CN104829728A - Construction and application of bispecific antibody HER2*CD3 - Google Patents

Abstract

The invention provides a bispecific antibody which is composed of a mono-valent unit and a single-chain unit, wherein the mono-valent unit, aiming to surface antigen CD3 of immune cells, has a specifically combining capability while the single-chain unit, aiming to surface antigen HER2 of tumor cells, has a specifically combining capability. The single-chain unit includes a single-chain variable fragment (ScFv) fused with an Fc fragment. The mono-valent unit includes a light chain-heavy chain pair. The application also provides a preparation of the bispecific antibody and medicinal applications of these antibodies.

Description

The construction and application of a kind of bi-specific antibody HER2XCD3

Technical field

The present invention relates to immunologic technical field.Specifically, structure and the preparation method of bi-specific antibody is related to.

Background technology

Bi-specific antibody (bispecific antibody, BiAb) is the artificial antibody containing two species-specific antigen binding sites, can erect bridge between target cell and functional molecular (cell), produces the effector function of guidance quality.BiAb, in biomedicine, particularly has broad application prospects in the immunotherapy of tumour.The focus that tumour cell is current immunotherapy applied research is killed by BiAb mediated cell toxic action, its principal feature is that BiAb can simultaneously in conjunction with the target molecule on tumor associated antigen and immune effector cell, and direct triggering immune effector cell is to the specific killing of tumour cell.Be below for studied Immune Cell Antigens and tumor-cell antigen, and some background technologies of correlation technique development are introduced.

1.CD3

CD3 molecule is made up of 4 subunits: δ, ε, γ, ζ, and its molecular mass is respectively 18.9kDa, 23.1kDa, 20.5kDa, 18.7kDa, and its length has 171,207,182,164 amino-acid residues respectively.They form 6 peptide chains together, and the normal TCR-CD3 complex body being formed and contain 8 peptide chains of combining closely with φt cell receptor (T cell receptor, TCR), structural representation is shown in Fig. 1.This complex body has T cell activation signal transduction, stablizes the function of TCR structure.CD3 kytoplasm section is containing immunoreceptor tyrosine-based activation motif (immunoreceptor tyrosine-basedactivation motif, ITAM), TCR identifies and combines the antigen peptide of being offered by MHC (major histo-compatibility complex) molecule, cause the tyrosine residues of the conserved sequence of the ITAM of CD3 by the tyrosine protein kinase p56lck phosphorylation in T cell, then can raise the tyrosine protein kinase (as ZAP-70) that other contain SH2 (Scr homology 2) structural domain.The phosphorylation of ITAM and be one of important biochemical reaction of T cell activation intracellular signaling process commitment with the combination of ZAP-70.Therefore, the function of CD3 molecule is that transduction TCR identifies the activation signals that antigen produces.

2.HER2

(the Shih C such as Shih in 1981, Padhy LC, Murray M, et al.Transforming genes ofcarcinomas and neuroblastomas introduced into mouse fibroblasts [J] .Nature, 1981,290 (5803): 261-264.) from rat neuroblastoma genome, oncogene neu is cloned first, (1987, the Science 2 such as Slamon; 35; 177-182) from human cDNA library, isolate HER2 gene.Sequential analysis subsequently and karyomit(e) spectrum analysis find that neu and HER2 is same gene, are called HER2/neu gene or c-erbB-2 gene traditionally.HER2 is the 2nd member of human epidermal growth factor acceptor family, this family belongs to I receptor Tyrosylprotein kinase, also known as ErbB receptor family, play important regulating and controlling effect in its growth at many normal and exception table chrotoplasts, differentiation and transfer process, the generation of many tumours, development and state of an illness weight size active with it are closely related.Four acceptors are had: HER1, HER2, HER3 and HER4 in family.These acceptors can interact and produce allos or homodimer, and many barss Signal Transduction Pathways in activating cells, wherein HER2 plays an important role in cell signalling process.The structure of HER2 comprises the land of born of the same parents' outgrowth factor, the cross-film district of lipophilic and the intracellular region with adjustment carboxy terminal fragment.HER2 acceptor intracellular region has tyrosine protein kinase PTK active, self also has some tyrosine residues Tyr phosphorylation sites.Can induce Dimerized after specificity growth factor and HER2 receptors bind and excite the cross phosphorylation of acceptor, the acceptor of phosphorylation can be transduceed rapidly in core extracellular growth signals, stimulates and controls the genetic expression relevant with cell fission.

HER2 is positioned human chromosome 17q21, and coding molecule amount is the transmembrane protein of 185kD, has Tyrosylprotein kinase RTK active, be in unactivated state under normal circumstances, participate in the adjustment of cell normal differentiation, usually only express in fetus period, after growing up, only trace expression in only a few healthy tissues.In normal cell, HER2 gene is 2 copies, transgenation can be activated, its amplification will cause transcriptional upregulation, albumen synthesis increases, thus inhibition tumor cell apoptosis, promote tumor cell proliferation, raise vascular endothelial growth factor VEGF/ vascular permeability factor VPF, promote tumor angiogenesis, increase invasive ability of tumor cell, destroy [the Artufel MV such as body tissue anti-invasion barrier, ValeroAC, Llado RR, etal.Molecular Protocol for Her-2/neu analysis in breastcarcinoma [J] .Clin Transl Oncol, 2005, 7. (11): 504-511.].The overexpression of HER2 albumen also plays a significant role [HynesNE in the transfer of the division of cell, propagation, conversion, promotion tumour, invasion and attack, adhesion, Stem DF.The biology of erbB-2/neu/HER-2and its role in cancer [J] .BiochemBiophys Acta, 1994,1198 (2-3): 165-184.].

Except can producer sudden change or amplification except, the expression of raising HER2 also can swash two key signal transduction approach in HER2 downstream: MAPK path, PI3K/Akt path, thus cause waterfall type chain reaction, regulate apoptosis-related genes, promote the differentiation of cell infinite multiplication, apoptosis inhibit, thus canceration occurs.The former mainly participates in the mitotic division of cell, the survival of the latter's major effect cell and apoptosis.HER2 is upper mediator's Telomere terminal transferase reverse transcriptase hTERT by MAPK pathway activation Ets transcription factor family member ER81; and then cause cell Telomere terminal transferase abnormal activation; make cell transformation and enter permanent vegetative state [GoueliBS; JanknechtR.Upregulation of thecatalytic telomerase subunit by the transcription factor ER81and oncogenicHER2/Neu; Ras; or Raf.Mol Cell Biol; 2004,24:25-35.].After PI3K activation, PIP2 and PIP3 can be generated by catalyze phospholipid acyl inositol PI, they are second messengers important in cell, the protein kinase A kt/PKB in downstream can be activated, cause the phosphorylation of downstream BAD albumen further, thus stop BAD and apoptotic proteins Bcl-2, Bcl-XL to form mixture, also induce jaw transcription factor 1 phosphorylation simultaneously, thus suppress the expression of former apoptogene.

In addition, HER2 oncogene is also metastases driving factors, HER2 process LAN increases Nasopharyngeal neoplasms ability by starting multiple transfer related mechanism, as cell migration rate, vitro invasion power, W Collagenase Type activity etc., the synthesis of some adhesion molecule as E-cadherin etc. can also be affected, thus promote transfer.(the CarterW such as Carter, Hoying J, Boswell C, et al.HER-2/neu over-expression inducesendothelial cell retraction [J] .Int Cancer, 2001,91 (3): 295-299.) study and think, HER2 process LAN can make endotheliocyte shrink, Dilated intercellular space, tumour cell is easy to pass through between endotheliocyte, and displacement or transfer occur tumour cell.Most research is thought, HER2 gene amplification and (or) protein overexpression often point out malignancy high, and transfer ability is strong.

