CN104774268B - The structure of bispecific antibody EGFR × CD3 a kind of and application - 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 for the surface antigen CD3 of immunocyte there is specific binding capacity, the unit price unit to have specific binding capacity for tumor cell surface antigen EGFR；The strand unit includes the single chain variable fragment (ScFv) with Fc segment compositions, which includes light chain and heavy chain pair.The application also provides the preparation method of bispecific antibody and the pharmaceutical use of these antibody.

Description

The structure of bispecific antibody EGFR × CD3 a kind of and application

Technical field

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

Background technology

Bispecific antibody (bispecific antibody, BiAb) is containing two species-specific antigen binding sites Artificial antibody can erect bridge between target cell and functional molecular (cell), generate the effector function of guidance quality.BiAb is in life In object medicine, especially have broad application prospects in the immunization therapy of tumour.It is killed by BiAb mediated cell toxic actions Dead tumour cell is the hot spot of current immunization therapy application study, and being mainly characterized by BiAb can be in combination with tumor associated antigen With the target molecule on immune effector cell, specific killing of the immune effector cell to tumour cell is directly triggered.It is needle below Some background technology introductions to Immune Cell Antigens and tumor-cell antigen and the relevant technologies development studied.

1.CD3

CD3 molecules are 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 complexs 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 structures.CD3 cytoplasm sections containing it is immune by Body tyrosine activation motifs (immunoreceptor tyrosine-based activation motif, ITAM), TCR identifications 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 phosphorylations 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 molecules is The TCR that transduces identifies activation signals caused by antigen.

2.EGFR

EGF-R ELISA (EGFR, Epidermal Growth Factor Receptor) is epidermal growth factor (EGF) receptor of cell Proliferation and signal transduction.EGFR belongs to one kind of ErbB receptor family, which includes EGFR (ErbB- 1), HER2/c-neu (ErbB-2), Her 3 (ErbB-3) and Her 4 (ErbB-4).EGFR is also referred to as HER1, ErbB1, dashes forward Tumour can generally be caused by becoming or being overexpressed.EGFR is a kind of glycoprotein, belongs to tyrosine kinase receptor, and cell membrane perforation divides Son amount 170KDa.EGFR is located at cell membrane surface, by being activated with ligand binding, including EGF and TGFα (transforming growth factor α).After activation, EGFR is converted into dimer by monomer, although also evidence suggests, activation before there is also Dimer.EGFR is also possible to polymerize to activate with other members of ErbB receptor family, such as ErbB2/Her2/neu.

After ligand is combined with EGFR, dimerization has occurred in receptor, and dimerization had both included two cognate receptor molecules Combination (homology dimerization), also include mankind EGF related receptors (HER) family tyrosine kinase in different members Combination (heterologous dimerization).It is the autophosphorylation effect of tyrosine residue after dimerization.The residue of these phosphorylations It is the binding site for raising adaptor protein and additional tyrosine kinase substrate.Protein is mutual in the receptor complex of activation Effect stimulation ras albumen, leads to the activation of the generation and mitogen-activated protein (MAP) kinases of phosphorylation cascade reaction.Or Transcription signal conducts and activation, (the PI3K)-Akt of phosphatidyl inositol kinase -3 and stress activated protein kinase (SAPK) signal transduction Access will be activated.These signal paths trigger genetic transcription successively, while controlling the access quilt of hyperplasia, differentiation and existence Activation.

EGFR mediates the property and four kinds of EGFR family members of the specificity and intensity of signal path depending on activator protein Level.The ligand combined with HER2 is unknown, but when HER2 and EGFR is co-expressed, the former often ties with the latter of ligand activation Conjunction forms dimer.This heterologous dimer often has higher reuse ratio, steady compared with EGFR homology dimers Qualitative and conducted signal ability.Dimerization can also occur with HER3 and HER4 for EGFR, and product has higher persistence With stronger PI3K activity.Once the EGFR of EGFR signal transduction pathway ligand bindings swallows cell by interior, signal will terminate, by Body will be degraded or be recycled to cell membrane surface, this depends on the property of ligand.For example, the receptor that EGF is combined will be degraded, And the receptor that TGF- α are combined then enters recycling.Different growth factors can influence EGFR signal paths quantity and it is lasting when Between.

EGFR signal paths have multiple biological action.For example, point of ras-MAPK signal transduction pathways stimulation cell It splits and migrates.EGFR is also the important mediator of a variety of receptor pathway, plays the role of signal convergent point, can signal is whole and with Diversification.For example, stress, film depolymerisation and some non-physiologic stimulants (including oxidant, radioactive ray and alkylating agent) Reaction in, Reverse Activity can induce the phosphorylation of EGFR tyrosine kinase and then occur signal transduction.EGFR families Member has played important role in normal development, but the often overexpression and out of hand in human tumor.

Research shows that the high expression there are EGFR or unconventionality expression in many entity tumors.The increasing of EGFR and tumour cell Grow, angiogenesis, tumor invasion, transfer and the inhibition of Apoptosis it is related.Its mechanism has：Under the high expression of EGFR causes Swim the enhancing of signal transduction；The increase of mutant egf R receptors or ligand expression leads to the continuous activation of EGFR；Autocrine loop Effect enhancing；The destruction of receptor down-regulated mechanism；The activation etc. of abnormal signal conduction path.The overexpression of EGFR is in malignant tumour It plays an important role in evolution, has EGFR's in the tissues such as spongiocyte, kidney, lung cancer, prostate cancer, cancer of pancreas, breast cancer It is overexpressed.The high expression of EGFR is mainly related with its gene magnification to be found to the research of spongiocytoma.But EGFR is expressed sometimes After horizontal dysregulation exists in translation and translation.High expression of the EGFR in tumour is also possible to reduce with degradation after activation Related, some researchs point out that c-Src can raise EGFR levels by inhibiting receptor ubiquitination and endocytosis.In many tumours With the presence of mutant egf R, it has now been found that many kinds of EGFR saltant types.The effect of mutant egf R may include：It is non-with ligand Rely on the cell continuous activation of receptor；Lead to the destruction, different of receptor down-regulated mechanism due to certain structural domains missing of EGFR The activation of regular signal conduction path, inhibition of Apoptosis etc..The generation of mutant be due to EGFR gene missing, mutation and It resets.The ligand of EGFR has a significant impact to Cellular Signaling Transduction Mediated.The ligand of EGFR activates EGFR to promote by autocrine form Into cell Proliferation, their coexpression often indicates that tumor prognosis is bad, for example, being sent out in the research of infiltration ductal carcinomas of breast Existing, TGFα is co-expressed with EGFR, and this coexpression is significantly correlated with the survival rate of patient.Kopp et al. is to knot/carcinoma of the rectum Research shows that the autocrine growth of tumour is the overexpression and its coefficient result of ligand expression of EGFR.

