcutaneous bearing mouse model of source of people liver cancer cells Huh-7, the growth of tumor tissues can be effectively suppressed.CG7-MICA's Design can be to avoid the caused tumor immune escape that falls off of MICA molecule in tumor tissues, and has the ADCC of Fc sections of guidance Effect is a kind of bispecific genetic engineering antibody with targeting CD24 molecule and enhancing NK cell function.

Background technique

The treatment of tumour is always our intractable problems for facing, due to chemotherapy and operative treatment it is big to body injury, Poor prognosis, tumour immunotherapy become the hot spot of contemporary oncotherapy.Immunotherapy of tumors is intended to remold body autoimmunity system The identification and killing ability united to tumour cell, utilize the guiding performance of the specific binding of antibody and antigen or ligand and receptor The naturally occurring immunocyte of body is raised to tumor focus position, the work of killing tumor cell is played using immunocyte Property.Therefore, possibility will be provided for the immunotherapy of tumors of realization specificity by finding effective molecular target.

(1) liver cancer and tumor related antigen CD24 molecule.

Liver cancer (Hepatocellular carcinoma；HCC it is) most common primary tumor, and affects in the world The health of many people.It is listed in the fifth-largest common cancer in the whole world and becomes second largest the reason of leading to cancer mortality.HCC phase Have the characteristics that high invasion and metastatic capacity for other mankind's solid malignants, therefore increases difficulty to the treatment of liver cancer Degree.Many effort have been put into treatment liver cancer, but all due to some adverse reactions, many is for treating advanced stage HCC Clinical medicine be abandoned.Single therapy drug, and including bevacizumab (Avastin) and Cetuximab (Cetuximab) therapeutic antibodies including also all show limited anti-HCC activity.

Tumor related antigen CD24 molecule is a kind of memebrane protein of high glycosylation, is expressed in kinds of tumor cells, CD24 has high expression probability in hepatocarcinoma.And the expression of CD24 is significantly higher than swollen in hepatocarcinoma Tumor adjacent tissue and normal hepatic tissue.In tumour cell, CD24 is by glycosyl-phosphatidyl inositol anchor due on cell membrane Lipid Rafts structure.The structure is the Important Platform of Src family tyrosine kinase signal approach.CD24 interacts and enhances with Src Src kinase activity, and then make STAT3 molecule (signal transducers and activators of Transcription activated state) is formed；The STAT3 of activation forms dimer and enters in nucleus, plays the work of transcription factor With.Illustrate that the expression of CD24 and the differentiation of tumour cell, invasion, transfer and the recurrence of tumour etc. are closely related.These all show CD24 molecule can be used as the specific marker molecule of tumour cell, may serve as the molecular target of liver cancer treatment.

(2) tumor immune escape and MICA/NKG2D signal path.

The important a part of immune system is immunosurveillance in human body, the important mechanisms of immunosurveillance first is that tumour is thin MHC-I class chain molecule (MICA/B, major histocompatibility complex class the I chain- of cellular surface Related gene A/B) high expression, can all express MICA/B molecule in kinds of tumor cells.MICA/B be NK cell and CD8⁺The ligand of T cell surface active receptor NKG2D, NK cell or CD8⁺T cell passes through NKG2D (NK cell receptor NK group 2, member D) with the interaction of MICA/B perforation is released and then close to tumour cell and in combination Element-granzyme, the cytokine profiles such as secretion of gamma-IFN, TNF-α induce the dissolution of tumour cell.

MICA is a kind of protein molecule of high glycosylation, belongs to non-classical HLA-I (human leucocyte Antigen-I) genoid family.Its structure is similar to classics HLA-I class molecule, including 3 extracellular domains: α 1, α 2 and α 3, Transmembrane region and intracellular region.α 1,2 region α then determine the interaction of MICA Yu NKG2D receptor.But studies have shown that in the life of tumour In long process, MICA molecule can be fallen off by followed by action of proteolytic enzymes, and tumor tissues is caused although to still fall within the MICA positive, but It can avoid immunosurveillance and immunologic escape occurs.Therefore, a kind of remodeling tumor cell surface MICA molecular function is found, it is avoided Fall off the drawbacks of bringing method will become immunotherapy of tumors new way.

(3) genetic engineering antibody.

