CN104558192B - A kind of building and application of bispecific antibody HER2XCD3 - Google Patents

Abstract

The present invention provides a kind of bispecific antibodies, the bispecific antibody of the application is made of strand unit and monovalent unit, wherein the strand unit has specific binding capacity for the surface antigen CD3 of immunocyte, which has specific binding capacity for tumor cell surface antigen HER2；The strand unit includes the single chain variable fragment ScFv with Fc segment composition, which includes light chain and heavy chain pair.The application also provides the preparation method of bispecific antibody, the pharmaceutical use of these antibody.

Description

A kind of building and application of bispecific antibody HER2XCD3

Technical field

The present invention relates to immunologic technical fields.Specifically, being related to the building and preparation method of bispecific antibody.

Background technique

Bispecific antibody (bispecificantibody, BiAb) is the people containing two species-specific antigen binding sites Work antibody can erect bridge between target cell and functional molecular (cell), generate the effector function of guiding performance.BiAb is in biology In medicine, especially have broad application prospects in the immunization therapy of tumour.It is killed by BiAb mediated cell toxic action Tumour cell is the hot spot of current immunization therapy application study, be mainly characterized by BiAb can in combination with tumor associated antigen and Target molecule on immune effector cell, directly specific killing of the triggering immune effector cell to tumour cell.It is directed to below The some background technique introductions for Immune Cell Antigens and tumor-cell antigen and the relevant technologies development studied.

1.CD3

CD3 molecule is made of 4 subunits: δ, ε, γ, ζ, molecular mass be respectively 18.9kDa, 23.1kDa, 20.5kDa, 18.7kDa, length have 171,207,182,164 amino acid residues respectively.They form 6 peptide chains together, Often combine closely to form the TCR-CD3 complex containing 8 peptide chains, structure with T cell receptor (T cell receptor, TCR) Schematic diagram is shown in Fig. 1.This complex has T cell activation signal transduction, stablizes the function of TCR structure.CD3 cytoplasm section containing it is immune by Body tyrosine activation motifs (immunoreceptor tyrosine-based activation motif, ITAM), TCR identification And combine by MHC (the major histo-compatibility complex) Antigenic Peptide that molecule is offered, lead to the ITAM of CD3 Conserved sequence tyrosine residue by the tyrosine protein kinase p56lck phosphorylation in T cell, then can raise other and contain There is the tyrosine protein kinase (such as ZAP-70) of SH2 (Scr homology 2) structural domain.The phosphorylation of ITAM and and ZAP-70 Combination be T cell activation signal transduction process early stage one of important biochemical reaction.Therefore, the function of CD3 molecule is The TCR that transduces identifies activation signals caused by antigen.

2.HER2

(Shih C, Padhy LC, Murray M, the et al.Transforming genes of such as Shih in 1981 carcinomas and neuroblastomas introduced into mouse fibroblasts[J].Nature, 1981,290 (5803): 261-264.) oncogene neu, Slamon are cloned from rat neuroblastoma genome for the first time Deng (1987, Science 2；35；HER2 gene 177-182) is isolated from human cDNA library.Subsequent sequence analysis and dye Colour solid spectrum analysis finds that neu and HER2 is the same gene, traditionally referred to as HER2/neu gene or c-erbB-2 gene.HER2 The 2nd member of human epidermal growth factor acceptor family, which belongs to I receptor tyrosine kinase, also known as ErbB by Body family plays important regulating and controlling effect in the growth, differentiation and transfer process of many normal and abnormal epidermal cells, perhaps Generation, development and the state of an illness weight of more tumours and its active size are closely related.There are four receptors altogether in family: HER1, HER2, HER3 and HER4.These receptors, which can interact, generates heterologous or homodimer, and a plurality of signal transduction is logical in active cell Road, wherein HER2 plays an important role during cell signalling.The structure of HER2 includes the combination of extracellular growth factor Area, the transmembrane region of lipophilic and the intracellular region with adjusting carboxy terminal fragment.HER2 receptor intracellular region has tyrosine protein kinase PTK activity, itself also has several tyrosine residue Tyr phosphorylation sites.After specificity growth factor is in conjunction with HER2 receptor It can induce cross phosphorylation that is Dimerized and exciting receptor, the receptor of phosphorylation rapidly can turn extracellular growth signals It is directed in core, stimulation controls gene expression related with cell division.

HER2 is positioned at human chromosome 17q21, and coding molecular weight is the transmembrane protein of 185kD, has tyrosine kinase RTK Activity is in unactivated state under normal circumstances, participates in the adjusting of cell normal differentiation, usually only expresses in foetal period, adult Afterwards, the only trace expression in only a few normal tissue.HER2 gene is 2 copies in normal cell, and gene mutation can be swashed Living, amplification will lead to transcriptional upregulation, and albumen synthesis increases, to inhibit apoptosis of tumor cells, promote tumor cell proliferation, on Vascular endothelial growth factor VEGF/ vascular permeability factor VPF is adjusted, tumor angiogenesis is promoted, increases tumour cell and invades Power is attacked, [Artufel MV, Valero AC, the Llado RR, etal.Molecular such as body tissue anti-invasion barrier are destroyed Protocol for Her-2/neu analysis in breast carcinoma[J].Clin Transl Oncol, 2005,7.(11):504-511.].The overexpression of HER2 albumen is also in the division of cell, proliferation, conversion, turn for promoting tumour It moves, invade, play a significant role [Hynes NE, Stem DF.The biology of erbB-2/neu/HER- in adherency 2and its role in cancer[J].Biochem Biophys Acta,1994,1198(2-3):165-184.]。

In addition to gene mutation or amplification can occur, the expression for raising HER2 can also swash two main signals turn in the downstream HER2 Lead approach: MAPK access, PI3K/Akt access adjust apoptosis-related genes to cause waterfall type chain reaction, promote cell Infinite multiplication differentiation, inhibits apoptosis, so that canceration occur.The former is primarily involved in the mitosis of cell, and the latter mainly influences carefully The survival of born of the same parents and apoptosis.HER2 can pass through mediator's telomerase on MAPK pathway activation Ets transcription factor family member ER81 Transferase reverse transcriptase hTERT, and then lead to cell telomerase abnormal activation, make cell transformation and enter permanent increase Grow state [GoueliBS, JanknechtR.Upregulation of the catalytic telomerase subunit by the transcription factor ER81and oncogenic HER2/Neu,Ras,or Raf.Mol Cell Biol,2004,24:25-35.].After PI3K activation, phosphatidylinositols PI can be catalyzed and generate PIP2 and PIP3, they are intracellular Important second messenger can activate the protein kinase A kt/PKB in downstream, further result in the phosphorylation of downstream BAD albumen, thus It prevents BAD and apoptotic proteins Bcl-2, Bcl-XL from forming compound, while also inducing 1 phosphorylation of plug transcription factor, to press down Make the expression of former apoptogene.

In addition, HER2 oncogene is also metastases driven factor, HER2 is overexpressed can be related by starting a variety of transfers Mechanism and increase Nasopharyngeal neoplasms ability, such as cell migration rate, vitro invasion power, W Collagenase Type activity can also influence The synthesis of certain adhesion molecules such as E-cadherin etc., to promote to shift.(CarterW, the Hoying such as Carter J,Boswel l C,et al.HER-2/neu over-expression induces endothelial cell Retraction [J] .Int Cancer, 2001,91 (3): 295-299.) it studies and thinks, HER2 overexpression can make endothelial cell It shrinks, Dilated intercellular space, tumour cell is easy to pass through between endothelial cell, and tumour cell is displaced or shifts.Majority is ground Study carefully and think, HER2 gene magnification and (or) protein overexpression often prompt malignancy high, and transfer ability is strong.