The process LAN of HER2 is normal relevant with the generation of tumour, such as:

(1) cancer of the stomach: cancer of the stomach is one of modal malignant tumour of China, poor prognosis, advanced gastric carcinoma 5 years survival rates are only 5% ~ 20%, and median survival time is no more than 1 year.The ratio variation that HER2 albumen process LAN in cancer of the stomach detects in different research groups is 7% ~ 43%.The positive expression of HER2 albumen in cancer of the stomach is relevant with tumor differentiation degree, Lauren somatotype and WHO somatotype, uncorrelated with age, sex, tumour happening part and clinical stages.

(2) mammary cancer: research shows, HER2 has the amplification of gene and the overexpression of albumen in the primary breast infitrating ductal carcinoma of 20% ~ 30%.The high expression level of HER2 often causes the pernicious transfer of cell, and therefore the Breast Cancer Infiltration of the HER2 positive is strong, and the disease free survival phase is short, poor prognosis.Experiment in vitro shows, and suppresses the expression of HER2 can cause the apoptosis of tumour cell.

(3) ovarian cancer: ovarian cancer is the major cause that gynecological tumor is lethal.In ovarian cancer, the process LAN of HER2 is similar to mammary cancer, accounts for 15% ~ 30%.(the Verri E such as Verri, Guglielmini P, Puntoni M, et al.HER2/neu oncoprotein overexpression in epithelial ovarian cancer:evaluationof its prevalence and prognostic significance [J] .Oncology, 2005,68:154-161.) research display, the HER2 positive (2+/3+) patient significantly reduces (29 months vs48 month, P<0.05) than negative patient (0/1+) Overall survival.Ovarian cancer 20 gonad cell strains of III, IV phase of observation find all there is HER2 protein overexpression.

(4) prostate cancer: belong to androgen-dependent when prostate cancer occurs, Tumor regression after accepting medicine or operation castration, but finally can change androgen independence into and continued growth, this is topmost problem in current prostate cancer therapy.Research shows, HER2 is prostate cancer changes hormone independent process into main mediation person from hormone-dependent type.(the Signoretti S such as Signoretti, Montironi R, Manola J, et al.Her-2-neu expression and progression toward androgen independence in humanprostate cancer [J] .J Natl Cancer Inst, 2000, 92:1918-1925.) research and analyse the DNA of different clinical stage tumor sample HER2, RNA and protein expression level, result shows, the patient (UNTtumor) of the only excision prostate cancer of 25%, the operation consent of 59% accept anti-androgen therapy patient (TAAtumor) and 78% androgen in treating failure after and there is the HER2 of process LAN in the patient that Bone tumour (androgen independent AI) occurs.

(5) lung cancer: in lung cancer, the process LAN of HER2 is main relevant with genetic transcription and post transcriptional modificaiton.Studies in China shows, and HER2 process LAN mainly occurs in nonsmall-cell lung cancer, and mainly gland cancer, instead of squama cancer.But the detected result display of external 88 routine Hungary Patients with Non-small-cell Lungs, only having 5 examples to there is HER2 process LAN, and be squamous cell carcinoma, there is difference in result of study.In addition, different conclusions is also had to HER2 process LAN in lung cancer from the relation of cell differentiation.

Antibody drug for HER2 target spot: bent appropriate pearl commodity are called Trastuzumab Herceptin take HER2 as the Humanized monoclonal antibodies of target spot.The antigenic determinant of the anti-HER2 protein I gG of the stable region of nonspecific human IgG and mouse to be entrenched togather by gene engineering method to obtain by Herceptin, it not only has high affinity to HER2 acceptor, also solve the immunogenicity problem that murine antibody is applied to human body simultaneously, the generation of human antimouse antibody can be reduced, thus avoid being removed by reticuloendothelial system.Experiment in vivo and vitro research shows, the expression that application Herceptin lowers, and Growth of Cells can be made to slow down, and can significantly improve its susceptibility to chemicotherapy.U.S. FDA in 1998 ratifies this medicine for the metastatic breast cancer two wires of HER2 process LAN and the treatment of three lines, to be first be also a unique Humanized monoclonal antibodies medicine being approved for treatment HER2/neu protein expression positive metastatic mammary cancer and breast carcinoma of early stage.

3. bi-specific antibody technical development

Bi-specific antibody, two antigen-binding sites in an antibody molecule can respectively in conjunction with the antibody of two kinds of different epitopes.

Antibody drug is the biopharmaceutical macromolecular drug prepared based on the antibody engineering technology of cell engineering and genetic engineering technique, has that specificity is high, character is homogeneous, can for advantages such as specific target spot directional preparations.Monoclonal antibody is mainly used in following three aspects clinically: oncotherapy, immunological disease treatment and anti-infective therapy.Wherein the treatment of tumour is the field that current monoclonal antibody is most widely used, and has entered in the monoclonal antibody product of clinical trial and listing at present, and the product amount accounting for oncotherapy is probably 50%.Mab treatment tumour be a kind of for sick cell specific target point stimulation immunity system to kill and wound the immunotherapy of target cell, in order to strengthen the effector function of antibody, the particularly effect of killing tumor cell, people attempt multiple method engineered antibody molecule, bi-specific antibody is one of developing direction improving Antybody therapy effect, now becomes the focus of antibody engineering research field.

Bi-specific antibody for immunotherapy is the artificial antibody containing 2 species-specific antigen binding sites, bridge can be erected between target cell and functional molecular (cell), excite the immune response with guidance quality, have broad application prospects in the immunotherapy of tumour.

4. bi-specific antibody preparation

Bi-specific antibody obtains by number of ways, and its preparation method mainly contains: chemical coupling method, hybridization-hybridoma and genetic engineering antibody preparation method.Chemical coupling method is linked together at the mode of 2 different monoclonal antibody chemical couplings, prepared bispecific monoclonal antibody, and this is bispecific monoclonal antibody concept the earliest.Hybridization-hybridoma produces bispecific monoclonal antibody by the mode of cell hybridization method or three way cross knurl, these quadromas or three way cross knurl are the hybridoma fusion by building up, or set up hybridoma and obtain from the lymphocyte cell that mouse obtains, can only produce the bi-specific antibody in mouse source, its application is greatly limited.And developing rapidly along with Protocols in Molecular Biology, there is the multiple forming types of genetically engineered humanization bi-specific antibody, and be mainly divided into dual specific miniantibody, double-chain antibody, strand bivalent antibody, multivalence bi-specific antibody four class.At present, existing Several gene engineering bispecific antibody drug enters clinical experimental stage in the world, and shows good application prospect.