Application of the antibody drug in antitumor field is now subjected to the favor of multi-field researcher, with its high targeting Upsurge with specific advantage as research, according to statistics, the antibody class drug of FDA approval at present is mainly used for treating cancer The diseases such as disease, autoimmunity disease, haematological disorders and virus infection.Antibody drug is in high speed development rank in antitumor field Section.Antibody drug and the combination for changing (putting) therapy and therapeutic method of surgery, can effectively reduce toxic side effect, improve treatment effect Rate has a extensive future.Antibody types include that bispecific antibody, single-chain antibody, nano antibody, chimeric antibody, humanization are anti- Body etc. respectively realizes different functions, can be transformed according to their own needs in gene level.And it is used to treat human body The antibody of disease must be the antibody of source of people, and the technology for now producing antibody is also increasingly mature, screen at present using phage library The antibody of source of people out, or carried out again using the antibody that hybridoma technology produces source of mouse humanization modified.With cell culture Horizontal raising, the small-scale building of antibody, expression, purifying are possibly realized.

Based on above-mentioned theory basis and research practice, source of mouse anti-CD24 single chain fusion of the present invention in laboratory independent development Genetic modification is carried out on the basis of MICA antibody rG7s-MICA and constitutes humanized chimeric antibody cG7, further in its Fc sections of C-terminal Pass through flexible peptide (Gly-Gly-Gly-Ser) with the extracellular area 1-3 source of people MICA to connect, designs a kind of novel bispecific antibodies cG7-MICA.The design of cG7-MICA selectively takes MICA on the surface of liver cancer cells using cG7 as effective carrier, MICA molecule activates NK cell, remolds the function of MICA in conjunction with the Activating receptor NKG2D of NK cell surface.And cG7- MICA can further activate NK cell by the Fc section of its fusion in conjunction with the Fc receptor of NK cell surface.NK after activation is thin Born of the same parents play ADCC effect, discharge perforin and granzyme, while secrete cytokines IFN-γ and TNF-α performance killing liver cancer are thin Cytoactive.Therefore, the exploitation of bispecific antibody cG7-MICA with independent intellectual property rights, effectively activation is in immunosurveillance system The NK cell to play a significant role in system provides possible clinical application scheme for immunotherapy of tumors system.

Summary of the invention

Goal of the invention: the present invention provides the bispecific antibody of targeting CD24 with tumour immunity curative effect a kind of.

Technical solution: a kind of bispecific antibody for targeting CD24 and activating NK cell, the bispecific antibody is with anti- CD24 humanized chimeric antibody cG7 with have activation NK cell activity source of people MICA molecule based on, with genetic recombination hand Section, utilizes flexible peptide (Gly₄Ser) two sections of albumen are connected and are built into cG7-MICA.The amino acid sequence of the heavy chain of the antibody Column are as shown in SEQ ID NO.1, and the nucleotide sequence of the heavy chain of the antibody is as shown in SEQ ID NO.2；The light chain of the antibody Amino acid sequence as shown in SEQ ID NO.3, the nucleotide sequence of the light chain of the antibody is as shown in SEQ ID NO.4.

Bispecific antibody in the present invention can simultaneously with leukocyte differentiation antigen CD24 and NK cell activation receptor The anti-CD24 humanized chimeric antibody of NKG2D specific binding merges the bispecific antibody cG7-MICA of source of people MICA molecule, It is selectively shown MICA using the function that the targeting of cG7 can remold MICA on liver cancer cells surface, is raised simultaneously Activate cell mediated cytotoxicity (the Antibody-Dependent Cell- of NK cells play antibody dependent mediated Cytotoxicity；ADCC).In CD24⁺It, can be effective in the subcutaneous bearing mouse model of source of people liver cancer cells Huh-7 Inhibit the growth of tumor tissues.The design of cG7-MICA can be exempted to avoid the caused tumour that falls off of MICA molecule in tumor tissues Epidemic disease escape, and have the Fc sections of ADCC guided effects, it is a kind of with pair for targeting CD24 molecule with enhancing NK cell function Specific gene engineered antibody.The feature of bispecific antibody of the present invention is specific binding people CD24 and NKG2D, in vitro can Enough enhancing NK cells discharge perforin and granzyme to the ADCC effects of liver cancer cells, at the same secrete cytokines IFN-γ and TNF-α kills human liver cancer cell Huh-7 and BEL-7402, remolds the immune surveillance function of NK cells against tumor cells；Suppression in vivo The growth of liver cancer cells Huh-7 processed, tumor killing effect are substantially better than the multiple target point selection inhibitor Sorafenib of FDA approval (Sorafenib)。

A kind of expression vector, containing one of nucleotide sequence shown in SEQ ID NO.2, SEQ ID NO.4 or Two kinds.

A kind of host cell, containing one of nucleotide sequence shown in SEQ ID NO.2, SEQ ID NO.4 or Two kinds.

Application of the bispecific antibody of above-mentioned targeting CD24 and activation NK cell in preparation treatment liver-cancer medicine.