In addition, finding that EGFR can pass through to the research of the angiogenesis of EGFR and tumour, high invasion and transfer relationship The adjusting of the factor levels such as Ang-1 and VEGF and influence Tumor Angiongesis.

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 biology prepared by the antibody engineering technology based on cell engineering and technique for gene engineering Macromolecular drug, have many advantages, such as specific high, property is uniform, specific target spot beam system can be directed to for.Monoclonal antibody is being faced Following three aspects are mainly used on bed：Oncotherapy, immunity disease treatment and anti-infective therapy.Wherein tumour is controlled Treatment is the field that current monoclonal antibody is most widely used, and is come at present in the monoclonal antibody product of clinical test and listing, for swelling The product quantity accounting of tumor treatment is about 50%.Mab treatment tumour is a kind of for sick cell specific target pricking method 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 guidance quality 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 It is to generate bispecific monoclonal antibody by way of cell hydridization method or three way cross tumor to hand over tumor method, these cell hydridizations Either 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 in mouse source 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, existing 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 allosome 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 cells, til cell, activation The immunization therapy of T lymphocytes and CIK cell.And immunotherapy can only remove a small amount of, scattered tumour cell, for late period Entity tumor curative effect it is limited.Therefore often combines with conventional methods such as operation, chemotherapy, radiotherapies as a kind of complementary therapy 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 coordinated 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 input is internal together therewith, and the other end of bispecific antibody can combine tumour cell well 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 are killed to tumour cell.Tumour cell can be effectively solved by this method Transfer and diffusion, overcome after operation, three great tradition therapeutic modality of chemicotherapy " be not thorough, easily transfer, side effect it is big " etc. disadvantages End.

Invention content

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 The initiative of recruit-bispecific antibody is mainly killed by CDC, ADCC and apoptosis capacity swollen in conventional monoclonal antibody On the basis of oncocyte, the immunotherapy of mediate T cell is increased, substantially increases the work(of immune system killing tumor cell Effect.

Specifically, the present invention provides technical solutions below：

In one embodiment, a kind of bispecific antibody is provided, which is characterized in that the described antibody includes：(a) single Valence unit is light-heavy chain pair, which has specific binding capacity for tumor cell surface antigen, preferably The ground tumor cell surface antigen is EGFR, CD20, CD30 and CD133, and the more preferably tumor cell surface antigen is EGFR； (b) strand unit, be fusogenic peptide, the fusogenic peptide include single chain variable fragment ScFv and with hinge area, CH2 structural domains and The Fc segments of CH3 structural domains, the immunocyte that wherein fusogenic peptide is directed to are selected from T cell, NKT cells or CIK cell；It is preferred that Ground, the fusogenic peptide have specific binding capacity to immune cell surface antigenic CD3.

In one embodiment, the CH2 structural domains of the strand unit of the bispecific antibody be located at ScFv segments and Between CH3 structural domains.

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 forms, they all target epitope CD3.

In one embodiment, in monovalent unit, light chain is combined by disulfide bond with heavy chain；Heavy chain by one or Multiple disulfide bond are combined with the fusogenic peptide.

In one embodiment, strand unit includes the anti-CD3 of antibody for people source CD3, and monovalent unit includes being directed to The antibody anti-EGFR of EGFR；Preferably, the amino acid sequence of the anti-EGFR heavy chain is amino acid sequence shown in sequence number 1 The amino acid sequence of row, the light chain of anti-EGFR is amino acid sequence and the anti-CD3ScFv-Fc shown in sequence number 3 Amino acid sequence is amino acid sequence shown in sequence number 9；And cysteine of the anti-EGFR heavy chain on 222 sites with it is anti- Cysteine on 215 site of light chain of EGFR is connected in the form of disulfide bond, and the anti-EGFR heavy chain is at 228 and 231 Cysteine on point is connected in the form of disulfide bond respectively with the cysteine on 255 and 258 sites of anti-CD3ScFv-Fc Connect, the anti-EGFR heavy chain on 394 and 411 sites with forming salt bridging on 428 and 397 sites of anti-CD3ScFv-Fc Connect, the anti-EGFR heavy chain on 368 sites with formed on 436 sites of anti-CD3ScFv-Fc knuckle-enter-cave connect；

Or the amino acid sequence of the anti-EGFR heavy chain is amino acid sequence shown in sequence number 5, the light chain of anti-EGFR Amino acid sequence be the amino acid sequence of amino acid sequence shown in sequence number 7 and the anti-CD3ScFv-Fc be sequence Amino acid sequence shown in numbers 9；And the light chain 214 of cysteine and anti-EGFR of the anti-EGFR heavy chain on 222 sites Cysteine on point is connected in the form of disulfide bond, cysteine of the anti-EGFR heavy chain on 228 and 231 sites It is connect in the form of disulfide bond respectively with the cysteine on 255 and 258 sites of anti-CD3ScFv-Fc, the anti-EGFR Heavy chain connects on 394 and 411 sites with forming salt bridging on 428 and 397 sites of anti-CD3ScFv-Fc, the anti-EGFR Heavy chain on 368 sites with formed on 436 sites of anti-CD3ScFv-Fc knuckle-enter-cave connect.