5. the adoptive immunotherapy of tumour

The adoptive immunotherapy of tumour is inputted after amplification in vitro in patient body by immunologically competent cell that is autologous or allosome, direct killing tumour cell, the immunologic function of adjustment and enhancing body, mainly comprises LAK cell, til cell, the T lymphocyte of activation and the immunotherapy of CIK cell.And immunotherapy can only remove a small amount of, scattered tumour cell, the entity tumor curative effect for late period is limited.Therefore often it can be used as the ordinary method combined utilization such as a kind of adjuvant therapy and operation, chemotherapy, radiotherapy.After first cleaning a large amount of tumour cells by ordinary method, then remove remaining tumour cell by immunotherapy, the effect of combined therapy of tumour can be improved.Wherein, adoptive immunotherapy, as the novel method of in combined therapy of tumour, is treated with routine operation, radiotherapy, chemotherapy and other cells and molecular therapy is extensively coordinated, illustrate application prospect widely in the treatment of kinds of tumors.But one more preferably mode should be, bi-specific antibody one end in conjunction with the surface antigen CD3 of cultured immunocyte, and can input in body thereupon together, and the other end of bi-specific antibody can well in conjunction with the surface antigen of tumour cell; Like this, bi-specific antibody just can erect the bridge between tumour cell and immunocyte in vivo, makes immunocyte concentrate near tumor cells, and then kills and wounds tumour cell.Effectively can solve the metastasis and extension of tumour cell by this method, overcome drawbacks such as " not thoroughly, easily transfers, side effect large " after operation, chemicotherapy three great tradition therapeutic modality.

Summary of the invention

Term and shortenings

BiAb: bi-specific antibody (bispecific antibody)

TA: tumour antigen (tumor antigen)

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

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

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

CDR: the abbreviation being English Complementarity determining regions (CDRs), refers to the antigen complementary determining region of antibody.

ScFv: Single chain antibody fragment (single-chain variable fragment), is also called single-chain antibody.

CLD: clone exploitation (cell line development)

FACS: fluorescence-activated cell sorting (Fluorescence-activated cell sorting), also referred to as Flow cytometry.

The present invention is directed to the weak point of conventional monoclonal antibody, the initiative of the recruit-bi-specific antibody undertaken by the method for genetically engineered and antibody engineering, at conventional monoclonal antibody mainly through CDC, ADCC and apoptosis capacity are come on the basis of killing tumor cell, add the immunotherapy of mediate T cell, substantially increase effect of immunity system killing tumor cell.

Particularly, the invention provides following technical scheme:

In one embodiment, provide a kind of bi-specific antibody, it is characterized in that, this antibody described comprises: (a) unit price unit, is light-heavy chain pair, and this light-heavy chain is to being selected from T cell, NKT cell or CIK cell for immunocyte; Preferably, this light-heavy chain has specific binding capacity to immune cell surface antigenic CD3; (b) strand unit, for fusogenic peptide, this fusogenic peptide comprises single chain variable fragment Scfv and has the Fc fragment of hinge area, CH2 structural domain and CH3 structural domain, wherein this fusogenic peptide has specific binding capacity for TCSA, preferably this TCSA is HER2, CD20, CD30 and CD133, and more preferably this TCSA is HER2.

In one embodiment, the CH2 structural domain of the strand unit of described bi-specific antibody is between ScFv fragment and CH3 structural domain.

In one embodiment, the single chain variable fragment of bi-specific antibody is made up of variable region of light chain and heavy chain variable domain, they all target in epitope HER2.

In one embodiment, in unit price unit, light chain is combined with heavy chain by disulfide linkage; Heavy chain is combined with described fusogenic peptide by one or more disulfide linkage.

In one embodiment, strand unit comprises the anti-HER2 of antibody for HER2, and unit price unit comprises the anti-CD3 of antibody for CD3; Preferably, the aminoacid sequence of described anti-CD3 heavy chain is the aminoacid sequence shown in sequence number 1, the aminoacid sequence of the light chain of anti-CD3 is the aminoacid sequence shown in sequence number 3, and the aminoacid sequence of described anti-HER2Scfv-Fc is the aminoacid sequence shown in sequence number 5; And the halfcystine of anti-CD3 heavy chain on 222 sites is connected with the form of disulfide linkage with the halfcystine on light chain 212 site of anti-CD3, described anti-CD3 heavy chain is connected with the form of disulfide linkage with the halfcystine on 258 and 261 sites of anti-HER2Scfv-Fc with the halfcystine on 231 sites respectively 228, described anti-CD3 heavy chain on 370 with 401 sites with 441 and 424 sites of anti-HER2Scfv-Fc form salt bridge be connected, described anti-CD3 heavy chain on 409 sites with 398 sites of anti-HER2Scfv-Fc are formed knuckle-enter-cave is connected.

In one embodiment, the heavy chain in unit price unit comprises people or humanized Fc fragment, and preferably, the Fc fragment of this heavy chain comprises human IgG1 Fc fragment; The Fc fragment of described fusogenic peptide comprises people or humanized Fc fragment, and preferably, the Fc fragment of this fusogenic peptide comprises human IgG1 Fc fragment.

In one embodiment, the human IgG1 Fc section of described unit price unit and the IgG1Fc of described strand unit are by salt bridge and knuckle-enter-cave anatomical connectivity.

In one embodiment, provide a kind of preparation method of bi-specific antibody, described method comprises:

(1) respectively heavy, the light chain of unit price unit are building up on the first expression vector, by strand cell formation on the second expression vector respectively;

(2) by the first and second expression vectors together cotransfection in cell, cultivate and get supernatant;

(3) bi-specific antibody after obtaining purifying is separated by expressing supernatant; Preferably, described cell is CHO-S cell; Or preferably, described separating step comprises: the antibody of all band Fc structural domains caught by protein A affinity chromatography post from expressing supernatant, by being separated of SP cation-exchange chromatography realize target bi-specific antibody and by product, after Q post, finally concentrated displacement damping fluid PBS.

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

In one embodiment, described unit price unit is anti-CD 3 antibodies, its light chain the primer that increases is Kozak (EcoR V) F, MK-Leader (EcoRV) F and hIgK (PacI) R, increased by over-lap PCR, Kozak sequence, leader sequence and restriction enzyme site EcoR V and PacI are introduced light chain; Its heavy chain the primer that increases is Kozak (Avr II) F, MK-Leader (AvrII) F, MK-Leader (AvrII) F and hIgG11 (BstZ17I) R, increased by over-lap PCR, Kozak sequence, leader sequence and restriction enzyme site AvrII and BstZl7I are introduced heavy chain; By the LC gene fragment increased with carry out homologous recombination with the pCHO1.0 expression vector that EcoR V and PacI enzyme cut through, the expression vector of acquisition loading AntiCD3 McAb light chain; Then carry out homologous recombination with HC again after cutting with AvrII and BstZl7I enzyme, obtain the pCHO1.0 expression vector of AntiCD3 McAb, plasmid called after pCHO1.0-L2K-HL-KKW;

Described strand unit is anti-HER2ScFv-Fc antibody, its the primer that increases is Kozak (Avr II) F, MK-Leader (AvrII) F, L2K-VH (MK) F1 and hIgG11 (BstZ17I) R, by the anti-HER2ScFv-Fc structural domain of pcr amplification, and by Kozak sequence, leader sequence and restriction enzyme site AvrII and BstZl7I introduce ScFv-Fc, the pCHO1.0-Totomycin expression vector gene fragment increased and enzyme cut through carries out homologous recombination, obtain the expression vector loading anti-HER2Scfv-Fc, plasmid called after pCHO1.0-Totomycin-Trastuzumab-ScFv-Fc-LDY.