Target the application mode of the bispecific antibody cG7-MICA of CD24: humanized chimeric antibody cG7 plays targeting It acts on and the MICA molecular specificity that can raise activated NK is showed in the highly expressed liver of CD24 as carrier molecule Cancer cell surfaces, MICA molecular selection in conjunction with NK cell surface activation receptor NKG2D, enhancing NK cell it is thin to liver cancer The targets identification ability and lethal effect of born of the same parents remolds the immune surveillance function of NK cells against tumor cells；And cG7-MICA can Through the Fc section of its fusion in conjunction with the Fc receptor of NK cell surface, NK cells play effect is further activated.

Invention further illustrates:

The bispecific antibody cG7-MICA that CD24 is targeted in the present invention is made of two chains, wherein a chain is by anti-CD24 Humanized chimeric antibody's heavy chain, flexible peptide (Gly₄Ser it) is formed with MICA albumen；Another chain is anti-CD24 humanization inosculating antibody Body light chain.

It is an object of the invention to construct the expression vector of the bispecific antibody cG7-MICA of targeting CD24, screening should The highly expressed engineering cell strain of antibody provides a kind of bispecific that can stablize high expression and the above-mentioned targeting CD24 of mass purification The method of antibody cG7-MICA；Another object of the present invention is to establish the body of the bispecific antibody cG7-MICA of targeting CD24 Inside and outside evaluating drug effect system assesses the feasibility of immunotherapy of tumors scheme.

The present invention is melted this laboratory patent using overlap PCR (polymerase chain reaction) technology Close the targeting CD24 humanized chimeric antibody cG7 constructed on the basis of antibody rG7s-MICA (patent No.: ZL2015106263749) Heavy chain constant region C-terminal and the extracellular area the 1-3 gene of source of people MICA be aided with flexible peptide (Gly₄Ser) it is connected, light chain remains unchanged, Clone's recombination is carried out, the bispecific antibody cG7-MICA recombinant vector of building targeting CD24 passes through electroporation stable transfection CHO-s cell, pressurize the stable operation cell strain for screening and obtaining correct expression cG7-MICA through two-wheeled neomycin G418；To stabilization Cell strain expands culture, and low-temperature centrifugation takes supernatant, supernatant is crossed Protein A column and is isolated and purified；SDS-PAGE(sodium Dodecyl sulphate polyacrylamide gel electrophoresis) and WB (Western Blot) identification table It is whether correct up to product expression and assembly；The parent of SPR (surface plasmon resonance) experimental analysis antibody and antigen And ability；FCM (flow cytometry) further discloses bispecific antibody and CD24⁺Liver cancer cells Huh-7 and BEL- 7402 combination situation；Cellular cytotoxicity cytolytic is tested with NK92-FcR or PBMCs (peripheral blood mononuclear It cells) is effector cell, hepatoma cell strain Huh-7 and BEL-7402 are target cell, and verifying cG7-MICA is mediated based on NK cell Immunocyte to the specific killing actions of liver cancer cells；Further flow cytometry and the verifying of ELISA test experience CG7-MICA induces NK92-FcR cell degranulation by MICA/NKG2D and Fc/FcR approach, while secreting large amount of cell factor IFN-γ and TNF-α are more effectively to kill target cell.

Experiment in vivo of the present invention verifies the antitumor work of cG7-MICA using lotus human liver cancer (Huh-7) nude mice model Property.Illustrate that cG7-MICA has nude mice Huh-7 liver cancer model by the measurement to subcutaneous transplantation knurl product, weight after administration Apparent growth inhibition effect.

The utility model has the advantages that

Effect of the NK cell in human immunity system is increasingly paid attention to, and research at present is dedicated to being immunized on NK cell The inhibition (inhibition of such as PD-1) of checkpoint is to restore NK cell function；But rarely has Activating receptor NKG2D ligand on NK cell The report of MICA molecule related fusion.The bispecific antibody cG7- of the selectively targeted CD24 of independent development in the present invention The MICA molecule of MICA fusion has the activity raised and activate NK cell；And " the biological bullet " in cG7-MICA is humanization Chimeric antibody cG7 rather than single-chain antibody comprising the Fc section of source of people can enhance its stability and further activation NK cell.And The engineering cell strain for stablizing high expression cG7-MICA is filtered out using lab platform, and passes through the inside and outside drug effect of independent development Appraisement system inhibits CD24 to its inside and outside⁺The activity of liver cancer cell growth is identified.

Detailed description of the invention

Fig. 1 is the bispecific antibody cG7-MICA genetic recombination analysis chart based on cG7, and the heavy chain gene of cG7-MICA is big Small about 2323bp, light chain gene size are about 775bp.VH and VL respectively represents the heavy chain variable region of cG7-MICA and light chain can Become region sequence, CH and CL respectively represent the heavy chain constant region and constant region of light chain of cG7-MICA；Sequence G₄S Linker is flexibility Peptide sequence is connected, SP is signal peptide sequence.Figure 1A and Figure 1B describes cG7 and the building of cG7-MICA expression vector is illustrated Figure.