The overexpression of HER2 is often related with the generation of tumour, such as:

(1) gastric cancer: gastric cancer is one of most common malignant tumour in China, poor prognosis, and 5 years survival rates of advanced gastric carcinoma are only 5%~20%, median survival time is no more than 1 year.The ratio that different research group's detection HER2 albumen is overexpressed in gastric cancer Rate variation is 7%~43%.Positive expression of the HER2 albumen in gastric cancer and tumor differentiation degree, Lauren parting and WHO divide Type is related, uncorrelated to age, gender, tumour happening part and clinical stages.

(2) breast cancer: research shows that, HER2 has gene in 20%~30% primary breast invasive ductal carcinoma The overexpression of amplification and albumen.The high expression of HER2 often results in the pernicious transfer of cell, therefore the breast cancer leaching of the HER2 positive Lubricant nature is strong, and DFS phase is short, poor prognosis.Experiment in vitro is shown, the expression of HER2 is inhibited to can lead to the apoptosis of tumour cell.

(3) oophoroma: oophoroma is the main reason for gynecological tumor is lethal.The overexpression and breast cancer of HER2 in oophoroma In it is similar, account for 15%~30%.(Verri E, Guglielmini P, Puntoni M, the et al.HER2/neu such as Verri oncoprotein overexpression in epithel ial ovarian cancer:evaluation of its Prevalence and prognostic significance [J] .Oncology, 2005,68:154-161.) research it is aobvious Show, the HER2 positive (2+/3+) patient significantly reduce than negative patient's (0/1+) Overall survival (29 months vs48 months, P < 0.05)., there is HER2 protein overexpression in the 20 gonad cell strain discoveries of oophoroma for observing for III, IV phase.

(4) prostate cancer: prostate cancer belongs to androgen-dependent when occurring, after receiving drug or operation castration Tumor regression, but eventually it is changed into androgen independence and continued growth, this is main in current prostate cancer therapy The problem of.Research shows that HER2 be prostate cancer from hormone-dependent type be changed into hormone independent during main mediation Person.(Signoretti S, Montironi R, Manola J, the et al.Her-2-neu expression such as Signoretti and progression toward androgen independence in human prostate cancer[J].J Natl Cancer Inst, 2000,92:1918-1925.) research and analyse DNA, RNA of different clinical stage tumor sample HER2 And the expression of protein, as the result is shown, the patient (UNTtumor) of 25% only operation excision prostate cancer, 59% Operation consent receive anti-androgen therapy patient (TAAtumor) and 78% androgen in treating failure after and Bone tumour occurs There is the HER2 being overexpressed in the patient of (androgen independent AI).

(5) lung cancer: the overexpression of HER2 is mainly related with genetic transcription and posttranscriptional modification in lung cancer.Studies in China is aobvious Show, HER2, which is overexpressed, occurs mainly in non-small cell lung cancer, and mainly gland cancer, rather than squamous carcinoma.But external 88 breast teeth The testing result of sharp Patients with Non-small-cell Lung shows that only 5 there are HER2 overexpressions, and are squamous cell carcinoma, research As a result there is difference.In addition, being overexpressed in lung cancer to HER2 also has different conclusions from the relationship of cell differentiation.

For the antibody drug of HER2 target spot: toltrazuril trade name Trastuzumab Herceptin, is using HER2 as target spot Humanized monoclonal antibodies.Herceptin is by gene engineering method by the anti-of the stable region of nonspecific human IgG and mouse The antigenic determinant of HER2 protein I gG is entrenched togather acquisition, not only has high affinity to HER2 receptor, while also solving Murine antibody of having determined is applied to the immunogenicity problem of human body, can be reduced the generation of human anti-mouse antibody, to avoid netted Endothelial system is removed.Inside and outside experimental study shows the expression lowered using Herceptin, can make cell decreased growth, and energy Significantly improve its sensibility to chemicotherapy.U.S. FDA in 1998 ratifies metastatic breast cancer of the medicine for HER2 overexpression It is also that only one is approved for treatment HER2/neu protein expression positive metastatic cream that two wires and the treatment of three lines, which are first, The Humanized monoclonal antibodies drug of gland cancer and early-stage breast cancer.

3. bispecific antibody technology develops

Bispecific antibody, two antigen-binding sites in an antibody molecule can be respectively in connection with two different antigens The antibody of epitope.

Antibody drug is the biology of the antibody engineering technology preparation based on cell engineering and technique for gene engineering Macromolecular drug, have many advantages, such as specific high, property is uniform, can for specific target spot beam system for.Monoclonal antibody is being faced Following three aspects: oncotherapy, immunity disease treatment and anti-infective therapy are mainly used on bed.Wherein tumour is controlled Treatment is the field that current monoclonal antibody is most widely used, and is come into the monoclonal antibody product of clinical test and listing at present, for swelling The product quantity accounting of tumor treatment is about 50%.Mab treatment tumour is a kind of for the pricking method of sick cell specific target Sharp immune system kills the immunotherapy of target cell, in order to enhance the effector function of antibody, especially killing tumor cell Effect, people attempt a variety of method engineered antibody molecules, bispecific antibody be improve Antybody therapy effect developing direction it One, become the hot spot of antibody engineering research field.

Bispecific antibody for immunization therapy is the artificial antibody containing 2 species-specific antigen binding sites, can be Bridge is erected between target cell and functional molecular (cell), the immune response with guiding performance is excited, in the immunization therapy of tumour In have broad application prospects.

4. prepared by bispecific antibody

Bispecific antibody can obtain through a variety of ways, and preparation method mainly has: chemical coupling method, hybridization-hybridization Tumor method and genetic engineering antibody the preparation method.Chemical coupling method is to connect the mode of 2 different monoclonal antibody chemical couplings It is connected together, has prepared bispecific monoclonal antibody, this is earliest bispecific monoclonal antibody concept.Hybridization-miscellaneous Handing over tumor method is to generate bispecific monoclonal antibody by way of cell hydridization method or three way cross tumor, these cell hydridizations Perhaps three way cross tumor is hybridoma fusion by building up or the hybridoma established and the lymphocyte obtained from mouse to tumor Obtained from fusion, the bispecific antibody of source of mouse can only be produced, its application is greatly limited.And with molecule There are a variety of forming types of genetic engineering humanization bispecific antibody, and mainly divides in the rapid development of biology techniques For bispecific miniantibody, double-chain antibody, single-stranded bivalent antibody, four class of multivalence bispecific antibody.Currently, having number in the world Kind genetic engineering double specific antibody drug enters clinical experimental stage, and shows preferable application prospect.

5. the adoptive immunotherapy of tumour

The adoptive immunotherapy of tumour is that self or allogeneic immunocompetent cell is inputted patient after amplification in vitro In vivo, direct killing tumour cell adjusts and enhances the immune function of body, and main includes LAK cell, til cell, activation The immunization therapy of T lymphocyte and CIK cell.And immunotherapy can only remove a small amount of, scattered tumour cell, for advanced stage Entity tumor curative effect it is limited.Therefore often combines as a kind of complementary therapy with conventional methods such as operation, chemotherapy, radiotherapies and answer With.After first cleaning a large amount of tumour cell with conventional method, then remaining tumour cell is removed with immunotherapy, tumour can be improved The effect of complex treatment.Wherein, adoptive immunotherapy is as a new method in combined therapy of tumour, with routine operation Treatment, radiotherapy, chemotherapy and other cells and molecular therapy are cooperated extensively, are illustrated in the treatment of kinds of tumors extensive Application prospect.However, a kind of more preferably mode should be that bispecific antibody one end can combine cultured immunocyte Surface antigen CD3, and in vivo, and the other end of bispecific antibody can be well in conjunction with tumour cell for input together therewith Surface antigen；In this way, bispecific antibody can erect the bridge between tumour cell and immunocyte in vivo, make immune thin Born of the same parents concentrate near tumor cells, and then kill to tumour cell.Tumour cell can be effectively solved by this method Transfer and diffusion, after overcoming operation, three great tradition therapeutic modality of chemicotherapy " be not thorough, easily transfer, side effect it is big " etc. disadvantages End.