In one embodiment, above-mentioned arbitrary bi-specific antibody or preparing the purposes in medicine according to bi-specific antibody prepared by above-mentioned either method, described medicine be used for the treatment of HER2 specific antigen express caused by tumour or relative disease, or express HER2 cell for killing.

In one embodiment, above-mentioned arbitrary bi-specific antibody or preparing the purposes in medicine according to bi-specific antibody prepared by above-mentioned either method, described medicine is used for screening in human tumor cell line and is used for the treatment of the drug effect that the medicine of the tumour cell relative disease of expressing HER2 specific antigen or evaluation be used for the treatment of the medicine of the tumour cell relative disease of expressing HER2 specific antigen.Present invention also offers following technical scheme:

The invention provides a kind of novel method and prepare bi-specific antibody SMBODY (ScFv and monomerbispecific antibody) (as shown in Figure 2), this bi-specific antibody comprises two groups of unit, wherein one group of unit is weight chain pair, be called unit price unit, a kind of antigen of specific combination, and carry out some transformations in its heavy chain Fc district, make its versus wild type, not easily self forms dimer; And another group unit is strand-heavy chain Fc district fusogenic peptide, be called strand unit, the another kind of antigen of specific combination, carry out other transformation equally in its heavy chain Fc district, also not easily self forms dimer, and is easy between these two groups of unit form heterozygosis dimer.And wherein the antibody structure of a group is unit price unit, another group is strand (ScFv-Fc), doing so avoids the possibility of respective light chain and the mispairing of the other side's heavy chain, thus forms the bi-specific antibody protein molecular of 125KD.After Fc transformation, the heavy chain of unit price unit and strand nature different dimerization, natural dimerization between CL and CH1, finally forms SMBODY simultaneously.The each domain arrangement order of SMBODY and structural representation are shown in Fig. 2.

Utilize the above method preparing bi-specific antibody in the present invention, prepare bi-specific antibody.Be wherein the bi-specific antibody that is target spot with HER2 and people source CD3, be named as HER2X CD3, as Fig. 2, anti-CD3 is here unit price unit form, comprise anti-CD3 heavy chain and light chain, anti-HER2 is here Scfv-Fc form, comprises anti-HER2VH, connection peptides, VL, Fc structural domain.Above bi-specific antibody is built by antibody genetic engineering method, the unit price unit heavy chain of bi-specific antibody SMBODY and unit price unit light chain double promoter expression vector, and ScFv-Fc expression vector.According to the multiple clone site design primer in LC, HC, ScFv, Fc gene order and carrier.Wherein unit price unit light chain (LC), unit price unit heavy chain (HC), ScFv and Fc carries out pcr amplification respectively, obtains gene fragment, then cloned by homologous recombination method by PCR or Overlap extension PCR method.Enzyme cuts pCHO1.0 or pCHO1.0-hygromycin vector, then purifying reclaim PCR primer and enzyme cut after carrier, point two steps are respectively by LC fragment, and HC fragment homologous recombination is cloned on pCHO1.0 carrier, ScFv-Fc fragment homologous recombination is cloned on pCHO1.0-hygromycin vector, and checks order.The expression of recombinant protein SMBODY in mammalian cell, detection, use transfection reagent by the plasmid of expressing unit price unit heavy chain and unit price unit light chain and express strand unit plasmid co-transfection in mammalian cell, regather supernatant and carry out the expression that SDS-PAGE and western blotting detect SMBODY.By centrifugal for the nutrient solution supernatant after transfection expression, filter, with the dilution of binding buffer liquid, cross affinity column, elution buffer wash-out, SDS-PAGE detects protein purification.

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

1. this application provides a kind of heterodimeric antibodies, this antibody comprises two different antigen-binding polypeptides unit.This heterodimer is different from its corresponding homodimer molecular size range, the size of molecular weight can be utilized to distinguish heterodimer and homodimer, thus more conveniently determine the purity of bi-specific antibody.One of these two antigen-binding polypeptides unit comprise the light-heavy chain pair being similar to wild-type antibodies, and in whole the application, this unit is also referred to as " unit price unit ".Another antigen-binding polypeptides unit comprises single chain variable fragment (ScFv).Such ScFv can merge the constant fragment (Fc) to antibody.In the application's full text, this fusogenic peptide is also referred to as " strand unit ".

2. the invention discloses foundation and the application thereof of a kind of outer effect experiment method of immunocyte contact element that novel bispecific antibodies SMBODY mediates.The present invention includes that the immunocyte mediated in bispecific antibody drug research process kills and wounds, the preparation of bi-specific antibody, and the foundation of bi-specific antibody pharmacy in vitro model and detection.Bi-specific antibody SMBODY comprises one group of strand unit (ScFv connects Fc combination), another group is then unit price unit (heavy light chain combination), the wherein tumor-cell antigen of a kind of people of strand unit specific combination, comprise a series of tumour cell film surface antigens such as HER2, and some transformations are carried out in its heavy chain Fc district, make its versus wild type, not easily self forms dimer; And the T cell antigen CD3 of another another kind of people of group unit price unit specific combination, carry out other transformation equally in its heavy chain Fc district, also not easily self form dimer, and be easy between these two groups of unit form heterodimer.Meanwhile, bi-specific antibody can erect bridge between target cell and functional molecular (cell), excite the immune response with guidance quality, in the presence of immunocyte, bi-specific antibody of the present invention has extremely strong fragmentation effect to tumour cell, has broad application prospects in the immunotherapy of tumour.

Surprisingly, the application proves that this asymmetrical antibody is stable and has high antigen joint efficiency.This makes us feeling surprised, even because the homodimer of verified single-chain antibody is in physiological conditions all unstable.Such as, Ahmad etc. " ScFv Antibody:Principles and ClinicalApplication; " Clinical and Developmental Immunology, 2012:980250 (2012), the IgG1 antibody-like shown based on ScFv is unstable, and needs transformation further assemble to reduce and improve stability.

In addition, because have asymmetry, heterodimer has and the homodimer difference iso-electric point be made up of wherein arbitrary antigen-binding polypeptides unit.Based on the iso-electric point difference between heterodimer and homodimer, easily the heterodimer of needs can be separated with homodimer, greatly reduce the difficulty that the ubiquitous downstream process exploitation of bi-specific antibody exists.

Accompanying drawing explanation

In order to be illustrated more clearly in the technical scheme in the embodiment of the present application, be briefly described to the accompanying drawing used required in embodiment below, apparently, the accompanying drawing that the following describes is only some embodiments recorded in the application, to those skilled in the art, under the prerequisite not paying creative work, other accompanying drawing can also be obtained according to these accompanying drawings, wherein:

Fig. 1 .CD3 schematic arrangement.

Fig. 2 .HER2X CD3 bi-specific antibody molecule schematic diagram.

Fig. 3. the double antibody electrophoresis of purifying and purity detecting result figure; (A) non denatured SDS-PAGE electrophoresis, M: protein markers; Swimming lane 1:S801; Swimming lane 2:S802; (B) the HPLC-SEC purity peak shape figure of the S802 of purifying.

Fig. 4. the HER2 × CD3 double antibody measured based on flow cytometric analysis and the avidity result figure of SK-BR-3 cell.

Fig. 5. the HER2 × CD3 double antibody measured based on flow cytometric analysis and the avidity result figure of Jurkat cell.

Fig. 6. the ELISA of dual anti-former HER2 and CD3 detects the binding activities result figure of S802.