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

In one embodiment, Fc sections of the human IgG of the monovalent unit and the IgG Fc 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, the method includes：

(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 to cell, cultivates and takes supernatant；

(3) the isolated bispecific antibody after purification of supernatant will be expressed；Preferably, the cell is CHO-S cells； Or preferably, the separating step includes：Protein A affinity chromatography column captures the anti-of all band Fc structural domains from expression supernatant Body realizes the separation of target bispecific antibody and by-product by SP cation-exchange chromatographies, and after Q columns, finally concentration is set Change buffer solution PBS.

In one embodiment, the first expression vector is pCHO1.0；Second expression vector is pCHO1.0- hygromycin.

In one embodiment, the monovalent unit is anti-EGFR-antibodies, and it is Kozak to expand its light chain the primer (EcoR V) F, MK-Leader (EcoRV) F and hIgK (PacI) R, is expanded by over-lap PCR, by Kozak sequences, targeting sequencing And restriction enzyme site EcoR V and PacI introduces light chain；It is Kozak (Avr II) F, MK-Leader to expand its heavy chain the primer (AvrII) F and hIgG1 (BstZ17I) R, is expanded by over-lap PCR, by Kozak sequences, targeting sequencing and restriction enzyme site AvrII Heavy chain is introduced with BstZl7I；By the LC genetic fragments expanded with the pCHO1.0 expression vectors of EcoR V and PacI digestions Homologous recombination is carried out, the expression vector for being packed into anti-EGFR light chain is obtained；Then use after AvrII and BstZl7I digestions again with HC into Row homologous recombination, obtains the pCHO1.0 expression vectors of anti-EGFR, and the plasmid with Erbitux antibody genes is named as pCHO1.0- ERB-HL-KKW, or the plasmid with Vectibix antibody genes are named as pCHO1.0-VEC-HL-KKW；

The strand unit is anti-CD3ScFv-Fc antibody, and it is Kozak (Avr II) F, L2K-VH to expand its primer (MK) F1 and hIgG1 (BstZ17I) R, by PCR amplification AntiCD3 McAb ScFv-Fc structural domains, and by Kozak sequences, targeting sequencing And restriction enzyme site AvrII and BstZl7I introduces ScFv-Fc, the genetic fragment expanded and the pCHO1.0- tides of digestion is mould Plain expression vector carries out homologous recombination, obtains the expression vector for being packed into AntiCD3 McAb ScFv-Fc, it is mould that plasmid is named as pCHO1.0- tides Element-L2K-ScFv-Fc-LDY.

In one embodiment, any of the above-described bispecific antibody or the double spies prepared according to any of the above-described method The purposes of heterogenetic antibody in medicine preparation, the drug are used to treat the caused tumour or correlation of EGFR specific antigens expression Disease, or for killing expression EGFR cells.

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

The present invention provides a kind of new methods to prepare bispecific antibody MSBODY (monomer and ScFv Bispecific antibody) (as shown in Figure 2), which includes that two groups of heavy and light chains combine, and one of which is special Some transformations are carried out in conjunction with a kind of antigen, and in the areas its heavy chain Fc, make its versus wild type, itself is not easy and forms dimer； And another group of specific bond another kind antigen, other transformation equally is carried out in the areas its heavy chain Fc, itself is not easy to and forms two Aggressiveness, and it is readily formed heterozygosis dimer between this two groups of heavy and light chains.And the antibody structure of one of which is single aggressiveness Ab, Another group is ScFv-Fc, avoids the possibility of respective light chain and other side's heavy chain mispairing in this way, to form the double of 125KD Specific antibody protein molecular.After Fc transformations, the heavy chain of single aggressiveness Ab and single-stranded natural heterodimerization, while between CL and CH1 certainly Right dimerization, eventually forms MSBODY, and each domain arrangement sequences 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.Wherein it is with EGFR With the bispecific antibody that people source CD3 is target spot, it is named as EGFRX CD3, such as Fig. 2, anti-EGFR is here IgG forms, packet Anti-EGFR heavy chain and light chain are included, anti-CD3 is here ScFv-Fc forms, including anti-CD3VH, VL, Fc structural domain.It is above double special Heterogenetic antibody is built by antibody genetic engineering method, single aggressiveness Ab heavy chains of bispecific antibody MSBODY and single aggressiveness Ab light chains binary expression vector and ScFv-Fc expression vectors.It is more in Fc gene orders and carrier according to LC, HC, ScFv Cloning site design primer.Wherein LC, HC, ScFv and Fc carry out PCR amplification respectively, are obtained by PCR or Overlap extension PCR method Then genetic fragment is cloned by homologous recombination method.Digestion pCHO1.0 or pCHO1.0- hygromycin vector, then purifies The carrier after PCR product and digestion is recycled, for point two steps respectively by LC segments, HC segment homologous recombinations are cloned into pCHO1.0 carriers On, ScFv-Fc segment homologous recombinations are cloned on pCHO1.0- hygromycin vectors, and are sequenced.Recombinant protein MSBODY is being fed Expression in newborn zooblast, detection will express monovalent unit heavy chain, monovalent unit light chain and single-stranded respectively using transfection reagent In the plasmid co-transfection to mammalian cell of unit, regathers supernatant and carry out SDS-PAGE and Western blotting detection The expression of MSBODY.By the culture solution supernatant centrifugation after transfection expression, filtering is diluted, excessively affine layer with combination buffer Column, elution buffer elution are analysed, SDS-PAGE detects protein purification.

The beneficial technique effect of technical scheme of the present invention has：

1. this application provides a kind of heterodimeric antibodies, which includes two different antigen-binding polypeptides units. The corresponding homodimer molecular size range of the heterodimer is different, can using the size of molecular weight come distinguish heterodimer and 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 It includes single chain variable fragment (ScFv) to close polypeptide unit.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. the invention discloses the immunocyte killing pharmacy in vitro that a kind of novel bispecific antibodies MSBODY is mediated is real The foundation and its application of proved recipe method.The present invention includes that the immunocyte mediated in bispecific antibody drug research process kills Wound, the preparation of bispecific antibody and the foundation and detection of bispecific antibody pharmacy in vitro model.Bispecific antibody MSBODY includes one group of unit price unit (combination of heavy and light chain), and another group is then strand unit (ScFv connections Fc combinations), wherein singly A kind of tumor-cell antigen of people of valence unit specific bond, a series of tumour cell film surface antigens such as including EGFR, and The areas its heavy chain Fc carry out some transformations, make its versus wild type, are not easy itself and form dimer；And another group of strand unit is special In conjunction with the T cell antigen CD3 of another people, other transformation equally is carried out in the areas its heavy chain Fc, itself is not easy to and forms two Aggressiveness, and it is readily formed heterodimer between this two groups of units.At the same time, bispecific antibody can be in target cell and function Bridge is erected between molecule (cell), excites the immune response with guidance quality, is had in the immunization therapy of tumour wide Application prospect.