Summary of the invention

Term and abbreviation

BiAb: bispecific antibody (bispecific antibody)

TA: tumour antigen (tumor antigen)

VH: heavy chain variable region (heavy chain variable region).

VL: light chain variable region (light chain variable region).

CL: constant region of light chain (constant region of light chain).

CDR: it is the abbreviation of English Complementarity determining regions (CDRs), refers to antibody Antigen complementary determining region.

ScFv: Single chain antibody segment (single-chain variable fragment), it is also known as single-stranded anti- Body.

CLD: cell line develops (cell line development)

FACS: fluorescence-activated cell sorting (Fluorescence-activated cell sorting), also referred to as streaming Cell sorting art.

The present invention is directed to the shortcoming of conventional monoclonal antibody, is carried out by genetic engineering and the method for antibody engineering new It is swollen to kill mainly to pass through CDC, ADCC and apoptosis capacity in conventional monoclonal antibody for the initiative of molecule --- bispecific antibody On the basis of oncocyte, the immunotherapy of mediate T cell is increased, substantially increases the function of immune system killing tumor cell Effect.

Specifically, the present invention provides technical solutions below: in one embodiment, it is anti-to provide a kind of bispecific Body, which is characterized in that the described antibody includes: (a) monovalent unit, is light-heavy chain pair, which is directed to tumour Cell surface antigen have specific binding capacity, preferably the tumor cell surface antigen be HER2, CD20, CD30 and CD133, the more preferably tumor cell surface antigen are HER2；(b) strand unit, is fusogenic peptide, which includes single Chain Fragment variable ScFv and Fc segment with hinge area, CH2 structural domain and CH3 structural domain, what wherein the fusogenic peptide was directed to exempts from Epidemic disease cell is selected from T cell, NKT cell or CIK cell；Preferably, which has immune cell surface antigenic CD3 special Property binding ability.

In one embodiment, the CH2 structural domain of the strand unit of the bispecific antibody be located at ScFv segment and Between CH3 structural domain；The strand unit does not include CH1 structural domain.

In one embodiment, the single chain variable fragment of bispecific antibody is by light chain variable region and heavy chain variable region knot Structure domain composition, they all target epitope CD3.

In one embodiment, in monovalent unit, the light chain constant domain and light chain variable region of the light chain Structural domain all targets tumour antigen epitope HER2；The heavy chain constant domain CH1 and heavy-chain variable domains of the heavy chain Target tumour antigen epitope HER2；Light chain is by disulfide bond in conjunction with heavy chain；Heavy chain passes through one or more disulfide bond and institute State fusogenic peptide combination, hinge of preferably one or more of disulfide bond formations between the CH1 (or VLs) and CH2 structural domain Between the amino acid residue of sequence.

In one embodiment, strand unit includes the anti-CD3 of antibody for CD3, and monovalent unit includes being directed to HER2 The anti-HER2 of antibody.

In one embodiment, the amino acid sequence of the heavy chain of the anti-HER2 of antibody is amino acid sequence shown in sequence number 1 Column, the amino acid sequence of the light chain of the anti-HER2 of antibody are amino acid sequence shown in sequence number 3 and described anti- The amino acid sequence of CD3ScFv-Fc is amino acid sequence shown in sequence number 5；And anti-HER2 heavy chain is on 223 sites Cysteine is connect in the form of disulfide bond with the cysteine on 214 site of light chain of anti-HER2, the anti-HER2 weight Cysteine of the chain on 255 and 258 sites of cysteine and anti-CD3ScFv-Fc on 229 and 232 sites is respectively with two The form of sulfide linkage connects, the anti-HER2 heavy chain on 395 and 412 sites with 428 and 397 sites of anti-CD3ScFv-Fc Upper forming salt bridging connects, the anti-HER2 heavy chain on 369 sites with formed on 436 sites of anti-CD3ScFv-Fc it is grand Dash forward-enter-cave connection.

In one embodiment, the heavy chain in monovalent unit includes the Fc segment of people or humanization, it is preferable that this is heavy The Fc segment of chain includes human IgG1 Fc segment；The Fc segment of the fusogenic peptide includes the Fc segment of people or humanization, it is preferable that The Fc segment of the fusogenic peptide includes human IgG1 Fc segment.

In one embodiment, Fc sections of the human IgG1 of the monovalent unit and the IgG1Fc of the strand unit pass through salt Bridge enters with knuckle-- and cave structure connects.

In one embodiment, a kind of preparation method of bispecific antibody is provided, which comprises

(1) weight of monovalent unit, light chain are building up to respectively on the first expression vector respectively, strand unit is building up to On two expression vectors；

(2) it by the first and second expression vectors together cotransfection into cell, cultivates and takes supernatant；

(3) the isolated bispecific antibody after purification of supernatant will be expressed；Preferably, the cell is CHO-S thin Born of the same parents；Or preferably, the separating step includes: that protein A affinity chromatography column captures all band Fc structures from expression supernatant The antibody in domain realizes the separation of target bispecific antibody and by-product by SP cation-exchange chromatography, after Q column, finally Concentration displacement buffer PBS.

In one embodiment, the first expression vector is pCHO1.0；Second expression vector is that pCHO1.0- tide is mould Element.

In one embodiment, the monovalent unit is anti-HER2 antibody, and expanding its light chain the primer is Kozak (EcoRV) F, MK-leader (EcoRV) F and hIgK (PacI) R, is expanded by over-lap PCR, by Kozak sequence, MK-leader And restriction enzyme site EcoRV and PacI introduces light chain；Expanding its heavy chain the primer is Kozak (AvrII) F, MK-leader (AvrI I) F and hIgG1 (BstZ17I) R, is expanded by over-lap PCR, by Kozak sequence, MK-leader and restriction enzyme site AvrI I and BstZl7I, which introduces heavy chain, to be seen；The LC genetic fragment that has expanded with the pCHO1.0 table of EcoRV and PacI digestion Homologous recombination is carried out up to carrier, obtains the expression vector for being packed into anti-HER2 light chain；Then with after AvrI I and BstZl7I digestion Homologous recombination is carried out with HC again, obtains the pCHO1.0 expression vector of anti-HER2, plasmid is named as the anti-HER2-HL- of pCHO1.0- KKW；

The strand unit is anti-CD3ScFv-Fc antibody, and expanding its primer is Kozak (AvrII) F, MK- Leader (AvrI I) F, L2K-VH (MK) F1 and hIgG1 (BstZ17I) R expands AntiCD3 McAb ScFv-Fc structure by over-lap PCR Domain, and Kozak sequence, MK-leader and restriction enzyme site AvrI I and BstZl7I are introduced into ScFv-Fc, the gene that will have been expanded Segment and the pCHO1.0- hygromycin expression vector of digestion carry out homologous recombination, obtain the expression load for being packed into AntiCD3 McAb ScFv-Fc Body, plasmid are named as pCHO1.0- hygromycin-L2K-ScFv-Fc-LDY.

In one embodiment, any of the above-described bispecific antibody or double spies according to the preparation of any of the above-described method The purposes of heterogenetic antibody in medicine preparation, the drug are used to treat the caused tumour or phase of HER2 specific antigen expression Related disorders, or for killing expression HER2 cell.