Fig. 7. flow cytometer detection heat challenge experiment process after double antibody to SK-BR-3 cell in conjunction with situation map.

Fig. 8. flow cytometer detection heat challenge experiment process after double antibody to human PBMC's cell in conjunction with situation map.

Fig. 9 .S802 and hPBMC is to the cell in vitro poison experimental result picture of BT-474 cell.

Figure 10 .S802 and hPBMC is to the cell in vitro poison experimental result picture of MCF-7 cell.

Figure 11 .S802 and hPBMC is to the cell in vitro poison experimental result picture of HEK293 cell.

Embodiment

Embodiment 1: the expression vector establishment (HER2 × CD3, S802) of bi-specific antibody

1. bi-specific antibody sequences Design

HER2 × CD3SMBODY is named as with the bi-specific antibody that HER2 and CD3 is target spot, wherein unit price unit is the heavy chain light chain pair of AntiCD3 McAb, variable region amino acid is with reference to the sequence of monoclonal antibody L2K, with reference to US20070123479 sequence 2, comprise AntiCD3 McAb heavy chain and light chain, containing Fab and Fc structural domain; Strand unit is the ScFv-Fc form of anti-HER2, and the sequence (PDB database No.1N8Z) of variable region amino acid sequence reference monoclonal antibody Herceptin, comprises VH, VL, Fc structural domain of Trastuzumab monoclonal antibody.Wherein the heavy chain Fc of unit price unit and the Fc (the heavy chain Fc with human IgG1 1) of strand unit all carries out amino acid mutation transformation, concrete Fc transformation process is see PCT/CN2012/084982, it is made not easily to form homodimer (homodimer) separately, and be easy to be formed heterodimer (heterodimer), this heterodimer is bi-specific antibody HER2 × CD3SMBODY, is numbered S802., in order to S802 can express in Chinese hamster ovary celI, and can be secreted in substratum meanwhile, have selected the leader peptide sequences of mouse source antibody kappa as secreting signal peptide.The aminoacid sequence of each structural domain and signal peptide and nucleotide sequence are shown in following sequence number: 1-8.Signal peptide is directly connected in the N end of antibody variable region.In patent PCT/CN2012/084982, the SMBODY of a kind of anti-Her2XCD3, unit price unit variable region is from Trastuzumab, and strand unit variable region is from humanization OKT3, and this SMBODY is numbered S801 in the present invention, as a comparison antibody.

L2K unit price unit heavy chain amino acid sequence (sequence number 1)

DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCRVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLASKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK-

L2K unit price unit heavy chain nucleic acid sequence (sequence number 2)

GACATCAAACTGCAGCAGTCAGGGGCTGAACTGGCAAGACCTGGGGCCTCAGTGAAGATGTCCTGCAAGACTTCTGGCTACACCTTTACTAGGTACACGATGCACTGGGTAAAACAGAGGCCTGGACAGGGTCTGGAATGGATTGGATACATTAATCCTAGCCGTGGTTATACTAATTACAATCAGAAGTTCAAGGACAAGGCCACATTGACTACAGACAAATCCTCCAGCACAGCCTACATGCAACTGAGCAGCCTGACATCTGAGGACTCTGCAGTCTATTACTGTGCAAGATATTATGATGATCATTACTGCCTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCGGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGAAGTCCGACGGCTCCTTCTTCCTCGCCAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA

L2K unit price unit light-chain amino acid sequence (sequence number 3)

DIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

L2K unit price unit light chain nucleic acid sequence (sequence number 4)

GACATTCAGCTGACCCAGTCTCCAGCAATCATGTCTGCATCTCCAGGGGAGAAGGTCACCATGACCTGCAGAGCCAGTTCAAGTGTAAGTTACATGAACTGGTACCAGCAGAAGTCAGGCACCTCCCCCAAAAGATGGATTTATGACACATCCAAAGTGGCTTCTGGAGTCCCTTATCGCTTCAGTGGCAGTGGGTCTGGGACCTCATACTCTCTCACAATCAGCAGCATGGAGGCTGAAGATGCTGCCACTTATTACTGCCAACAGTGGAGTAGTAACCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG

Trastuzumab strand unit aminoacid sequence (sequence number 5)

EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRGAAAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK-

Trastuzumab strand unit nucleotide sequence (sequence number 6)

GAAGTGCAGCTGGTGGAAAGCGGCGGCGGCCTGGTGCAGCCGGGCGGATCCCTGCGCCTGAGCTGCGCGGCGAGCGGCTTTAACATTAAAGATACCTATATTCATTGGGTGCGCCAGGCGCCGGGCAAAGGCCTGGAATGGGTGGCGCGCATTTATCCGACCAACGGCTATACCCGCTATGCGGATAGCGTGAAAGGCCGCTTTACCATTAGCGCGGATACCAGCAAAAACACCGCGTATCTGCAGATGAACAGCCTGCGCGCGGAAGATACCGCGGTGTATTATTGCAGCCGCTGGGGCGGCGATGGCTTTTATGCGATGGATTATTGGGGCCAGGGCACCCTGGTGACCGTGAGCTCAGGAGGCGGCGGTTCAGGCGGAGGTGGAAGTGGTGGAGGAGGTTCTGATATTCAGATGACCCAGAGCCCGTCAAGCTTAAGCGCGAGCGTGGGCGATCGCGTGACCATTACCTGCCGCGCGAGCCAGGATGTGAACACCGCGGTGGCGTGGTATCAGCAGAAACCGGGCAAAGCGCCGAAACTGCTGATTTATAGCGCGAGCTTTCTGTATAGCGGCGTGCCGAGCCGCTTTAGCGGCAGCCGCAGCGGCACCGATTTTACCCTGACCATTAGCAGCCTGCAGCCGGAAGATTTTGCGACCTATTATTGCCAGCAGCATTATACCACCCCGCCGACCTTTGGCCAGGGTACCAAAGTGGAAATTAAACGAGGTGCGGCCGCAGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACGATACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCGATCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA

The leader peptide sequences aminoacid sequence (sequence number 7) of mouse kappa

METDTLLLWVLLLWVPGSTG

The leader peptide sequences nucleotide sequence (sequence number 8) of mouse kappa

atggagacagacacactcctgctatgggtactgctgctctgggttccaggttccactggt

2. bi-specific antibody gene clone

Select pCHO 1.0Expression Vector (is called for short pCHO1.0, purchased from the test kit of Life technologies kit, article No. A13696-01) heavy chain and the light chain gene of cloning and expressing unit price unit is removed as expression vector, pCHO1.0-Totomycin expression vector is the tetracycline genetic modification by replacing by hygromycin gene in pCHO1.0 carrier, is selected to cloning and expressing strand unit.After primer in table 1 designs according to cloning approach, be sent to Jin Weizhi bio tech ltd, Suzhou and synthesize.Pcr amplification is carried out with the primer in table 1, template is gene chemical synthesis or the gene plasmid that is subcloned on pCDNA3.1 or pUC57 in earlier trials, PCT/CN2012/084982 patent has a detailed description, then different promoters downstream on the expression vector respectively weight of unit price unit, light chain cdna being building up to respectively pCHO1.0, is building up to strand unit cDNA on the expression vector of pCHO1.0-Totomycin.