Fig. 2 is the expression and purification figure of cG7 and cG7-MICA, and the cG7 and cG7-MICA that Fig. 2A describes fermentation expression pass through SDS-PAGE that Protein A column is isolated and purified is as a result, illustrate that the molecular weight of cG7 and cG7-MICA are correct, the molecule of cG7 Amount is about 150kD, and the molecular weight of cG7-MICA is about 210kD, and to reach electrophoresis pure for purity；Fig. 2 B and Fig. 2 C are described after purification The reduction of cG7 and cG7-MICA and non-reduced WB are as a result, illustrate that the equipment of cG7 and cG7-MICA are correct；It is incubated in Fig. 2 B and Fig. 2 C The primary antibody educated is goat anti-human igg (H+L)；M is albumen Marker.

Fig. 3 is that the binding test using SPR technique of cG7-MICA and CD24 and NKG2D tests (Biacore), and Fig. 3 A is surveyed The affinity constant for determining humanized chimeric antibody cG7 and antigens c D24 is 1.91E-10 (ka (1/Ms): 1.76E+06；kd(1/ s):3.36E-04；KD(M):1.91E-10)；Fig. 3 B measures bispecific antibody cG7-MICA and the affinity of antigens c D24 is normal Number is 2.42E-10 (ka (1/Ms): 1.40E+06；kd(1/s):3.39E-04；KD(M):2.42E-10)；Fig. 3 C measurement is double special The affinity constant of heterogenetic antibody cG7-MICA and NKG2D are 5.76E-07 (ka (1/Ms): 2.62E+03；kd(1/s):1.5E- 03；KD(M):5.76E-07).

Fig. 4 is cG7-MICA and source of people CD24⁺Hepatoma cell strain Huh-7 and BEL-7402 streaming binding assay figure, Fig. 4 A The Percentage bound of display cG7 and Huh-7 and BEL-7402 cell surface CD24 molecule, respectively 88.0% and 80.7%；Fig. 4 B is aobvious Show the Percentage bound of cG7-MICA Yu Huh-7 and BEL-7402 cell surface CD24 molecule, respectively 80.3% and 79.3%；In conjunction with Fig. 3 can illustrate not influencing cG7-MICA and the respective affinity of CD24 and NKG2D in the Fc section terminal fusion MICA molecule of cG7.

Fig. 5 is cG7-MICA enhancing NK92-FcR and PBMCs cell to source of people CD24⁺Hepatoma cell strain Huh-7 and BEL- 7402 cytotoxicity experiment result figure, Fig. 5 A's the experimental results showed that cG7-MICA can be activated in PBMCs based on NK cell Immunocyte cytotoxicity, Fig. 5 B be Fig. 5 A T-test significance analysis result；The result of Fig. 5 C and the knot of Fig. 5 A Fruit is consistent, and Fig. 5 D is the T-test significance analysis result of Fig. 5 C；CG7-MICA can activate NK92-FcR cell killing about 60% Huh-7 and BEL-7402 cell, and its cytotoxicity is substantially better than cG7 and rG7s-MICA, recombinates in source of people In the presence of hMICA, which is suppressed.

Fig. 6 is the release experiment testing result of activation threshing and cell factor that cG7-MICA promotes NK92-FcR cell Figure, Fig. 6 A is the flow cytometer detection figure of CD107a, when NK cell is activated, can discharge perforin and granzyme isoreactivity substance, Thus the surface that CD107a can be transported to NK cell membrane keeps the NK cell CD107a of activation positive, the positive rate of CD107a and The ratio that NK cell is activated is directly proportional, incubates 10 μ g/mL cG7-MICA altogether with NK92-FcR, Huh7 cell with this principle The expression quantity of rear Flow cytometry NK92-FcR cell surface CD107a is educated, cG7-MICA activates NK92-FcR as the result is shown The effect that cell expresses CD107a is most significant, 31.4% NK92-FcR can be made to be activated, threshing occurs；Fig. 6 B and Fig. 6 C is ELISA testing result figure, the burst size and cG7-MICA for describing NK92-FcR cell IFN-γ and TNF-α have concentration dependent (effect is more excellent compared with cG7, rG7s-MICA, hMICA).

Fig. 7 is lotus human liver cancer Huh-7 transplanted tumor in nude mice Cell suppression test result figure, and Fig. 7 A is tumor growth curve figure, Fig. 7 B is knurl weight figure, and description cG7-MICA has significant inhibiting effect to the growth of hepatocellular carcinoma in nude mice transplantable tumor, and better than the positive Compare Sorafenib group.