In one embodiment, any of the above-described bispecific antibody or double spies according to the preparation of any of the above-described method The purposes of heterogenetic antibody in medicine preparation, it is special for treating expression HER2 that the drug is used for the screening in tumor cell line The drug of the tumour cell related disease of antigen or evaluation are for treating the tumour cell correlation disease of expression HER2 specific antigen The drug effect of the drug of disease.The present invention also provides technical solutions below:

The present invention provides a kind of novel antibodies for being referred to as bispecific antibody, and establish and a kind of utilize the immune of human body System carries out immunization therapy and carries out the method for the drug efficacy study of bispecific antibody.This bispecific antibody, as one kind Novel antibodies are simultaneously used for pharmacophore model, introduce T cell to specific cytotoxic effect of the tumour antigens such as HER2.

The present invention provides a kind of new methods to prepare bispecific antibody MSBODY (monomer and ScFv-Fc Bispecific antibody) (as shown in Figure 2), which includes two groups of heavy and light chain combinations, wherein one group is special Some transformations are carried out in conjunction with a kind of antigen, and in the area its heavy chain Fc, makes its versus wild type, is not easy itself and forms dimer； And another group of specific bond another kind antigen, other transformation equally is carried out in the area its heavy chain Fc, itself is also not easy and forms two Aggressiveness, and heterozygosis dimer is readily formed between this two groups of heavy and light chains.And wherein one group of antibody structure is monovalent unit, Another group is single-stranded (ScFv-Fc), a possibility that respective light chain is with the mispairing of other side's heavy chain is avoided in this way, to be formed The bispecific antibody protein molecular of 125KD.After Fc transformation, the heavy chain of monovalent unit and single-stranded natural heterodimerization, while CL The natural dimerization between CH1, eventually forms MSBODY, and each domain arrangement sequence of MSBODY and structural schematic diagram are shown in Fig. 2.

The method that bispecific antibody made above is utilized in the present invention, prepares bispecific antibody.It is wherein with HER2 It is the bispecific antibody of target spot with CD3, is named as HER2XCD3, such as Fig. 2, anti-HER2 is here monovalent unit form, packet Anti- HER2 heavy chain and light chain are included, anti-CD3 is here ScFv-Fc form, including anti-CD3VH, VL, Fc structural domain.It is above double special Heterogenetic antibody is constructed by antibody genetic engineering method, the monovalent unit heavy chain and unit price list of bispecific antibody MSBODY First light chain double promoter expression vector and ScFv-Fc expression vector.According to monovalent unit light chain (LC), monovalent unit heavy chain (HC), the multiple cloning sites design primer in ScFv, Fc gene order and carrier.Wherein LC, HC, ScFv and Fc are carried out respectively PCR amplification obtains genetic fragment by PCR or Overlap extension PCR method, is then cloned by homologous recombination method.Digestion PCHO1.0 or pCHO1.0- hygromycin vector, the then carrier after purification and recovery PCR product and digestion, point two steps are respectively by LC Segment, HC segment homologous recombination are cloned on pCHO1.0 carrier, and it is mould that ScFv-Fc segment homologous recombination is cloned into pCHO1.0- tide On plain carrier, and it is sequenced.Recombinant protein MSBODY expression in mammalian cells, detection, using transfection reagent by table Up to monovalent unit heavy chain and monovalent unit light chain plasmid and expression strand unit plasmid co-transfection into mammalian cell, Regather the expression that supernatant carries out SDS-PAGE and Western blotting detection MSBODY.By the culture solution after transfection expression Supernatant centrifugation, filtering are diluted with combination buffer, cross affinity column, elution buffer elution, SDS-PAGE detection purifying Protein.

The beneficial technical effect of technical solution of the present invention has:

1. the antibody includes two different antigen-binding polypeptides units this application provides a kind of heterodimeric antibodies. The corresponding homodimer molecular size range of the heterodimer is different, can be distinguished using the size of molecular weight heterodimer with Homodimer, to more conveniently determine the purity of bispecific antibody.One of the two antigen-binding polypeptides units include class It is similar to the light-heavy chain pair of wild-type antibodies, in entire the application, which is also referred to as " monovalent unit ".Another antigen knot Closing polypeptide unit includes single chain variable fragment (ScFv).Such ScFv can be fused to the constant fragment (Fc) of antibody.In this Shen " strand unit " please be also referred to as by this fusogenic peptide in full text.

2. internal medicine outside the immunocyte contact element mediated the invention discloses a kind of novel bispecific antibodies MSBODY Imitate the foundation and its application of experimental method.The present invention includes the immunocyte mediated in bispecific antibody drug research process The foundation and detection of killing, the preparation of bispecific antibody and bispecific antibody internal pharmacophore model in vitro.Bispecific Antibody MSBODY includes one group of strand unit (ScFv connection Fc combination), and another group is then monovalent unit (combination of heavy and light chain), A kind of tumor-cell antigen of people of middle unit price unit specific bond, a series of tumour cell film surface antigens such as including HER2, and And some transformations are carried out in the area its heavy chain Fc, make its versus wild type, is not easy itself and forms dimer；And another group of strand unit The T cell antigen CD3 of specific bond another kind people equally carries out other transformation in the area its heavy chain Fc, is also not easy from figure At dimer, and heterodimer is readily formed between this two groups of units.At the same time, bispecific antibody can in target cell and Bridge is erected between functional molecular (cell), excites the immune response with guiding performance, in the presence of immunocyte, the present invention Bispecific antibody have extremely strong fragmentation effect to tumour cell, in the immunization therapy of tumour with wide application before Scape.

It is surprising that the application proves that this asymmetrical antibody is stable and imitates with high antigen binding Rate.This makes us feeling surprised, even as it have been shown that the homodimer of single-chain antibody is all not in physiological conditions Stable.For example, " ScFv Antibody:Principles and Clinical Application, " (Cl of Ahmad etc. Inical and Developmental Immunology, 2012:980250 (2012)), IgG class of the display based on ScFv is anti- Body is unstable, and needs further transformation to reduce aggregation and improve stability.

In addition, heterodimer has and is made of any antigen-binding polypeptides unit because having asymmetry The different isoelectric point of homodimer.Based on the isoelectric point difference between heterodimer and homodimer, will can easily need The heterodimer wanted is separated with homodimer, is greatly reduced existing for the generally existing downstream process exploitation of bispecific antibody It is difficult.

Detailed description of the invention

It in order to more clearly explain the technical solutions in the embodiments of the present application, below will be to needed in the embodiment Attached drawing is briefly described, it should be apparent that, the accompanying drawings in the following description is only some embodiments as described in this application, right For those of ordinary skill in the art, without creative efforts, it can also be obtained according to these attached drawings Its attached drawing, in which:

Fig. 1 .CD3 schematic arrangement.

Fig. 2 .HER2 × CD3 bi-specific antibody molecule schematic diagram.

The double antibody electrophoresis and purity detecting result figure of Fig. 3 purifying, (A) non-reduced SDS-PAGE electrophoresis, M: protein molecular Amount label；1:M802；(B) the HPLC-SEC purity peak shape figure of M802.

The affinity feelings of HER2 × CD3 double antibody and SK-BR-3 cell that Fig. 4 is measured based on flow cytometric analysis Condition figure.

Affinity situation of Fig. 5 based on the flow cytometric analysis HER2 × CD3 double antibody measured and Jurkat cell Figure.

Fig. 6 .HER2 positive cell NCI-N87 (CFSE dyeing) and Jurkat cell (PKH26 dyeing) are dissipated without antibody streaming Point diagram.

Fig. 7 .HER2 positive cell NCI-N87 (CFSE dyeing) and Jurkat cell (PKH26 dyeing) have M802 antibody total It is incubated for streaming scatter plot.