The primer used in the gene clone of table 1 bi-specific antibody

The template DNA of initial PCR amplification template DNA: 35ng, e.g., the light chain of target antibody and heavy chain; 10 μMs of forward primers of 1 μ l and reverse primer; The 10x PCR Buffer damping fluid of 2.5 μ l; The 10mM dNTP of 1 μ l; 2.5 units/μ l Pyrobest archaeal dna polymerase (Takara, R005A) of 1 μ l; Softly mix in 200 μ L PCR pipe to 25 μ l cumulative volumes with distilled water, and in Eppendorf centrifuge fast rotational to collect at the bottom of reaction mixture to pipe.GeneAmp PCR System 9700 (Applied Biosystem) and following setting is used to carry out PCR reaction: 95 DEG C, 5 minutes; 25 following circulations: 95 DEG C, each 30 seconds; 56 DEG C, 30 seconds; With 72 DEG C, 1 minute.

Take turns overlapping pcr amplification by several, Kozak sequence, leader sequence and restriction enzyme site EcoR V and PacI are introduced light chain; And Kozak sequence, leader sequence and restriction enzyme site AvrII and BstZl7I are introduced heavy chain by corresponding primer.First by the LC gene fragment increased with carry out homologous recombination with the pCHO1.0 expression vector that EcoR V and PacI enzyme cut through, the expression vector of acquisition loading AntiCD3 McAb light chain; Then carry out homologous recombination with HC again after cutting with AvrII and BstZl7I enzyme, obtain the pCHO1.0 expression vector of AntiCD3 McAb, plasmid called after pCHO1.0-L2K-HL-KKW.

To be increased anti-HER2ScFv-Fc structural domain by over-lap PCR, and Kozak sequence, leader sequence and restriction enzyme site AvrII and BstZl7I are introduced ScFv-Fc, the pCHO1.0-Totomycin expression vector gene fragment increased and enzyme cut through carries out homologous recombination, obtain the expression vector loading anti-HER2 Scfv-Fc, plasmid called after pCHO1.0-Totomycin-Trastuzumab-ScFv-Fc-LDY.

Embodiment 2: bi-specific antibody expression and purification

1. the expression of bi-specific antibody S802

Utilization is carried out plasmid without the large extraction reagent kit of intracellular toxin (Qiagen, 12391) and is carried greatly, and the specification sheets that concrete operations provide according to manufacturer carries out.The specification sheets that CHO-S cell cultures provides according to manufacturer, in CD FortiCHO substratum (Invitrogen, article No. A11483-1), is placed in 37 DEG C, 5%CO ₂cultivate in cell culture incubator, after getting out cell, according to the specification sheets (Maxcyte) of manufacturers, use Maxcyte STX electroporation that plasmid pCHO1.0-L2K-HL-KKW cotransfection together with pCHO1.0-Totomycin-Trastuzumab-ScFv-Fc-LDY, in CHO-S cell, is expressed the bi-specific antibody S802 of anti-HER2 × CD3.Cultivate after 14 days, 800 × g harvested by centrifugation expresses supernatant.The expression experimental procedure of S801 is identical with S802.

2. the purifying of bi-specific antibody S802

Express supernatant 0.22 μM of membrane filtration, utilize Mabselect SuRe affinity column (purchased from GE company, post article No. 18-1153-45, filler article No. 17-5438-01) from expressing supernatant the antibody of catching all band Fc structural domains, with level pad (9.5mM NaH ₂pO ₄+ 40.5mM Na ₂hPO ₄, pH7.0) balance chromatography column after, cross affinity column, with elution buffer (50mM citric acid+100mM arginine, pH3.2) wash-out.By SP cation-exchange chromatography, realize target bi-specific antibody is separated with by product, cationic exchange coloum purchased from GE company (post article No. 18-1153-44, filler article No. 17-1087-01), with level pad A (43.8mM NaH ₂pO ₄+ 6.2mM Na ₂hPO ₄, pH 6.0) and after balance chromatography column, sample, with between two pure water dilution conductance to 3.0-3.5ms, is crossed after SP pillar combines, with elution buffer B (43.8mMNaH ₂pO ₄+ 6.2mM Na ₂hPO ₄+ 1M NaCl, pH 6.0) 20 column volume linear elutions; Finally concentrated displacement phosphate buffered saline buffer (PBS).The purification experiment step of S801 is identical with S802.Bi-specific antibody after purifying carries out SDS-PAGE, SEC and detects, and purity is (see Fig. 3) more than 95%.

Embodiment 3: the binding activities of bi-specific antibody and cell measures (FACS)

The target antigen of bi-specific antibody of the present invention on corresponding cell is combined.The present invention is using SK-BR-3 (purchased from China typical culture collection center) as the cell of the HER2 positive, Jurkat (American Type Culture collection warehousing (ATCC), TIB-152) as the cell of the CD3 positive, and its cell-bound activity is measured with double antibody prepared by the present invention.

1. utilize flow cytometer showed method to detect the binding activities of bi-specific antibody and SK-BR-3 cell

Cultivate enough SK-BR-3 cells, with 0.25% trysinization, centrifugal collecting cell.Dilute bi-specific antibody, concentration is from 160nmol, and 4 times of gradient dilutions, obtain 6 concentration gradients, for subsequent use simultaneously.The cell PBS+1%FBS of collection is washed twice, then adds PBS+1%FBS re-suspended cell to 4 × 10 ⁶individual cell/ml, plating cells in 96 orifice plates, every hole 50ul (2 × 10 ⁵individual cell), add 50ul PBS to be diluted to the antibody of different concns (Trastuzumab is humanization OKT3 prepared by laboratory purchased from Genentech, HOKT3, and sequence is shown in PCT/CN2012/084982 sequence number 2, the same S802 of expression and purification method), incubated at room 1 hour; Centrifugally remove supernatant, cell is washed twice with PBS, again with the anti-human igg 1 FC antibody (Biolegend of the PE mark diluted, 409304) re-suspended cell, room temperature lucifuge hatches 30 minutes, and PBS washes twice, use 100ul PBS resuspended again, upper machine testing, then with average fluorescent strength, by carrying out the binding affinity KD value of analytical calculation double antibody and SK-BR-3 with software GraphPad Prism5.0.The SK-BR-3 cell of result display S802 double antibody and the HER2 positive has good binding activities, is better than S801, sees Fig. 4.With HER2 positive cell SK-BR-3 in conjunction with situation: the KD value of S801 is the KD value of 9.592nM, S802 is 6.365nM, and the KD value of Trastuzumab is 2.283nM.

2. flow cytometer showed method detects the binding activities of bi-specific antibody and Jurkat cell

Cultivate enough Jurkat suspension cells, centrifugal collecting cell.Ensuing experimentation is same as the previously described embodiments, and by cell resuspended for 100ul PBS, upper machine testing, with average fluorescent strength, by carrying out the binding affinity KD value of analytical calculation double antibody and Jurkat cell with software GraphPad Prism5.0.The Jurkat cell of result display HER2 × CD3 double antibody and the CD3 positive has good binding activities, sees Fig. 5.With CD3 positive cell Jurkat in conjunction with situation: the KD value of the KD value of S801 to be the KD value of 0.5965nM, S802 be 0.2374nM, L2K is 0.1860nM.