Specific embodiment

Embodiment 1 targets the building of the bispecific antibody cG7-MICA of CD24.

Heavy and light chain variable region gene is transferred by template design primer of the rG7s-MICA of laboratory independent research first, and And the IgG1 constant region gene to be protected by laboratory transfers weight light chain constant region gene as template design primer, by heavy and light chain Variable region passes through overlap round pcr respectively with constant region and connect the heavy chain (H-chain) and light chain (L- for being built into cG7 Chain) gene.Continue the gene for transferring the extracellular area 1-3 source of people MICA using rG7s-MICA as template design primer simultaneously, and sets Meter primer transfers the heavy chain gene of cG7, passes through flexible peptide (Gly with overlap round pcr₄Ser) this two sections of genes are connected It picks up to constitute the heavy chain of cG7-MICA (H '-chain), cG7-MICA is consistent with the light chain of cG7.With H '-chain and L- Chain is that template carries out PCR, and PCR product and pMH3/pCApuro distinguish double digestion, target gene and plasmid piece recycled after digestion Section.The connection overnight of 16 DEG C of T4 ligase, obtaining four kinds of recombinant plasmids is H '-pMH3, H '-pCApuro, L-pMH3, L- pCApuro。CaCl₂Connection product is converted and is expanded and saved into bacillus coli DH 5 alpha by method.

Embodiment 2 targets expression and purification and the identification of the bispecific antibody cG7-MICA of CD24.

Four kinds of recombinant plasmid H '-pMH3, H '-pCApuro, L-pMH3 and L-pCApuro are led in a manner of equal proportion first It crosses electric shocking method (0.4cm shocks by electricity in cup, 160V, 15ms) and imports CHO-s cell, and carry out two-wheeled pressurization screening (Dot Blot point Marking sxemiquantitative screening), obtain the cell strain for stablizing high expression cG7-MICA.The overexpression cell line that screening is obtained, step by step Expand, collects cell culture fluid, carry out Protein A affinitive layer purification after 0.22 μm of membrane filtration sample, finally largely obtain Obtain purpose antibody.10%SDS-PAGE protein electrophoresis identifies molecular weight and purity, and Western blot carries out just destination protein Step card sees whether assemble correctly.

Embodiment 3 targets the SPR experiment of the bispecific antibody cG7-MICA of CD24.

The measurement of the interaction of cG7-MICA and antigens c D24 and MICA receptor NKG2D uses in the experiment BiacoreX100 is detected as the biosensor that SPR is relied on.Kit is captured by 25 μ g/ using Human Antibody The CM5 chip surface of mL anti-human igg (Fc) antibody coupling extremely activation (coupling only limits No. two channels).Adjust cG7 and cG7-MICA Concentration be 10 μ g/mL, utilize anti-human igg (Fc) antibody specificity on CM5 chip to capture cG7 and cG7-MICA.It is sharp later With the naked peptide of buffer doubling dilution CD24 (from 100nM to 0.39nM).Under 25 DEG C, 30 μ L/min working environment of flow velocity, sample introduction Detect combination/dissociation capability between cG7 and cG7-MICA and CD24.Meanwhile No.1 channel be not coupled as reference channel or Capture any albumen.Therefore the signal that No.1 Air conduct measurement obtains will be used as reference data.Between cG7 and cG7-MICA and CD24 Naturally it after dissociation reaches preset time, using the antigen antibody complex of 3M MgCl2 regeneration buffer elution chip surface, washes De- target is that baseline response value returns the initial communication value level after coupling.Biacore is utilized after obtaining sensing diagram data X100Evaluation software is analyzed binding constant (ka) and dissociation constant (kd), and fitting preferably combines dissociation curve, is calculated Obtain the equilibrium dissociation constant KD (kd/ka) between antibody antigen.The CM5 chip that wherein coupling has cG7-MICA is carried out again sharp With mobile phase is changed to the recombined human rNKG2D molecule of buffer doubling dilution (from 100nM to 6.25nM).Similarly calculate its KD (kd/ka)。

The bispecific antibody cG7-MICA and CD24 of 4 Flow cytometry of embodiment targeting CD24⁺Hepatoma cell strain The binding ability of Huh-7 and BEL-7402.

First by 2 × 10 in the experiment⁵A CD24⁺Hepatoma cell strain Huh-7's and BEL-7402 is resuspended in 250 μ L PBS In, prepare single cell suspension.Total incubation is carried out then to isometric 10 μ g/mL cG7-MICA of concentration is added in each cell suspension 1h.It is thin using anti-human igg (H+L) antibody and tumour that have FITC label after PBS washing removes unbonded cG7-MICA Born of the same parents are incubated for, and flow cytomery tumor cell surface is combined with the cell proportion of cG7-MICA after PBS washing, are calculated The Percentage bound of cG7-MICA and Huh-7 and BEL-7402.