Combination situation map of the double antibody to SK-BR-3 cell after Fig. 8 flow cytometer detection heat challenge experiment process.

Combination situation map of the double antibody to human PBMC's cell after Fig. 9 flow cytometer detection heat challenge experiment process.

Cell in vitro poison experimental result picture of Figure 10 .M802 and hPBMC to SK-BR-3 cell.

Cell in vitro poison experimental result picture of Figure 11 .M802 and hPBMC to NCI-N87 cell.

Cell in vitro poison experimental result picture of Figure 12 .M802 and hPBMC to MDA-MB-231 cell.

Cell in vitro poison experimental result picture of Figure 13 .M802 and hPBMC to HEK-293 cell.

The interior animal experiment result figure of Figure 14 .M802.

Specific embodiment

Embodiment 1: the expression vector establishment (HER2 × CD3, M802) of bispecific antibody

1. bispecific antibody sequence design

It is named as HER2 × CD3MSBODY by the bispecific antibody of target spot of HER2 and CD3, wherein monovalent unit is The heavy chain light chain pair of anti-HER2, sequence (the PDB database of variable region amino acid sequence reference monoclonal antibody Herceptin No.1N8Z), including anti-HER2 heavy chain and light chain, contain Fab and Fc structural domain；Strand unit is the ScFv-Fc form of AntiCD3 McAb, Variable region amino acid with reference to monoclonal antibody L2K sequence (refer to US20070123479 sequence number 2), including AntiCD3 McAb VH, VL, Fc structural domain.Wherein the heavy chain Fc of monovalent unit and the Fc (with the heavy chain Fc of human IgG1) of strand unit carry out amino acid mutation Transformation, specific Fc transformation process make it respectively be not easy to form homodimer (homodimer) referring to PCT/CN2012/084982, And it is easily formed heterodimer (heterodimer), which is bispecific antibody HER2 × CD3MSBODY, is compiled Number be M802.Meanwhile in order to M802 can Chinese hamster ovary cell (CHO (Cricetulusgriseus, hamster, Chinese, ovary) it expresses in cell, and can be secreted into culture medium, select the leader peptide sequences of source of mouse antibody kappa As secreting signal peptide.The amino acid sequence and nucleic acid sequence of each structural domain and signal peptide are shown in following sequence number: 1-8.Signal Peptide is directly connected in the N-terminal of antibody variable region.In patent PCT/CN2012/084982, a kind of anti-HER2 × CD3's MSBODY, monovalent unit variable region come from Trastuzumab, and strand unit variable region comes from humanization OKT3, and the MSBODY is in the present invention Middle number is M801, as a comparison antibody.

Monovalent unit heavy chain amino acid sequence (Trastuzumab, sequence number 1)

EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKN TAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFP EPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPP CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWE SNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK-

Monovalent unit heavy chain nucleic acid sequence (Trastuzumab, sequence number 2)

GAAGTGCAGCTGGTGGAAAGCGGCGGCGGCCTGGTGCAGCCGGGCGGATCCCTGCGCCTGAGCTGCGCG GCGAGCGGCTTTAACATTAAAGATACCTATATTCATTGGGTGCGCCAGGCGCCGGGCAAAGGCCTGGAATGGGTGGC GCGCATTTATCCGACCAACGGCTATACCCGCTATGCGGATAGCGTGAAAGGCCGCTTTACCATTAGCGCGGATACCA GCAAAAACACCGCGTATCTGCAGATGAACAGCCTGCGCGCGGAAGATACCGCGGTGTATTATTGCAGCCGCTGGGGC GGCGATGGCTTTTATGCGATGGATTATTGGGGCCAGGGCACCCTGGTGACCGTGAGCTCAGCCTCCACCAAGGGCCC ATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACT ACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTA CAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTG CAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACAT GCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTC ATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTG GTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGG TCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTC CCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATC CCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGG AGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACGATACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTC CTCTACAGCGATCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGC TCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA

Monovalent unit light-chain amino acid sequence (Trastuzumab, sequence number 3)

DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISS LQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNAL QSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC-

Monovalent unit light chain nucleic acid sequence (Trastuzumab, sequence number 4)

GATATTCAGATGACCCAGAGCCCGTCAAGCTTAAGCGCGAGCGTGGGCGATCGCGTGACCATTACCTGC CGCGCGAGCCAGGATGTGAACACCGCGGTGGCGTGGTATCAGCAGAAACCGGGCAAAGCGCCGAAACTGCTGATTTA TAGCGCGAGCTTTCTGTATAGCGGCGTGCCGAGCCGCTTTAGCGGCAGCCGCAGCGGCACCGATTTTACCCTGACCA TTAGCAGCCTGCAGCCGGAAGATTTTGCGACCTATTATTGCCAGCAGCATTATACCACCCCGCCGACCTTTGGCCAG GGTACCAAAGTGGAAATTAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAA ATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATA ACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGC ACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTC GCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG

Single-stranded strand unit amino acid sequence (L2K, sequence number 5)

DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSS TAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSGGGGSGGGGSGGGGSDIQLTQSPAIMSASPGEKVT MTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTF GAGTKLELKGAAAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRD ELTKNQVSLTCRVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLASKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPGK-

Single-stranded strand unit nucleic acid sequence (L2K, sequence number 6)

GACATCAAACTGCAGCAGTCAGGGGCTGAACTGGCAAGACCTGGGGCCTCAGTGAAGATGTCCTGCAAG ACTTCTGGCTACACCTTTACTAGGTACACGATGCACTGGGTAAAACAGAGGCCTGGACAGGGTCTGGAATGGATTGG ATACATTAATCCTAGCCGTGGTTATACTAATTACAATCAGAAGTTCAAGGACAAGGCCACATTGACTACAGACAAAT CCTCCAGCACAGCCTACATGCAACTGAGCAGCCTGACATCTGAGGACTCTGCAGTCTATTACTGTGCAAGATATTAT GATGATCATTACTGCCTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCAGGAGGCGGCGGTTCAGGCGG AGGTGGAAGTGGTGGAGGAGGTTCTGACATTCAGCTGACCCAGTCTCCAGCAATCATGTCTGCATCTCCAGGGGAGA AGGTCACCATGACCTGCAGAGCCAGTTCAAGTGTAAGTTACATGAACTGGTACCAGCAGAAGTCAGGCACCTCCCCC AAAAGATGGATTTATGACACATCCAAAGTGGCTTCTGGAGTCCCTTATCGCTTCAGTGGCAGTGGGTCTGGGACCTC ATACTCTCTCACAATCAGCAGCATGGAGGCTGAAGATGCTGCCACTTATTACTGCCAACAGTGGAGTAGTAACCCGC TCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAAGGTGCGGCCGCAGAGCCCAAATCTTGTGACAAAACTCACACA TGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCT CATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACT GGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTG GTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCT CCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCAT CCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCGGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTG GAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGAAGTCCGACGGCTCCTTCTT CCTCGCCAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGG CTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA

The leader peptide sequences (amino acid sequence, sequence number 7) of mouse kappa

METDTLLLWVLLLWVPGSTG

The leader peptide sequences (nucleic acid sequence, sequence number 8) of mouse kappa

ATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCAGGTTCCACTGGT

2. bispecific antibody gene cloning

Selection(abbreviation pCHO1.0 is purchased from Lifetechnologies to 0ExpressionVector KitArticle No. A13696-01) it goes to clone and express the weight of monovalent unit as expression vector Chain and light chain gene, pCHO1.0- hygromycin expression vector are by being replaced in pCHO1.0 carrier with hygromycin gene Puromycin genetic modification is selected to clone and express strand unit.Primer in table 1 is designed according to cloning approach After good, it is sent to Suzhou Jin Weizhi Biotechnology Co., Ltd and is synthesized.PCR amplification is carried out with the primer in table 1, template is Gene chemical synthesis or the gene plasmid being subcloned on pCDNA3.1 or pUC57, PCT/CN2012/084982 patent in earlier trials It has a detailed description, then the weight of monovalent unit, light chain cdna is building up to difference on the expression vector of pCHO1.0 respectively respectively and opened Strand unit cDNA is building up on the expression vector of pCHO1.0- hygromycin by mover downstream.