3. pair antigen ELISA detects two target binding abilities of S802

1) antigen preparation: HER2 antigen design of primers is with reference to Genbank SeqNo.M11730.1, and primer sequence is in Her2-F and Her2-R of table 1; MRNA extracts from SK-BR-3 cell, and the reagent of use is for Trizol (Invitrogen) and reverse transcription becomes cDNA, and the Reverse Transcription box of use is rT-PCR Kit (TaKaRa), primer is Her2-R, with primer Her2-F and Her2-R, the ectodomain of this antigen is increased out again, then primer Her2-F and Histag-R is used to add histidine-tagged (7XHis) with PCR method, build up in expression vector pcDNA3.1/Hygro (+) (Invitrogen), transfection expression method is with the transfection expression (see embodiment 2) of S802, and purifying adopts the nickel post (1mL specification prepacked column) of GE company.

2) one of antigen used is HRP mark CD3 antigen (CD3 antigen is self-control, refers to patent CN201310399169), and two of antigen is HER2 (see step 1).

3) envelope antigen: HER2 is diluted to 1 μ g/mL adds in enzyme plate hole with coating buffer (pH=9.6,0.05mol/L sodium carbonate buffer), the 100 every holes of μ l, 4 DEG C spend the night or 37 DEG C hatch 2 hours.PBST (PBS+0.1%Tween20) washes plate once, 100 μ L/ holes;

4) close: add confining liquid, 100 μ L/ holes, hatch 1 hour for 37 DEG C; PBST washes plate once, 100 μ L/ holes;

5) typical curve: add different antibody 100 μ L/ holes respectively, concentration is 20 μ g/mL, often kind of antibody does 3 multiple holes; Hatch 1 hour for 37 DEG C; PBST washes plate 3 times, 100 μ L/ holes; Negative control is PBS;

6) enzyme-labelled antigen is added: add HRP again and mark CD3 antigen (PBS is diluted to 1 μ g/mL), 100 μ L/ holes, hatch 30 minutes for 37 DEG C; PBST washes plate 5 times, 100 μ L/ holes;

7) develop the color: add nitrite ion, 100 μ L/ holes, 37 DEG C of lucifuge colour developing 1-10 minute;

8) stop: every hole adds 100 μ l stop buffer (2M hydrochloric acid) color development stopping reactions, and microplate reader reads the value of each hole reaction solution OD450nm.

As shown in Figure 6, S802 can simultaneously in conjunction with HER2 and CD3 two kinds of antigens.

Embodiment 4: the thermal stability determination of bi-specific antibody

1. the hot challenge experiment of bi-specific antibody

Antibody PBS is diluted to 0.5mg/mL, is dispensed in PCR pipe with the specification of 50 μ L/ pipes, at the upper thermal treatment 60min of PCR instrument (ABI PCRsystem9700).PCR instrument is set temperature gradient from left to right, from 37 DEG C to 82 DEG C, and the corresponding temperature of each sample.After processing, the sample of cooling is transferred in 96 orifice plates (Corning) at the bottom of V-type, 4 DEG C, the centrifugal 30min of 2000rpm.Get supernatant for SK-BR-3 cell or human PBMC's cell binding assay.Cell and supernatant at room temperature hatch 30min altogether, wash twice with the 1%FBS-PBS of precooling on ice, then resist (Sigma, P9170) room temperature dyeing 30min with the goat-anti people two of the PE mark of 50 times of dilutions.The 1%FBS-PBS of the cell precooling after dyeing washes 3 times, is resuspended in PBS and analyzes with flow cytometer (FC500, Beckman): 100,000 cell countings.S shape dose response (a sigmoidaldose response with variable slope) model with GraphPad Prism5 software with variable slope is analyzed.The temperature mid point value of thermomechanical curve is T ₅₀.

Single chain antibody fragments (ScFv) is by a connection peptides (Gly ₄ser) ₃variable region of heavy chain and variable region of light chain are coupled together and is formed.But there is the unstable of report ScFv inherence may affect the quality (MichaelsonJS1 of antibody drug, Demarest SJ, Miller B, Amatucci A, Snyder WB, Wu X, Huang F, Phan S, GaoS, Doern A, Farrington GK, Lugovskoy A, Joseph I, Bailly V, Wang X, Garber E, Browning J, Glaser SM.Anti-tumor activity of stability-engineered IgG1-likebispecific antibodies targeting TRAIL-R2and LTbetaR.MAbs.2009Mar-Apr; 1 (2): 128-41.The strand unit of S802 and S801 completely the same, the T50 that both are combined with SK-BR-3 is (Fig. 7) also closely, the T50=59.96 DEG C of the T50=60.21 DEG C of S802, S801; But, the unit price unit of S802 uses the variable region of L2K, the unit price unit of S801 uses the variable region of humanization OKT3, the T50 value difference be combined with T cell not comparatively large (Fig. 8), the T50=62.89 DEG C of S802, the T50=59.33 DEG C of S801, S802 thermostability is better than S801.

Embodiment 5: the cell in vitro of double antibody mediation kills and wounds detection

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

Get fresh anti-freezing human blood, the centrifugal 5min of 400g, abandons supernatant.Add the erythrocyte cracked liquid of 10 times of cell volumes, blow and beat mixing gently, room temperature or on ice cracking 4-5 minute.Should suitably shake to promote erythrocyte splitting in cracking process.4 DEG C of centrifugal 5min of 400 × g, abandon red supernatant.If erythrocyte splitting is incomplete, repeating step 2 and 3 once.Wash 1-2 time.Add the PBS of 5 times of cell precipitation volumes, resuspended precipitation, 4 DEG C of centrifugal 2-3 minute of 400 × g, abandon supernatant.1 time can be repeated again, wash 1-2 time altogether.Experimentally need namely to obtain hPBMC with after suitable 4 DEG C of precooling PBS re-suspended cells precipitation, can carry out the subsequent experimental such as counting.

2. double antibody effectively mediates the detection of PBMC cell killing HER2 positive tumor cell

(the BT-474 breast cancer cell of HER2 high expression level is comprised with trysinization target cell, the MCF-7 breast cancer cell of the low expression of HER2 and the HEK293 HEKC of HER2 feminine gender, all purchased from China typical culture collection center), prepare single cell suspension.With final concentration be 5 μMs CFSE dye target cell, after dyeing with the 10%FBS-1640 of this cell cultures by resuspended for cell to 2 × 10 ⁵/ ml, according to 2 × 10 ⁴/ hole, namely 100 μ l/ holes add 96 orifice plate overnight incubation.Experimental design adds 5 times of effector cells to target cell number (hPBMC), and 50 μ l/ holes, arrange control wells, and the substratum without the need to the Kong Zeyong same volume adding PBMC cell fills into.Empirically design while adding PBMC cell and add corresponding antibodies, 50ul/ hole, the substratum without the need to the Kong Zeyong same volume adding antibody fills into.Taking out 96 orifice plates after 48h, is single cell suspension with each porocyte of trysinization, and all supernatants in this process and the equal correspondence of cell suspension are collected in 1.5ml centrifuge tube, the centrifugal 5min of 500 × g.Abandon supernatant, each hole adds the resuspended mixing cell of 150ul 1%FBS-PBS.Each pipe in streaming before machine 10-15min to add in PI (final concentration is 1 μ g/ml) streaming the two positive cell of machine testing CFSE, PI and account for the mortality ratio that CFSE positive cell ratio is target cell.