Embodiment 5LDH discharges cytotoxicity experiment detection cG7-MICA activation NK92-FcR and PBMCs cell to source of people CD24⁺The cytotoxic effect of hepatoma cell strain Huh-7 and BEL-7402.

It is thin to target that 96 non-radioactive cell toxicity detection method of CytoTox detects cG7-MICA activation NK92-FcR and PBMCs The splitting action of born of the same parents Huh-7 and BEL-7402, NK92-FcR and PBMCs are as effector cell, and Huh-7 and BEL-7402 are as target Cell.The IL-2 stimulating effect cell of 1000U/mL is added in the culture supernatant of NK92-FcR and PBMCs for 24 hours in advance first, A series of NK92-FcR cell numbers and target cell number effect target ratio (10:1,5:1,1:1), PBMCs cell number and target cell number are set It imitates target ratio (100:1,30:1,5:1) detection 10 μ g/mL cG7-MICA under different effect target ratios and activates NK92-FcR and PBMCs pairs The lethal effect of target cell.

6 flow cytometry of embodiment and enzyme-linked immunization detection cG7-MICA to the activation of NK92-FcR cell and it is related carefully The influence of intracellular cytokine release.

Streaming is thin after 10 μ g/mL cG7-MICA processing with Huh-7 liver cancer cells (effect target ratio 10:1) for NK92-FcR cell Born of the same parents' art detects the expression quantity of NK92-FcR cell surface CD107a, shows that cG7-MICA can effectively activate NK92-FcR cell. NK92-FcR cell is mixed with Huh-7 and BEL-7402 cell by effect target ratio 10:1, and cG7-MICA concentration gradient (0.1 μ g/ is arranged ML, 1 μ g/mL, 10 μ g/mL) it is added in cell mixing liquid, 37 DEG C of reaction 4h, supernatant, which is collected, with ELISA kit detects cell The burst size of factor IFN-γ and TNF-α, release have dose dependent.

Growth inhibition effect of the embodiment 7cG7-MICA to lotus human liver cancer Huh-7 transplanted tumor in nude mice.

5 week old BALB/c Female nude mices are selected to establish lotus human liver cancer Huh-7 Xenografts in nude mice model.By 1 × 10⁷It is a Logarithmic growth phase Huh-7 cell is resuspended in 200 μ L PBS, and syringe is implanted into oxter on the left of nude mice, daily to observe nude mice tumor formation Situation.About 100mm is grown to transplanted tumor in nude mice³Afterwards, tumor-bearing mice is grouped at random, group is respectively control group, cG7 Group (5.0mg/kg), rG7s-MICA group (5.0mg/kg), cG7-MICA group (5.0mg/kg) and positive controls Sorafenib Group (20.0mg/kg), every group six.Tail vein administration is carried out every other day, and gross tumor volume is measured, and is calculated as follows Knurl accumulates (V=LW²/ 2, wherein L represents tumour longest diameter, and W is represented as the maximum transverse diameter of tumour vertical direction), it is raw to draw tumour Long curve.Tumor bearing nude mice was put to death in a manner of from neck at the 17th day and solution takes tumor, is weighed to the tumor tissue taken off and is drawn tumor Redistribution figure.

Sequence table

<110>China Medicine University

<120>bispecific antibody and its application of a kind of targeting CD24 and activation NK cell

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Leu Trp Val Leu Leu Leu Trp Val Pro Gly Ser Thr Gly Gln Val Lys

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Ser Lys Val Arg Tyr Glu Asp Thr Ser Leu Tyr Tyr Cys Ala Arg Gln

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Gly Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Ala Ser

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Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

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Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

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Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

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Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

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Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

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Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

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Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

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Ser Glu Pro His Ser Leu Arg Tyr Asn Leu Thr Val Leu Ser Trp Asp