Primer used in 1 bispecific antibody gene cloning of table

Initial PCR amplification template DNA: the template DNA of 35ng, e.g., the light chain and heavy chain of target antibody；10 μM of 1 μ l are just To primer and reverse primer；The 10xPCRBuffer buffer of 2.5 μ l；The 10mMdNTP of 1 μ l；2.5 units/μ of 1 μ l LPyrobestDNA polymerase (Takara, R005A)；It is softly mixed in 200 μ LPCR pipes with distilled water to 25 μ l total volumes, And it is quickly rotated in microcentrifuge to collect reaction mixture to tube bottom.Use Gene Amp PCR System 9700 (Applied Biosystem) and progress PCR reaction arranged below: 95 DEG C, 5 minutes；25 circulations below: 95 DEG C, every time 30 seconds；56 DEG C, 30 seconds；With 72 DEG C, 1 minute.

It is expanded, Kozak sequence, MK-leader and restriction enzyme site EcoRV and PacI is introduced light by a few wheel over-lap PCRs Chain；And Kozak sequence, MK-leader and restriction enzyme site AvrII and BstZl7I are introduced heavy chain by corresponding primer.To first it expand The LC genetic fragment increased carries out homologous recombination with the pCHO1.0 expression vector of EcoRV and PacI digestion, and it is anti-to obtain loading The expression vector of HER2 light chain；Then with homologous recombination is carried out with HC again after AvrII and BstZl7I digestion, obtain anti-HER2's PCHO1.0 expression vector, plasmid are named as pCHO1.0-- Trastuzumab-HL-KKW.

AntiCD3 McAb ScFv-Fc structural domain is expanded by over-lap PCR, and by Kozak sequence, MK-leader and restriction enzyme site AvrII and BstZl7I introduces ScFv-Fc, by the pCHO1.0- hygromycin expression vector of the genetic fragment expanded and digestion Homologous recombination is carried out, the expression vector for being packed into AntiCD3 McAb Scfv-Fc is obtained, plasmid is named as pCHO1.0- hygromycin-L2K- ScFv-Fc-LDY。

Embodiment 2: bispecific antibody expression and purification

1. the expression of bispecific antibody

It carries out plasmid using the big extraction reagent kit of endotoxin-free (Qiagen, 12391) to mention greatly, concrete operations are provided according to manufacturer Specification carry out.The specification that CHO-S cell culture is provided according to manufacturer CDFortiCHO culture medium (Invitrogen, Article No. A11483-1) in, 37 DEG C are placed in, 5%CO₂It is cultivated in cell incubator, after getting out cell, according to manufacturer Specification (Maxcyte), it is using MaxcyteSTX electroporation that plasmid pCHO1.0- Trastuzumab-HL-KKW and pCHO1.0- tide is mould Cotransfection expresses the bispecific antibody M802 of anti-HER2 × CD3 into CHO-S cell to element-L2K-ScFv-Fc-LDY together. After culture 14 days, expression supernatant is harvested by centrifugation in 800Xg.

2. the purifying of bispecific antibody

Supernatant 0.22uM membrane filtration is expressed, (is purchased from GE company, column goods using MabselectSuRe affinity column Number 18-1153-45, filler article No. 17-5438-01) antibody of all band Fc structural domains is captured from expression supernatant, it is slow with balance Fliud flushing (9.5mM NaH₂PO₄+40.5mM Na₂HPO₄, pH7.0) balance chromatographic column after, cross affinity column, use elution buffer (50mM citric acid+100mM arginine, pH3.2) elution.By SP cation-exchange chromatography, target bispecific antibody is realized With the separation of by-product, cation exchange column is purchased from GE company (column article No. 18-1153-44, filler article No. 17-1087-01), uses Equilibration buffer A (43.8mM NaH₂PO₄+6.2mM Na₂HPO₄, pH6.0) and after balance chromatographic column, the double pure water dilution electricity of sample It is directed between 3.0-3.5ms, after crossing the combination of SP pillar, with elution buffer B (43.8mM NaH₂PO₄+6.2mM Na₂HPO₄+1M NaCl, pH6.0) 20 column volume linear elutions；Finally concentration displacement BufferPBS.Bispecific antibody after purification carries out SDS-PAGE, SEC detection, purity are shown in Fig. 3 95% or more.

Embodiment 3: the combination determination of activity (FACS) of bispecific antibody and cell

Bispecific antibody of the invention is in conjunction with the target antigen on corresponding cell.The present invention is with SK-BR-3 (purchased from China Type Tissue Collection) cell as the HER2 positive, Jurkat (American Type Culture collection warehousing (ATCC), TIB- 152) cell as the CD3 positive, and its cell-bound activity is measured with double antibody prepared by the present invention.

1. utilizing the combination activity of flow cytometer showed method detection bispecific antibody and SK-BR-3 cell

Enough SK-BR-3 cells are cultivated, with the digestion of 0.25% pancreatin, cell is collected by centrifugation.Bispecific is diluted simultaneously Antibody, concentration is since 160nmol, 4 times of gradient dilutions, obtains 6 concentration gradients, spare.By the cell PBS+1% of collection FBS is washed twice, then plus PBS+1%FBS cell is resuspended to 4 × 10⁶A cell/ml, plating cells are in 96 orifice plates, every hole 50ul (2×10⁵A cell), the bispecific antibody that 50ul has diluted is added, is incubated at room temperature 1 hour；Supernatant is removed in centrifugation, is washed with PBS Twice of cell, then cell is resuspended with the anti-human igg FC antibody (Biolegend, 409304) of the PE label diluted, room temperature is protected from light It is incubated for 30 minutes, PBS is washed twice, then is resuspended with 100ulPBS, upper machine testing, then with average fluorescent strength, by with software The binding affinity KD value of GraphPadPrism5.0 progress analytical calculation double antibody and SK-BR-3.HER2 × CD3 as the result is shown Double antibody and the SK-BR-3 cell of the HER2 positive have good combination activity, see Fig. 4.It is tied with HER2 positive cell SK-BR-3 Close situation: the KD value of M801 is 14.84nM, and the KD value of M802 is 10.61nM, and the KD value of Trastuzumab is 3.772nM.

2. the combination activity that flow cytometer showed method detects bispecific antibody and Jurkat cell

Enough Jurkat suspension cells are cultivated, cell is collected by centrifugation.Next experimentation and above-described embodiment phase Together, cell 100ulPBS being resuspended, upper machine testing, with average fluorescent strength, by with software GraphPad Prism 5.0 Carry out the binding affinity KD value of analytical calculation double antibody and Jurkat cell.HER2 × CD3 double antibody and CD3 sun as the result is shown Property Jurkat cell there is good combination activity, see Fig. 5.The situation in conjunction with CD3 positive cell Jurkat: the KD value of M801 It is 7.25nM, the KD value of M802 is 6.61nM, and the KD value of Trastuzumab is 0.39nM.