It is surprising that the application proves that this asymmetrical antibody is stable and is imitated 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, " the ScFv Antibody of Ahmad etc.:Principles and Clinical Application,” Clinical and Developmental Immunology,2012:980250 (2012), IgG class of the display based on ScFv are anti- Body is unstable, and needs further transformation to reduce aggregation and improve stability.

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

Description of the drawings

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 described in the application, right For those of ordinary skill in the art, without creative efforts, it can also be obtained according to these attached drawings Other attached drawings, wherein：

Fig. 1 .CD3 schematic arrangements.

Fig. 2 .EGFR X CD3 bi-specific antibody molecule schematic diagrames.

The non denatured SDS-PAGE electrophoresis of double antibody and purity detecting result figure of Fig. 3 purifying；M：Protein markers； 1：M1001；2：M1003.

The bis- double targeting capability result figures of antigen ELISA detection antibody of Fig. 4 .EGFR and CD3.

Combination situation map of the double antibody to HCT116 cells after Fig. 5 flow cytometer detection heat challenge experiment process.

Combination situation map of the double antibody to human PBMC's cell after Fig. 6 flow cytometer detection heat challenge experiment process.

Cell in vitro poison experimental result pictures of Fig. 7 .EGFR × CD3MSBODY and hPBMC to HCT116 cells.

Cell in vitro poison experimental result pictures of Fig. 8 .EGFR × CD3MSBODY and hPBMC to MDA-MB-453 cells.

Cell in vitro poison experimental result pictures of Fig. 9 .EGFR × CD3MSBODY and hPBMC to HEK293 cells.

Specific implementation mode

Embodiment 1：The expression vector establishment (EGFR × CD3, M1001, M1003) of bispecific antibody

1. bispecific antibody sequence design

It is named as EGFR × CD3MSBODY by the bispecific antibody of target spot of EGFR and CD3, wherein monovalent unit is The heavy chain light chain pair of anti-EGFR, sequence (the PDB database sequences of variable region amino acid sequence reference monoclonal antibody Erbitux Number 1YY8) and Vectibix sequence (source is the sequence number 38 and 49 in patent US6235883), including anti-EGFR heavy chains with Light chain contains Fab and Fc structural domains；Strand unit is the ScFv-Fc forms of AntiCD3 McAb, and variable region amino acid is anti-with reference to monoclonal The sequence (referring to US20070123479 sequence numbers 2) of body L2K, including AntiCD3 McAb VH, VL, Fc structural domain.Wherein monovalent unit The heavy chain Fc and Fc (with the heavy chain Fc of human IgG1) of strand unit carries out amino acid mutation transformation, specific Fc transformation process referring to PCT/CN2012/084982 makes it respectively be not easy to form homodimer (homodimer), and is easily formed heterodimer (heterodimer), which is bispecific antibody EGFR × CD3MSBODY, and monovalent unit derives from Erbitux EGFR × CD3MSBODY number be M1001, monovalent unit from Vectibix EGFR × CD3MSBODY number is M1003.It meanwhile in order to which EGFR × CD3MSBODY can be expressed in Chinese hamster ovary celI, and can be secreted into culture medium, select mouse source The leader peptide sequences of antibody kappa are as secreting signal peptide.The amino acid sequence and nucleic acid sequence of each structural domain and signal peptide Row are shown in following sequence number:1-12.Signal peptide is directly connected in the N-terminal of antibody variable region.

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

QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSI NKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGC LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPS DIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

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

caggtgcagctgaaacagagcggcccgggcctggtgcagccgagccagagcctgagcattacctgcaccgtgagcgg ctttagcctgaccaactatggcgtgcattgggtgcgccagagcccgggcaaaggcctggaatggctgggcgtgattt ggagcggcggcaacaccgattataacaccccgtttaccagccgcctgagcattaacaaagataacagcaaaagccag gtgttttttaaaatgaacagcctgcagagcaacgataccgcgatttattattgcgcgcgcgcgctgacctattatga ttatgaatttgcgtattggggccagggcaccctggtgaccgtgagcgcggcgtcgaccaagggcccatcggtcttcc ccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaa ccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcagg actctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatc acaagcccagcaacaccaaggtggacaagaaagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgc ccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccg gacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacg gcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctc accgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccat cgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtctacaccctgcccccatcccgggatgagc tgaccaagaaccaggtcagcctgtggtgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagc aatgggcagccggagaacaactacgataccacgcctcccgtgctggactccgacggctccttcttcctctacagcga tctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaacc actacacgcagaagagcctctccctgtctccgggtaaatga

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

DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGT DFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

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

gatattctgctgacccagagcccggtgattctgagcgtgagcccgggcgaacgcgtgagctttagctgccgcgcgag ccagagcattggcaccaacattcattggtatcagcagcgcaccaacggcagcccgcgcctgctgattaaatatgcga gcgaaagcattagcggcattccgagccgctttagcggcagcggcagcggcaccgattttaccctgagcattaacagc gtggaaagcgaagatattgcggattattattgccagcagaacaacaactggccgaccacctttggcgcgggcaccaa actggaactgaaacgccgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctg gaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgcc ctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccct gacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccg tcacaaagagcttcaacaggggagagtgttag

Monovalent unit heavy chain amino acid sequence (Vectibix, sequence number 5)