To the tumour cell BT-474 fragmentation effect of HER2 high expression level clearly, most High Fragmentation rate reaches 80% to S802, and using dosage is lower than Trastuzumab, HOKT3 and S801 (Fig. 9); Also have the tumour cell MCF-7 of the low expression of HER2 and significantly kill and wound, and effect is better than S801 and HOKT3 (Figure 10) greatly.But for the cell HEK293 that HER2 is completely negative, S802 does not show fragmentation effect (Figure 11).S802 double antibody is described in vitro in cellulotoxic experiment, in the presence of immunocyte, the tumour cell different to HER2 positive expression amount all has good fragmentation effect and is better than S801, and does not substantially have toxicity for the cell that HER2 does not express.

Should be understood that the present invention of disclosure is not limited only to specific method, scheme and the material described, because these equal alterable.Will also be understood that terminology used here is only used to describe the object of specific embodiment scheme, instead of be intended to limit the scope of the invention, scope of the present invention is only limited to appended claim.

Those skilled in the art also will recognize, or can confirm that use is no more than normal experiment, in this article many Equivalents of described specific embodiment of the present invention.These Equivalents are intended to comprise in the appended claims.

Claims (12)

1. bi-specific antibody, is characterized in that, this antibody described comprises: (a) unit price unit, is light-heavy chain pair, and this light-heavy chain is to being selected from T cell, NKT cell or CIK cell for immunocyte; Preferably, this light-heavy chain has specific binding capacity to immune cell surface antigenic CD3; (b) strand unit, for fusogenic peptide, this fusogenic peptide comprises single chain variable fragment Scfv and has the Fc fragment of hinge area, CH2 structural domain and CH3 structural domain, wherein this fusogenic peptide has specific binding capacity for TCSA, preferably this TCSA is HER2, CD20, CD30 and CD133, and more preferably this TCSA is HER2.

2. bi-specific antibody according to claim 1, is characterized in that: the CH2 structural domain of strand unit is between hinge area and CH3 structural domain; Do not comprise CH1 structural domain; The hinge area of strand unit is between ScFv and CH2 structural domain.

3. bi-specific antibody according to claim 1, is characterized in that: described single chain variable fragment is made up of variable region of light chain and heavy chain variable domain, they all target in epitope HER2.

4. bi-specific antibody according to claim 1, is characterized in that: in described unit price unit, light chain is combined with heavy chain by disulfide linkage; Described heavy chain is combined with described fusogenic peptide by one or more disulfide linkage.

5. bi-specific antibody described in claim 1, is characterized in that: strand unit comprises the anti-HER2 of antibody for HER2, and unit price unit comprises the anti-CD3 of antibody for CD3;

Preferably, the aminoacid sequence of described anti-CD3 heavy chain is the aminoacid sequence shown in sequence number 1, the light-chain amino acid sequence of anti-CD3 is the aminoacid sequence shown in sequence number 3, and the aminoacid sequence of described anti-HER2Scfv-Fc is the aminoacid sequence shown in sequence number 5; And the halfcystine of anti-CD3 heavy chain on 222 sites is connected with the form of disulfide linkage with the halfcystine on light chain 212 site of anti-CD3, described anti-CD3 heavy chain is connected with the form of disulfide linkage with the halfcystine on 258 and 261 sites of anti-HER2Scfv-Fc with the halfcystine on 231 sites respectively 228, described anti-CD3 heavy chain on 370 with 401 sites with 441 and 424 sites of anti-HER2Scfv-Fc form salt bridge be connected, described anti-CD3 heavy chain on 409 sites with 398 sites of anti-HER2Scfv-Fc are formed knuckle-enter-cave is connected.

6. bi-specific antibody according to claim 1, it is characterized in that: the heavy chain in described unit price unit comprises people or humanized Fc fragment, preferably, the Fc fragment of this heavy chain comprises human IgG1 Fc fragment; The Fc fragment of described fusogenic peptide comprises people or humanized Fc fragment, and preferably, the Fc fragment of this fusogenic peptide comprises human IgG1 Fc fragment.

7. bi-specific antibody according to claim 6, is characterized in that: the human IgG1 Fc section of described unit price unit and the IgG1Fc of described strand unit are by salt bridge and knuckle-enter-cave anatomical connectivity.

8. prepare the method for bi-specific antibody according to any one of claim 1-7, it is characterized in that, described method comprises step:

(1) respectively heavy, the light chain of unit price unit are building up on the first expression vector, by strand cell formation on the second expression vector respectively;

(2) by the first and second expression vectors together cotransfection in cell, cultivate and get supernatant;

(3) bi-specific antibody after obtaining purifying is separated by expressing supernatant; Preferably, described cell is CHO-S cell; Or preferably, described separating step comprises: the antibody of all band Fc structural domains caught by protein A affinity chromatography post from expressing supernatant, by being separated of SP cation-exchange chromatography realize target bi-specific antibody and by product, after Q post, finally concentrated displacement damping fluid PBS.

9. method according to claim 8, described first expression vector is pCHO1.0; Described second expression vector is pCHO1.0-Totomycin.

10. method according to claim 8, is characterized in that, in the step (1) of described method:

Described unit price unit is anti-CD 3 antibodies, described unit price unit is anti-CD 3 antibodies, its light chain the primer that increases is Kozak (EcoR V) F, MK-Leader (EcoRV) F and hIgK (PacI) R, increased by over-lap PCR, Kozak sequence, leader sequence and restriction enzyme site EcoR V and PacI are introduced light chain; Its heavy chain the primer that increases is Kozak (Avr II) F, MK-Leader (AvrII) F and hIgG11 (BstZ17I) R, increased by over-lap PCR, Kozak sequence, leader sequence and restriction enzyme site AvrII and BstZl7I are introduced heavy chain; By the LC gene fragment increased with carry out homologous recombination with the pCHO1.0 expression vector that EcoR V and PacI enzyme cut through, the expression vector of acquisition loading AntiCD3 McAb light chain; Then carry out homologous recombination with HC again after cutting with AvrII and BstZl7I enzyme, obtain the pCHO1.0 expression vector of AntiCD3 McAb, plasmid called after pCHO1.0-L2K-HL-KKW;

Described strand unit is anti-HER2ScFv-Fc antibody, its the primer that increases is Kozak (Avr II) F, MK-Leader (AvrII) F, L2K-VH (MK) F1 and hIgG11 (BstZ17I) R, by the anti-HER2ScFv-Fc structural domain of pcr amplification, and by Kozak sequence, leader sequence and restriction enzyme site AvrII and BstZl7I introduce ScFv-Fc, the pCHO1.0-Totomycin expression vector gene fragment increased and enzyme cut through carries out homologous recombination, obtain the expression vector loading anti-HER2Scfv-Fc, plasmid called after pCHO1.0-Totomycin-Trastuzumab-ScFv-Fc-LDY.

Bi-specific antibody prepared by bi-specific antibody according to any one of 11. claim 1-7 or the method according to the bi-specific antibody prepared any one of claim 8-10 is preparing the purposes in medicine, described medicine be used for the treatment of HER2 specific antigen express caused by tumour or relative disease, or express HER2 cell for killing.

Bi-specific antibody prepared by bi-specific antibody according to any one of 12. claim 1-7 or the method according to the bi-specific antibody prepared any one of claim 8-10 is preparing the purposes in medicine, and described medicine is used for the treatment of the drug effect of the medicine of the tumour cell relative disease of expressing HER2 specific antigen for the medicine or evaluation screening the tumour cell relative disease being used for the treatment of expression HER2 specific antigen in tumor cell line.