3. the total Binding experiment of immunocyte and tumour cell that double antibody mediates

By cultured NCI-N87 (HER2 positive gastric carcinoma cell, be purchased from China typical culture collection center) and Jurkat cell is collected by centrifugation and is washed 2 times with PBS, dyed respectively with CFSE and PKH-26.M802 is diluted to 160nM simultaneously.It will Supernatant is removed in NCI-N87 and the Jurkat cell centrifugation dyed, and is washed twice with PBS+1%FBS, then plus PBS+1%FBS be resuspended it is thin Born of the same parents are to 4 × 10⁶A cell/ml is uniformly mixed by 1:1, by plating cells in 96 orifice plates, every hole 50ul (2 × 10⁵A cell), The bispecific antibody that 50ul has diluted is added, is incubated at room temperature 1 hour；Supernatant is removed in centrifugation, is washed twice of cell with PBS, is finally used 100ulPBS is resuspended, and the ratio of double positive cells is analyzed in the detection of upper machine (FC500, Beckman), by with software GraphPadPrism5.0 carries out analytical calculation.As a result in the case where being displayed without M802, the ratio of the double fluorescence of flow cytometer detection Very low (Fig. 6)；In the case where HER2 × CD3 double antibody M802 is added, the ratio of the double fluorescence of flow cytometer detection reaches 26.3%, Show that M802 can promote being total to for two kinds of cells in combination with the NCI-N87 cell of the HER2 positive and the Jurkat cell of the CD3 positive In conjunction with (see Fig. 7).

Embodiment 4: the thermal stability determination of bispecific antibody

1. the hot challenge of bispecific antibody is tested

Antibody is diluted to 0.5mg/mL with PBS, is dispensed into PCR pipe with the specification of 50 μ L/ pipes, in PCR instrument (ABIPCRsystem9700) 60min is heat-treated on.Temperature gradient is arranged in PCR instrument from left to right, from 37 DEG C to 82 DEG C, each sample Product correspond to a temperature.After having handled, cooling sample is transferred in 96 orifice plate of V-type bottom (Corning), 4 DEG C, 2000rpm from Heart 30min.Take supernatant for SK-BR-3 cell or human PBMC's cell binding assay.Cell is incubated for altogether at room temperature with supernatant 30min is washed twice with the 1%FBS-PBS being pre-chilled on ice, then with the goat-anti people secondary antibodies of 50 times of diluted PE labels (Sigma, P9170) room temperature dyes 30min.The 1%FBS-PBS of cell pre-cooling after dyeing is washed 3 times, is resuspended in PBS and is used fluidic cell Instrument (FC500, Beckman) analysis: 100,000 cell counts.With 5 software of GraphPad Prism S-shaped having a variable slope Dose response (a sigmoidal dose response with variable slope) model is analyzed.Thermal denaturation is bent The temperature midrange of line is T₅₀。

Single chain antibody fragments (ScFv) pass through a link peptide (Gly₄Ser)₃Heavy chain variable region is connected with light chain variable region Get up and is formed.But have been reported that in ScFv unstability may will affect the quality (Michaelson of antibody drug JS,etc.,Farrington GK,LugovskoyA,Joseph I,Bailly V,Wang X,Garber E,Browning J,Glaser SM.Anti-tumoractivity of stability-engineered IgG-like bispecific antibodies targeting TRAIL-R2and LTbetaR.MAbs.2009Mar-Apr；1(2):128-41.).M802 Monovalent unit and M801 it is completely the same, T of the two in conjunction with SK-BR-3₅₀It is also very close to (Fig. 8), the T of M802₅₀= 60.60 DEG C, the T of M801₅₀=57.97 DEG C；But the strand unit of M802 uses the variable region of L2K, the single-stranded list of M801 Member uses the variable region of humanization OKT3, the T in conjunction with T cell₅₀Value difference is not larger (Fig. 9), the T of M802₅₀=59.98 DEG C, the T of M801₅₀=48.79 DEG C, M802 thermal stability is substantially better than M801.

Embodiment 5: the cell in vitro that double antibody mediates kills detection

The separation of human peripheral blood mononuclear cell 1. (hPBMC) cell

Fresh anticoagulant people's blood is taken, 400g is centrifuged 5min, abandons supernatant.The erythrocyte cracked liquid of 10 times of cell volumes is added, gently Mixing, room temperature or on ice cracking 4-5 minutes are beaten in featheriness.It is preferably appropriate in cracking process to shake to promote erythrocyte splitting.4℃ 400g is centrifuged 5min, abandons red supernatant.If erythrocyte splitting is incomplete, step 2 and 3 is repeated once.Washing 1-2 times.It is added 5 times Precipitating is resuspended in the PBS of cell precipitation volume, and 4 DEG C of 400g are centrifuged 2-3 minutes, abandons supernatant.It can repeat 1 time, wash 1-2 times altogether. It needs to be resuspended after cell precipitation with appropriate 4 DEG C of pre-cooling PBS up to hPBMC according to experiment, can carry out the subsequent experimental such as counting.

2. double antibody effectively mediates PBMC cell killing HER2 positive tumor cell to detect

With pancreatin digestion target cell (including the highly expressed SK-BR-3 breast cancer cell of HER2, the highly expressed NCI- of HER2 N87 stomach cancer cell, the HEK-293 human embryonic kidney cells of MDA-MB-231 breast cancer cell and the HER2 feminine gender of HER2 low expression, Purchased from China typical culture collection center), prepare single cell suspension.Target cell, dyeing are dyed with final concentration of 5 μM of CFSE Cell is resuspended to 2 × 10 with the 10%FBS-1640 of the cell culture afterwards⁵/ ml, according to 2 × 10⁴/ hole, i.e. 100 holes μ l/ are added 96 orifice plate overnight incubations.The effector cell (hPBMC) of 5 times of target cell numbers is added in experimental design, and control wells are arranged in 50 holes μ l/, Culture medium it is not necessary that the Kong Zeyong same volume of PBMC cell is added fills into.It is added while PBMC cell is added by experimental design Corresponding antibodies, the hole 50ul/ are filled into it is not necessary that the culture medium of Kong Zeyong same volume of antibody is added.96 orifice plates are taken out after 48h, are used It is single cell suspension that pancreatin, which digests each hole cell, all supernatants and cell suspension during this it is corresponding be collected into 1.5ml from In heart pipe, 500 × g is centrifuged min.Supernatant is abandoned, each hole is added 150 μ l1%FBS-PBS and mixing cell is resuspended.Each pipe is in streaming PI (final concentration of 1 μ g/ml) dyeing is added in 10-15min before machine.Machine testing CFSE, PI double positive cells account for CFSE sun in streaming Property cell proportion is the death rate of target cell.

Clearly to the highly expressed tumor cytotoxicity effect of HER2, highest killing rate uses agent up to 80% to M802 Amount is far below Trastuzumab and L2K (Figure 10,11)；Also there is significant killing to the tumour cell of HER2 low expression, and effect is significantly excellent In Trastuzumab and L2K (Figure 12).But the cell completely negative for HER2, M802 do not show fragmentation effect (Figure 13). Illustrate M802 double antibody in vitro in cellulotoxic experiment, in the presence of immunocyte, different to HER2 positive expression amount is swollen Oncocyte has good fragmentation effect, and the cell that do not express for HER2 does not have toxicity substantially.

Embodiment 6: the Composition analyzed of bispecific antibody killing subcutaneous transplantation tumor

The culture of CIK cell: CIK cell Primary culture liquid (serum-free X-Vivo cell culture fluid+750IU/mlIFN- is used ± 2% autologous plasma of γ) every part of cell filled into 30ml, it is added to 75cm²In culture bottle, it is placed in saturated humidity, 37 DEG C, 5.0% CO₂Incubator culture.After culture 24 hours, CIK cell stimulating factor mixed liquor 1ml (serum-free X-Vivo cell culture is added Liquid+75ng/ml OKT3 monoclonal antibody (self-control), 750IU/ml interleukin-22 (IL-2), 0.6ng/ml interleukin-11 (IL-1 α), continue to set In saturated humidity, 37 DEG C, 5.0%CO₂Culture in incubator.Following step determines to mend according to the growing state of CIK cell Liquid (± 2% autologous plasma of serum-free X-Vivo culture solution+750IU/ml IL-2), the thing passed on, will substantially maintain cell In 2*10⁶The density growth of/ml or so.Phenotypic examination finally is carried out with CIK cell of the flow cytometer FC500 to collection, is wrapped Include: CD3, CD56, CD4, CD8 detect these cell surface antigens in the expression of CIK cell.