QVQLQESGPGLVKPSETLSLTCTVSGGSVSSGDYYWTWIRQSPGKGLEWIGHIYYSGNTNYNPSLKSRL TISIDTSKTQFSLKLSSVTAADTAIYYCVRDRVTGAFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGC LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPS DIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Monovalent unit heavy chain nucleic acid sequence (Vectibix, sequence number 6)

caggtgcagctgcaggagtcgggcccaggactggtgaagccttcggagaccctgtccctcacctgcactgtctctgg tggctccgtcagcagtggtgattactactggacctggatccggcagtccccagggaagggactggagtggattggac acatctattacagtgggaacaccaattataacccctccctcaagagtcgactcaccatatcaattgacacgtccaag actcagttctccctgaagctgagttctgtgaccgctgcggacacggccatttattactgtgtgcgagatcgagtgac tggtgcttttgatatctggggccaagggacaatggtcaccgtctcttcagcgtcgaccaagggcccatcggtcttcc ccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaa ccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcagg actctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatc acaagcccagcaacaccaaggtggacaagaaagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgc ccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccg gacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacg gcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctc accgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccat cgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtctacaccctgcccccatcccgggatgagc tgaccaagaaccaggtcagcctgtggtgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagc aatgggcagccggagaacaactacgataccacgcctcccgtgctggactccgacggctccttcttcctctacagcga tctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaacc actacacgcagaagagcctctccctgtctccgggtaaatga

Monovalent unit light-chain amino acid sequence (Vectibix, sequence number 7)

DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGT DFTFTISSLQPEDIATYFCQHFDHLPLAFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Monovalent unit light chain nucleic acid sequence (Vectibix, sequence number 8)

gacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttgccaggcgag tcaggacatcagcaactatttaaattggtatcagcagaaaccagggaaagcccctaaactcctgatctacgatgcat ccaatttggaaacaggggtcccatcaaggttcagtggaagtggatctgggacagattttactttcaccatcagcagc ctgcagcctgaagatattgcaacatatttctgtcaacactttgatcatctcccgctcgctttcggcggagggaccaa ggtggagatcaaacgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaa ctgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctc caatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgac gctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtca caaagagcttcaacaggggagagtgttag

Strand unit amino acid sequence (L2K, sequence number 9)

DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKAT LTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSGGGGSGGGGSGGGGSDIQLTQSPAIMS ASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQ WSSNPLTFGAGTKLELKGAAAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV YTLPPSRDELTKNQVSLTCRVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLASKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPGK

Strand unit nucleic acid sequence (L2K, sequence number 10)

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 11) of mouse kappa

METDTLLLWVLLLWVPGSTG

The leader peptide sequences nucleic acid sequence (sequence number 12) of mouse kappa

atggagacagacacactcctgctatgggtactgctgctctgggttccaggttccactggt

2. bispecific antibody gene cloning

PCHO1.0 is selected to go to clone and express the heavy chain and light chain gene of anti-EGFR, pCHO1.0- tides as expression vector Mycin expression vector is by replacing the puromycin genetic modification in pCHO1.0 carriers, quilt with hygromycin gene Selection is used for cloning and expressing the ScFv-Fc fusions of AntiCD3 McAb.After primer in table 1 is designed according to cloning approach, send It is synthesized to the Suzhou bio tech ltd Jin Weizhi.PCR amplification is carried out with the primer in table 1, template is by Jin Weizhi Gene chemical synthesis is simultaneously cloned into the gene plasmid on pUC57, includes the variable region of Erbitux and Vectibix, then respectively will be anti- EGFR weights, light chain are building up on the expression vector of pCHO1.0 respectively, and AntiCD3 McAb ScFv-Fc is building up to pCHO1.0- hygromycin On expression vector.

The primer used in 1. bispecific antibody gene cloning of table

Initial PCR amplification template DNA：M1001 and M1003 uses identical primer；Expanding its light chain the primer is Kozak (EcoR V) F, MK-Leader (EcoRV) F and hIgK (PacI) R, is expanded by over-lap PCR, by Kozak sequences, preceding It leads sequence and restriction enzyme site EcoR V and introduces light chain with PacI；It is Kozak (Avr II) F, MK- to expand its heavy chain the primer Leader (AvrII) F and hIgG1 (BstZ17I) R, is expanded by over-lap PCR, by Kozak sequences, targeting sequencing and digestion position Point AvrII and BstZl7I introduces heavy chain；The strand unit is anti-CD3ScFv-Fc antibody, and expanding its primer is Kozak (Avr II) F, L2K-VH (MK) F1 and hIgG1 (BstZ17I) R, by PCR amplification AntiCD3 McAb ScFv-Fc structural domains, and Kozak sequences, targeting sequencing and restriction enzyme site AvrII and BstZl7I are introduced into ScFv-Fc；The dosage of template DNA is 35ng/ Pipe, e.g., the light chain and heavy chain of target antibody；The 10 μM of forward primers and reverse primer of 1 μ l；The 10x PCR Buffer of 2.5 μ l Buffer solution；The 10mM dNTP of 1 μ l；2.5 units of 1 μ l/μ l Pyrobest archaeal dna polymerases (Takara, R005A)；And distillation Water is softly mixed to 25 μ l total volumes in microfuge pipes, and is quickly rotated in microcentrifuge to collect reaction mixing Object is to tube bottom.Use GeneAmp PCR System 9700 (Applied Biosystem) and progress PCR reactions arranged below： 95 DEG C, 5 minutes；25 cycles below：95 DEG C, every time 30 seconds；56 DEG C, 30 seconds；With 72 DEG C, 1 minute.

It is expanded, Kozak sequences, targeting sequencing and restriction enzyme site EcoR V and PacI is introduced light by a few wheel over-lap PCRs Chain；And Kozak sequences, targeting sequencing and restriction enzyme site AvrII and BstZl7I are introduced heavy chain by corresponding primer.To first it expand The LC genetic fragments increased carry out homologous recombination with EcoR V and the pCHO1.0 expression vectors of PacI digestions, are packed into The expression vector of anti-EGFR light chain；Then it with homologous recombination is carried out with HC again after AvrII and BstZl7I digestions, obtains anti- The pCHO1.0 expression vectors of EGFR, the plasmid with Erbitux antibody genes are named as pCHO1.0-ERB-HL-KKW, band The plasmid of Vectibix antibody genes is named as pCHO1.0-VEC-HL-KKW.