485 490 495

Gly Ser Val Gln Ser Gly Phe Leu Ala Glu Val His Leu Asp Gly Gln

500 505 510

Pro Phe Leu Arg Tyr Asp Arg Gln Lys Cys Arg Ala Lys Pro Gln Gly

515 520 525

Gln Trp Ala Glu Asp Val Leu Gly Asn Lys Thr Trp Asp Arg Glu Thr

530 535 540

Arg Asp Leu Thr Gly Asn Gly Lys Asp Leu Arg Met Thr Leu Ala His

545 550 555 560

Ile Lys Asp Gln Lys Glu Gly Leu His Ser Leu Gln Glu Ile Arg Val

565 570 575

Cys Glu Ile His Glu Asp Asn Ser Thr Arg Ser Ser Gln His Phe Tyr

580 585 590

Tyr Asp Gly Glu Leu Phe Leu Ser Gln Asn Leu Glu Thr Glu Glu Trp

595 600 605

Thr Val Pro Gln Ser Ser Arg Ala Gln Thr Leu Ala Met Asn Val Arg

610 615 620

Asn Phe Leu Lys Glu Asp Ala Met Lys Thr Lys Thr His Tyr His Ala

625 630 635 640

Met His Ala Asp Cys Leu Gln Glu Leu Arg Arg Tyr Leu Glu Ser Gly

645 650 655

Val Val Leu Arg Arg Thr Val Pro Pro Met Val Asn Val Thr Arg Ser

660 665 670

Glu Ala Ser Glu Gly Asn Ile Thr Val Thr Cys Arg Ala Ser Ser Phe

675 680 685

Tyr Pro Arg Asn Ile Ile Leu Thr Trp Arg Gln Asp Gly Val Ser Leu

690 695 700

Ser His Asp Thr Gln Gln Trp Gly Asp Val Leu Pro Asp Gly Asn Gly

705 710 715 720

Thr Tyr Gln Thr Trp Val Ala Thr Arg Ile Cys Arg Gly Glu Glu Gln

725 730 735

Arg Phe Thr Cys Tyr Met Glu His Ser Gly Asn His Ser Thr His Pro

740 745 750

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

755 760 765

<210> 2

<211> 2323

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 2

cccccctcat ggaattcgat atctactatg gaaaccgata cactgctgct gtgggtgctg 60

ctgctgtggg tgcctgggtc aactggtcag gtgaaactgg aagagagcgg aggtgggctg 120

gtgcagccag gcggatcact gaaaatctcc tgcgccgcct ccggtttcga cttttctagg 180

tactggatga gttgggtccg acaggcccct ggcaagggtc tggagtggat cggcgaaatt 240

aaccccgatt ccagcactat taattatacc ccttcactga gagacaagtt catcatttcc 300

cgcgataacg caaaaaatac actgtacctc cagatgagca aggtgcgata tgaggacacc 360

tctctgtact attgtgccag gcagggagat tactggggcc agggaaccag tgtgacagtc 420

tctagtgcta gcgcctccac caagggccca tcggtcttcc ccctggcacc ctcctccaag 480

agcacctctg ggggcacagc ggccctgggc tgcctggtca aggactactt ccccgaaccg 540

gtgacggtgt cgtggaactc aggcgccctg accagcggcg tgcacacctt cccggctgtc 600

ctacagtcct caggactcta ctccctcagc agcgtggtga ccgtgccctc cagcagcttg 660

ggcacccaga cctacatctg caacgtgaat cacaagccca gcaacaccaa ggtggacaag 720

agagttgagc ccaaatcttg tgacaaaact cacacatgcc caccgtgccc agcacctgaa 780

ctcctggggg gaccgtcagt cttcctcttc cccccaaaac ccaaggacac cctcatgatc 840

tcccggaccc ctgaggtcac atgcgtggtg gtggacgtga gccacgaaga ccctgaggtc 900

aagttcaact ggtacgtgga cggcgtggag gtgcataatg ccaagacaaa gccgcgggag 960

gagcagtaca acagcacgta ccgtgtggtc agcgtcctca ccgtcctgca ccaggactgg 1020

ctgaatggca aggagtacaa gtgcaaggtc tccaacaaag ccctcccagc ccccatcgag 1080

aaaaccatct ccaaagccaa agggcagccc cgagaaccac aggtgtacac cctgccccca 1140

tcccgggagg agatgaccaa gaaccaggtc agcctgacct gcctggtcaa aggcttctat 1200

cccagcgaca tcgccgtgga gtgggagagc aatgggcagc cggagaacaa ctacaagacc 1260

acgcctcccg tgctggactc cgacggctcc ttcttcctct acagcaagct caccgtggac 1320

aagagcaggt ggcagcaggg gaacgtcttc tcatgctccg tgatgcatga ggctctgcac 1380

aaccactaca cgcagaagag cctctccctg tctccgggta aaggaggtgg tggaagtgga 1440

tccgagcccc acagcctgcg gtacaacctg accgtcctgt cctgggacgg aagcgtccag 1500

agcggctttc tggctgaggt gcacctggac ggccagcctt tcctgcggta cgaccggcag 1560

aaatgtcggg ctaaacccca gggccagtgg gctgaggatg tcctgggcaa caagacatgg 1620

gaccgggaga ccagggacct cacaggcaac ggcaaggacc tccggatgac actggcccac 1680

atcaaggacc agaaggaagg cctgcacagc ctgcaggaga tccgggtgtg cgaaatccac 1740

gaggacaact ccacccggtc ctcccagcac ttctactacg acggcgaact cttcctgtcc 1800

cagaatctgg agaccgaaga gtggacagtg cctcagagca gcagggccca aaccctcgcc 1860

atgaacgtgc ggaacttcct gaaggaggac gccatgaaga ccaagaccca ctaccatgcc 1920

atgcatgccg actgtctgca ggaactgagg aggtacctgg agtccggcgt ggtcctcagg 1980

aggacagtgc ctcccatggt caacgtgaca cggagcgaag cctccgaggg aaacatcacc 2040