Tumor inoculation and CIK are injected while being carried out, 5 × 10⁶NCI-N87 tumour cell and 5 × 10⁶It is infused after CIK cell mixing It penetrates in the right back side of female NOD/SCID mouse.Mouse is grouped to (8/group of mouse) at random in two hours, is infused by tail vein Administration is penetrated, dosage is respectively the M802 of 4,2 and 1mg/kg.Control group are as follows: (1) dosage is the Trastuzumab of 4mg/kg, and (2) are given Pharmaceutical quantities are the MCO101 of 4mg/kg.MCO101 is also MSBODY, and strand unit is completely the same with M802, and monovalent unit is variable Area is that 4420 (a kind of anti-fluorescein antibody is shown in Kranz DM, Voss EW Jr., Partial elucidation of an anti-hapten repertoire in BALB/c mice:comparative characterization of several monoclonal anti fluorescyl antibodies.MolImmunol.1981；18 (10): 889-898) it is variable Area.(3) negative control group only injects PBS.The administration same day is the 0th day.Continue to be administered within 2nd day and the 4th day, dosage is constant.Every 3 The volume of its tumour of measurement, calculating cubature formula is 1/2 × length × wide × wide (mm³)。

In Figure 14, tumour cell NCI-N87 and immunocyte CIK is with identical quantity co-injection in female NOD/ SCID mice is subcutaneous；It is administered after two hours, tail vein injection；It is administered once respectively again within 3 days and 5 days after administration, dosage is constant, often 3 staggering amount tumors are primary.Figure 14 shows that the M802 administration of various dose shows the good curative effect for inhibiting tumour growth, wherein 2 Hes All mouse (totally 16) tumour of 4mg/kg dosage treatment group was suppressed completely at 53 days even to disappear, and 1mg/kg The mouse Partial tumors of dosage treatment group also completely inhibit (3/8), remaining 5 mouse also only has lesser tumor mass to exist (<150mm³).In control group, Trastuzumab treatment group tumors are suppressed, and have a small amount of growth after 44 days；MCO101 treatment group tumors Do not inhibit, tumor mass volume reaches 300mm³.The tumour growth situation of feminine gender group is normal, and 53 days whens reach 800mm³Left and right.

It should be understood that the present invention disclosed is not limited only to specific method, scheme and the substance of description, because these It is alterable.It will also be understood that purpose of the terminology used here just for the sake of the specific embodiment scheme of description, rather than It is intended to limit the scope of the invention, the scope of the present invention is limited solely by the attached claims.

Those skilled in the art, which will also be appreciated that or be able to confirm that, uses no more than routine experiment, institute herein The many equivalents for the specific embodiment of the present invention stated.These equivalents are intended to comprising in the appended claims.

Claims (6)

1. bispecific antibody, which is characterized in that the antibody includes: (a) monovalent unit, is light-heavy chain pair, light chain is sequence Amino acid sequence shown in row number 3, heavy chain are amino acid sequence shown in sequence number 1, which is directed to tumour cell Surface antigen HER2 has specific binding capacity；(b) strand unit, is fusogenic peptide, which is shown in sequence number 5 Amino acid sequence, wherein the fusogenic peptide has specific binding capacity for immune cell surface antigenic CD3；

Strand unit includes single chain variable fragment ScFv and the Fc segment with hinge area, CH2 structural domain and CH3 structural domain, Middle CH2 structural domain does not include CH1 structural domain between hinge area and CH3 structural domain, and the hinge area of strand unit is located at ScFv Between CH2 structural domain；The single chain variable fragment is connected with heavy chain variable domain by link peptide by light chain variable region Composition；

Cysteine of the anti-HER2 heavy chain on 214 site of light chain of cysteine and anti-HER2 on 223 sites is with two sulphur The form of key connects, and the 255 of cysteine of the anti-HER2 heavy chain on 229 and 232 sites and anti-CD3 ScFv-Fc It is connected in the form of disulfide bond respectively with the cysteine on 258 sites, the anti-HER2 heavy chain is on 395 and 412 sites Connect with forming salt bridging on 428 and 397 sites of anti-CD3 ScFv-Fc, the anti-HER2 heavy chain on 369 sites with Knuckle-is formed on 436 sites of anti-CD3 ScFv-Fc enters-cave connection.

2. the method for preparing bispecific antibody described in claim 1, which is characterized in that the method comprising steps of

(1) weight of monovalent unit, light chain are building up to respectively on the first expression vector respectively, strand unit is building up to the second table Up on carrier；

(2) it by the first and second expression vectors together cotransfection into CHO-S cell, cultivates and takes supernatant；

(3) the isolated bispecific antibody after purification of supernatant will be expressed；The separating step includes: the affine layer of albumin A Analysis column captures the antibody of all band Fc structural domains from expression supernatant, realizes target bispecific by SP cation-exchange chromatography The separation of antibody and by-product, after Q column, finally concentration displacement buffer PBS.

3. according to the method described in claim 2, first expression vector is pCHO1.0；Second expression vector It is pCHO1.0- hygromycin.

4. according to the method described in claim 2, it is characterized in that, the step of the described method in (1): the unit price unit is Anti- HER2 antibody, expand its light chain the primer be Kozak EcoRV F, MK-leader EcoRV F and hIgK PacI R, It is expanded by over-lap PCR, Kozak sequence, MK-leader and restriction enzyme site EcoRV and PacI is introduced into light chain；Expand its heavy chain The primer is Kozak AvrII F, MK-leader AvrII F and hIgG1 BstZ17I R, is expanded by over-lap PCR, will Kozak sequence, MK-leader and restriction enzyme site AvrII and BstZl7I introduce heavy chain；By the light chain gene segment expanded with Homologous recombination is carried out with the pCHO1.0 expression vector of EcoRV and PacI digestion, obtains the expression load for being packed into anti-HER2 light chain Body；Then with homologous recombination is carried out with heavy chain again after AvrII and BstZl7I digestion, the pCHO1.0 expression for obtaining anti-HER2 is carried Body, plasmid are named as pCHO1.0-- Trastuzumab-HL-KKW；

The strand unit is anti-CD3 ScFv-Fc antibody, and expanding its primer is Kozak AvrIIF, MK-leader AvrII F, L2K-VH MK F1 and hIgG1 BstZ17I R expand AntiCD3 McAb ScFv-Fc structural domain by over-lap PCR, and will Kozak sequence, MK-leader and restriction enzyme site AvrII and BstZl7I introduce ScFv-Fc, by the genetic fragment expanded and enzyme The pCHO1.0- hygromycin expression vector cut through carries out homologous recombination, obtains the expression vector for being packed into AntiCD3 McAb ScFv-Fc, plasmid It is named as pCHO1.0- hygromycin-L2K-ScFv-Fc-LDY.

5. the purposes of bispecific antibody described in claim 1 in medicine preparation, the drug is special for treating HER2 Tumor disease caused by antigen presentation, or the cell for killing expression HER2.

6. the purposes of bispecific antibody described in claim 1 in medicine preparation, the drug is used in tumor cell line Expression HER2 specific antigen is treated in the drug of the tumour cell disease of middle screening treatment expression HER2 specific antigen or evaluation The drug effect of the drug of tumour cell disease.