AntiCD3 McAb ScFv-Fc structural domains are expanded by over-lap PCR, and by Kozak sequences, targeting sequencing and restriction enzyme site AvrII and BstZl7I introduces ScFv-Fc, by the pCHO1.0- hygromycin expression vectors 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

Plasmid is carried out using the big extraction reagent kit of endotoxin-free (Qiagen, 12391) to carry greatly, concrete operations are provided according to manufacturer Specification carry out.The specification that CHO-S cell culture is provided according to manufacturer CD FortiCHO culture mediums (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) is damp by plasmid pCHO1.0-Erbitux-HL-KKW and pCHO1.0- using Maxcyte STX electroporations Mycin-L2K-ScFv-Fc-LDY in cotransfection to CHO-S cells, expresses the bispecific antibody of anti-EGFR × CD3 together M1001；Using Maxcyte STX electroporations by plasmid pCHO1.0-Vectibix-HL-KKW and pCHO1.0- hygromycin-L2K- Together in cotransfection to CHO-S cells, method is same as above ScFv-Fc-LDY, expresses the bispecific antibody of anti-EGFR × CD3 M1003.After culture 14 days, expression supernatant is harvested by centrifugation in 800Xg.

2. the purifying of bispecific antibody

Supernatant 0.22uM membrane filtrations are expressed, (GE companies, column goods are purchased from using Mabselect SuRe affinity columns 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) is eluted.By SP cation-exchange chromatographies, target bispecific antibody is realized With the separation of by-product, cation exchange column is purchased from GE companies (column article No. 18-1153-44, filler article No. 17-1087-01), uses Equilibration buffer A (43.8mM NaH₂PO₄+6.2mM Na₂HPO₄, pH 6.0) and after balance chromatographic column, sample is diluted with double pure water Conductance is to after between 3.0-3.5ms, crossing the combination of SP pillars, with elution buffer B (43.8mM NaH₂PO₄+6.2mM Na₂HPO₄+ 1M NaCl, pH 6.0) 20 column volume linear elutions；Finally concentration displacement Buffer PBS.Bispecific antibody after purification SDS-PAGE, SEC detection are carried out, two kinds of MSBODY purity are shown in Fig. 3 80% or so.

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

The bispecific antibody of the present invention is combined with the target antigen on corresponding cell.The present invention is with HT29 (purchased from Chinese allusion quotation Type culture collection) cell as the EGFR positives, cells of the Jurkat (ATCC, TIB-152) as the CD3 positives, and Dual anti-body measurement its cell-bound activity prepared with the present invention.

1. utilizing the combination activity of flow cytometer showed method detection bispecific antibody and HT29 cells

Enough HT29 cells are cultivated, with the digestion of 0.25% pancreatin, cell is collected by centrifugation.It is anti-that bispecific is diluted simultaneously Body, concentration is since 160nM, 4 times of gradient dilutions, obtains 6 concentration gradients, spare.By the cell PBS+1%FBS of collection It washes twice, then adds PBS+1%FBS that cell is resuspended to 4 × 10⁶A cell/ml, plating cells are in 96 orifice plates, per 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 carefully with PBS Twice of born of the same parents, then cell is resuspended with the anti-human igg FC antibody (Biolegend, 409304) of the PE labels diluted, room temperature is protected from light It is incubated 30 minutes, PBS is washed twice, then with 100ul PBS resuspensions, upper machine testing, then with average fluorescent strength, by with software GraphPad Prism5.0 carry out the binding affinity KD values that analysis calculates double antibody and HT29.As a result show that EGFR × CD3 is bis- Antibody and the HT29 cells of the EGFR positives have good combination active (being shown in Table 2).With EGFR positive cell HT29 combination situations： The KD values of M1001 are 0.65 ± 0.27nM, and the KD values of M1003 are 0.65 ± 0.18nM, the KD values of Erbitux are 0.12 ± 0.03nM.The affinity of two kinds of EGFR × CD3MSBODY is close, and is not much different with the affinity of monoclonal antibody Erbitux.

Affinity of 2 EGFR of the table × CD3MSBODY to HT29 cells

2. flow cytometer showed method detects the combination activity of 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 100ul PBS being resuspended, upper machine testing, with average fluorescent strength, by with software GraphPad Prism5.0 Carry out the binding affinity KD values that analysis calculates double antibody and Jurkat cell.As a result EGFR × CD3 double antibodies and CD3 sun are shown Property Jurkat cell there is preferable combine active (being shown in Table 3).With CD3 positive cell Jurkat combination situations：The KD of M1001 Value is 8.73 ± 5.29nM, and the KD values of M1003 are 14.36 ± 5.79nM, and affinity is below monoclonal antibody L2K, and (KD values are 0.42±0.12nM).Illustrate that the structure of strand unit can reduce the affinity of antibody.

Affinity of 3 EGFR of the table × CD3MSBODY to Jurkat cell

3. double targeting binding abilities of couple antigen ELISA detection S802

1) prepared by antigen：EGFR antigen design of primers refers to Genbank SeqNo.NM_005228.3, and primer sequence is shown in Table 2 EGFR-F and EGFR-R；MRNA is extracted from HT29 cells, and the reagent used is Trizol (Invitrogen) and reverse transcription At cDNA, the reverse transcription reagent box used isRT-PCR Kit (TaKaRa), primer EGFR-R, then use Primer EGFR-F and EGFR-R by the extracellular domain of the antigen amplify come, then use primer EGFR-F and Histag-R with PCR method adds histidine tag (7 × His), builds into expression vector pcDNA3.1/Hygro (+) (Invitrogen), Transfection expression method purifies the nickel column (1mL specifications prepacked column) using GE companies with the transfection expression (see embodiment 2) of M1001.