gtgacctgca gggcctcctc cttctacccc aggaacatca tcctgacctg gaggcaagac 2100

ggcgtgagcc tctcccatga cacccagcag tggggcgatg tgctgcctga cggcaacggc 2160

acataccaaa cctgggtggc tacccggatt tgtaggggcg aagagcagcg gttcacctgc 2220

tacatggaac acagcggaaa ccactccaca caccctgtcc ccagcggcaa agtcctggtg 2280

ctgcagagcc actggtagta agctctagag cttgcggccg caa 2323

<210> 3

<211> 249

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 3

Pro Pro Ser Trp Asn Ser Ile Ser Thr Met Glu Thr Asp Thr Leu Leu

1 5 10 15

Leu Trp Val Leu Leu Leu Trp Val Pro Gly Ser Thr Gly Asp Ile Val

20 25 30

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

35 40 45

Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu His Ser Asp Gly Lys

50 55 60

Thr Tyr Leu Asn Trp Leu Leu Gln Arg Pro Gly Gln Ser Pro Lys Arg

65 70 75 80

Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro Asp Arg Phe

85 90 95

Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Val Ser Arg Val

100 105 110

Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Trp Gln Gly Ala His Phe

115 120 125

Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Val Asp Thr

130 135 140

Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu

145 150 155 160

Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro

165 170 175

Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly

180 185 190

Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr

195 200 205

Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His

210 215 220

Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val

225 230 235 240

Thr Lys Ser Phe Asn Arg Gly Glu Cys

245

<210> 4

<211> 775

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 4

cccccctcat ggaattcgat atctactatg gaaaccgata cactgctgct gtgggtcctg 60

ctgctgtggg tccccggatc tactggtgat attgtgatga ctcagactcc tctgtcactg 120

tccgtgacaa tcggccagcc cgcatctatt agttgcaagt ccagccagag cctgctgcac 180

tctgacggca aaacttacct gaactggctg ctccagaggc ccggacagtc tcctaagcgg 240

ctgatctatc ttgtgagtaa actggactca ggggtcccag atagattcac cggttcaggc 300

tccggaaccg actttacact gaaggtgtcc agggtcgagg cagaggacct gggggtctac 360

tattgttggc agggtgctca tttcccctac acttttggcg gagggaccaa gctggagatt 420

aaagtcgaca ccgtggccgc tccatccgtg ttcatctttc cccctagcga cgagcagctg 480

aagagcggca cagcctctgt ggtgtgcctg ctgaacaatt tctaccccag ggaggccaag 540

gtgcagtgga aggtggataa cgctctgcag agcggcaatt ctcaggagtc cgtgaccgag 600

caggacagca aggattctac atattccctg tccagcaccc tgacactgtc taaggccgac 660

tacgagaagc acaaggtgta tgcttgtgag gtgacccatc agggcctgtc ttcccccgtc 720

acaaagtcct ttaaccgggg cgagtgttga taagctctag agcttgcggc cgcaa 775

Claims

1. a kind of targeting CD24 and the bispecific antibody for activating NK cell, which is characterized in that the amino acid of the heavy chain of the antibody Sequence is as shown in SEQ ID NO.1.

2. targeting CD24 according to claim 1 and the bispecific antibody for activating NK cell, which is characterized in that the antibody Light chain amino acid sequence as shown in SEQ ID NO.3.

3. targeting CD24 according to claim 1 and the bispecific antibody for activating NK cell, which is characterized in that described anti- The nucleotide sequence of the heavy chain of body is as shown in SEQ ID NO.2.

4. targeting CD24 according to claim 1 and the bispecific antibody for activating NK cell, which is characterized in that described anti- The nucleotide sequence of the light chain of body is as shown in SEQ ID NO.4.

5. a kind of expression vector contains one of nucleotide sequence shown in SEQ ID NO.2, SEQ ID NO.4 or two Kind.

6. a kind of host cell contains one of nucleotide sequence shown in SEQ ID NO.2, SEQ ID NO.4 or two Kind.

7. the bispecific antibody of any targeting CD24 of Claims 1 to 4 and activation NK cell treats liver cancer medicine in preparation Application in object.