2) one of antigen used is that (CD3 antigens are self-control to HRP label CD3 antigens, refer to patent CN201310399169), the two of antigen are EGFR (see step 1).

3) envelope antigen：EGFR is diluted to 1 μ g/mL with coating buffer (pH=9.6,0.05mol/L sodium carbonate buffer) It is added in ELISA Plate hole, 100 μ l are per hole, and 4 DEG C overnight or 37 DEG C are incubated 2 hours.PBST (PBS+0.1%Tween20) board-washing Once, 100 holes μ L/；

4) it closes：Confining liquid is added, 100 holes μ L/, 37 DEG C are incubated 1 hour；PBST board-washings are primary, 100 holes μ L/；

5) standard curve：Different 100 holes μ L/ of antibody are separately added into, concentration is 20 μ g/mL, each antibody does 3 again Hole；37 DEG C are incubated 1 hour；PBST board-washings 3 times, 100 holes μ L/；Negative control is PBS；

6) enzyme-labelled antigen is added：HRP label CD3 antigens (PBS is diluted to 1 μ g/mL) are added, 100 holes μ L/, 37 DEG C incubate It educates 30 minutes；PBST board-washings 5 times, 100 holes μ L/；

7) it develops the color：Developing solution is added, 100 holes μ L/, 37 DEG C are protected from light colour developing 1-10 minutes；

8) it terminates：The reaction of 100 μ l terminate liquids (2M hydrochloric acid) color development stoppings is added per hole, each hole reaction is read in microplate reader The value of liquid OD450nm.

As shown in Figure 4, M1001 and M1003 can be in combination with two kinds of antigens of EGFR and CD3.

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 (ABI PCRsystem9700 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 pair Answer a temperature.After having handled, cooling sample is transferred in 96 orifice plate of V-type bottom (Corning), 4 DEG C, 2000rpm centrifugations 30min.Take supernatant for HCT116 cells (being purchased from China typical culture collection center) or human PBMC's cell binding assay.Carefully Born of the same parents are incubated 30min altogether at room temperature with supernatant, are washed twice with the 1%FBS-PBS being pre-chilled on ice, then are marked with 50 times of diluted PE Goat-anti people secondary antibody (Sigma, P9170) room temperature dye 30min.The 1%FBS-PBS of cell precooling after dyeing is washed 3 times, weight It is suspended from PBS and is analyzed with flow cytometer (FC500, Beckman)：100000 cell counts.With GraphPad Prism5 softwares S-shaped dose response (a sigmoidal dose response with variable slope) model with variable slope It is analyzed.The temperature midrange of thermomechanical curve is T₅₀。

Single chain antibody fragments (ScFv) connect heavy chain variable region and light chain variable region by a connection peptide (Gly4Ser) 3 It picks up and comes and formed.But have been reported that in ScFv unstability may influence the quality of antibody drug (Michaelson JS1,etc.,Farrington GK,Lugovskoy A,Joseph I,Bailly V,Wang X, Garber E,Browning J,Glaser SM.Anti-tumor activity of stability-engineered IgG-like bispecific antibodies targeting TRAIL-R2 and LTbetaR.MAbs.2009Mar- Apr；1(2):128-41).The strand unit of M1001 and M1003 are completely the same, the T that the two is combined with Jurkat₅₀Also it connects very much Closely (Fig. 5), the T of M1001₅₀=60.26 ± 0.24 DEG C, the T of M1003₅₀=59.71 ± 0.64 DEG C；The monovalent unit of M1001 makes It is the light chain and heavy chain of Erbitux, the monovalent unit of M1003 uses the light chain and heavy chain of Vectibix, with The T that HCT116 is combined₅₀The other very little (Fig. 6) of value difference, the T of M1001₅₀=59.66 ± 0.50 DEG C, the T of M1003₅₀=59.20 ± 0.70℃.The thermal stability difference of two kinds of MSBODY is little.

Embodiment 5：The cell in vitro killing detection that double antibody mediates

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

Fresh anti-freezing people blood, 400g is taken to centrifuge 5min, abandon 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 centrifuges 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 Precipitation is resuspended in the PBS of cell precipitation volume, and 4 DEG C, 400 × g is centrifuged 2-3 minutes, abandons supernatant.It can repeat 1 time, wash 1-2 altogether It is secondary.It needs to be resuspended after cell precipitation up to hPBMC with appropriate 4 DEG C precooling PBS according to experiment, can carry out the subsequent experimental such as counting.

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

(include the HCT116 colon cancer cells of EGFR high expression, the MDA-MB- of EGFR low expressions with pancreatin digestion target cell The HEK-293 human embryonic kidney cells of 453 breast cancer cells and EGFR feminine genders, are purchased from China typical culture collection center), it prepares Single cell suspension.Target cell is dyed with final concentration of 5 μM of CFSE, it will be thin with the 10%FBS-1640 of the cell culture after dyeing Born of the same parents are resuspended to 2 × 10⁵/ ml, according to 2 × 10⁴96 orifice plate overnight incubations are added in/hole, i.e. 100 holes μ l/.Experimental design is added 5 times Control wells are arranged, it is not necessary that the Kong Zeyong same volumes of PBMC cells are added in the effector cell (hPBMC) of target cell number, 50 holes μ l/ Culture medium fill into.Empirically corresponding antibodies, the holes 50ul/, it is not necessary that antibody is added is added in design while PBMC cells are added The culture medium of Kong Zeyong same volumes fills into.96 orifice plates are taken out after 48h, it is single cell suspension to digest each hole cell with pancreatin, this The correspondence of all supernatants and cell suspension in the process is collected into 1.5ml centrifuge tubes, centrifuges 500g × 5min.Supernatant is abandoned, respectively Hole is added 150ul 1%FBS-PBS and mixing cell is resuspended.PI (final concentration of 1 μ are added in each pipe 10-15min before machine in streaming G/ml) machine testing CFSE, PI double positive cells account for the death rate that CFSE positive cell ratios are target cell in streaming.

The tumour cell HCT116 fragmentation effects that M1001 and M1003 expresses EGFR high clearly, highest killing rate Up to 60% or more, and dosage is far below monoclonal antibody Erbitux and L2K (Fig. 7)；It is thin to the tumour of EGFR low expressions Born of the same parents MDA-MB-453 also has notable killing, and effect is significantly better than Erbitux and L2K (Fig. 8).But it is complete for EGFR Negative cell HEK293, M1001 and M1003 does not show fragmentation effect (Fig. 9).Illustrate EGFR × CD3MSBODY in body In outer cellulotoxic experiment, in the presence of immunocyte, the tumour cell different to EGFR positive expression amounts has to kill well Hinder effect, and the cell that do not expressed for EGFR does not have toxicity substantially.

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 State many equivalents of specific embodiment of the present invention.These equivalents are intended to comprising in the appended claims.

Claims (5)

1. bispecific antibody, which is characterized in that the described antibody includes：(a) monovalent unit, is light-heavy chain pair, this is light Chain-heavy chain has specific binding capacity to being directed to tumor cell surface antigen, which is EGFR；With (b) strand unit is fusogenic peptide, and the fusogenic peptide is comprising single chain variable fragment ScFv and has hinge area, CH2 structural domains and CH3 The Fc segments of structural domain, the wherein fusogenic peptide have specific binding capacity for immune cell surface antigenic CD3；

Wherein, strand unit includes the anti-CD3 of antibody for people source CD3, monovalent unit include for EGFR antibody it is anti- EGFR；And

The amino acid sequence of the anti-EGFR heavy chain is amino acid sequence shown in sequence number 1, the amino of the light chain of anti-EGFR Acid sequence is that the amino acid sequence of amino acid sequence shown in sequence number 3 and the anti-CD3ScFv-Fc are 9 institute of sequence number The amino acid sequence shown；And on 215 site of light chain of cysteine and anti-EGFR of the anti-EGFR heavy chain on 222 sites Cysteine is connected in the form of disulfide bond, cysteine of the anti-EGFR heavy chain on 228 and 231 sites with it is anti- The 255 of CD3ScFv-Fc are connected in the form of disulfide bond respectively with the cysteine on 258 sites, the anti-EGFR heavy chain It is connect with forming salt bridging on 428 and 397 sites of anti-CD3ScFv-Fc on 394 and 411 sites, the anti-EGFR heavy chain On 368 sites with formed on 436 sites of anti-CD3ScFv-Fc knuckle-enter-cave connect；

Or the amino acid sequence of the anti-EGFR heavy chain is amino acid sequence shown in sequence number 5, the ammonia of the light chain of anti-EGFR Base acid sequence is that the amino acid sequence of amino acid sequence shown in sequence number 7 and the anti-CD3ScFv-Fc are sequence number 9 Shown in amino acid sequence；And on 214 site of light chain of cysteine and anti-EGFR of the anti-EGFR heavy chain on 222 sites Cysteine connected in the form of disulfide bond, cysteine of the anti-EGFR heavy chain on 228 and 231 sites with it is anti- The 255 of CD3ScFv-Fc are connected in the form of disulfide bond respectively with the cysteine on 258 sites, the anti-EGFR heavy chain It is connect with forming salt bridging on 428 and 397 sites of anti-CD3ScFv-Fc on 394 and 411 sites, the anti-EGFR heavy chain On 368 sites with formed on 436 sites of anti-CD3ScFv-Fc knuckle-enter-cave connect.

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

(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, first expression vector is pCHO1.0；Second expression vector is pCHO1.0- hygromycin；

(2) in the first and second expression vectors together cotransfection to cell, will cultivate and take supernatant, the cell is CHO-S thin Born of the same parents；

(3) the isolated bispecific antibody after purification of supernatant will be expressed；The separating step includes：Protein A affinity chromatography Column captures the antibody of all band Fc structural domains from expression supernatant, realizes that target bispecific is anti-by SP cation-exchange chromatographies The separation of body and by-product, after Q columns, buffer solution PBS is replaced in finally concentration.

3. method according to claim 2, which is characterized in that the step of the method in (1)：

It is described unit price unit be anti-EGFR-antibodies, expand its light chain the primer be KozakF, MK-LeaderF and hIgKR, It is expanded by over-lap PCR, Kozak sequences, targeting sequencing and restriction enzyme site EcoR V and PacI is introduced into light chain；Expand its heavy chain The primer is KozakF, MK-LeaderF and hIgG1R, is expanded by over-lap PCR, by Kozak sequences, targeting sequencing and enzyme Enzyme site AvrI I and BstZl7I introduces heavy chain；By the LC genetic fragments expanded with EcoR V and PacI digestions PCHO1.0 expression vectors carry out homologous recombination, obtain the expression vector for being packed into anti-EGFR light chain；Then use AvrII with Homologous recombination is carried out with HC again after BstZl7I digestions, obtains the pCHO1.0 expression vectors of anti-EGFR, band Erbitux antibody bases The plasmid of cause is named as pCHO1.0-ERB-HL-KKW, or the plasmid with Vectibix antibody genes is named as pCHO1.0- VEC-HL-KKW；

The strand unit be anti-CD3ScFv-Fc antibody, expand its primer be KozakF, L2K-VH (MK) F1 and HIgG1R, by PCR amplification AntiCD3 McAb ScFv-Fc structural domains, and by Kozak sequences, targeting sequencing and restriction enzyme site AvrII with BstZl7I introduces ScFv-Fc, the pCHO1.0- hygromycin expression vectors of the genetic fragment expanded and digestion is carried out homologous Recombination, obtains the expression vector for being packed into AntiCD3 McAb ScFv-Fc, and plasmid is named as pCHO1.0- hygromycin-L2K-ScFv-Fc-LDY.

4. the purposes of bispecific antibody described in claim 1 in medicine preparation, the drug specifically resists for treating EGFR The caused tumor disease of original expression, or for killing expression EGFR cells.

5. the purposes of bispecific antibody described in claim 1 in medicine preparation, the drug is used in tumor cell line The drug of tumour cell disease of the screening for treating expression EGFR specific antigens or evaluation are special for treating expression EGFR The drug effect of the drug of the tumour cell disease of antigen.