The construction and application of a kind of bi-specific antibody HER2XCD3Technical 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 (bispecificantibody, 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, Boswel l 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 epithel ial 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, recruit is carried out by the method for genetically engineered and antibody engineering---the initiative of bi-specific antibody, 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, a kind of bi-specific antibody is provided, it is characterized in that, this antibody described comprises: (a) unit price unit, and be light-heavy chain pair, this light-heavy chain has specific binding capacity to for TCSA, preferably this TCSA is HER2, CD20, CD30 and CD133, and more preferably this TCSA is HER2; (b) strand unit is fusogenic peptide, and 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 for immunocyte be selected from T cell, NKT cell or CIK cell; Preferably, this fusogenic peptide has specific binding capacity to immune cell surface antigenic CD3.
In one embodiment, the CH2 structural domain of the strand unit of described bi-specific antibody is between ScFv fragment and CH3 structural domain; Described strand unit does not comprise CH1 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 CD3.
In one embodiment, in unit price unit, the light chain constant domain of described light chain and light chain variable domain all target in tumour antigen epi-position HER2; The heavy chain constant domain CH1 of described heavy chain and heavy-chain variable domains also target in tumour antigen epi-position HER2; Light chain is combined with heavy chain by disulfide linkage; Heavy chain is combined with described fusogenic peptide by one or more disulfide linkage, between the amino-acid residue of the preferably hinge area of described one or more disulfide formation between this CH1 (or VLs) and CH2 structural domain.
In one embodiment, strand unit comprises the anti-CD3 of antibody for CD3, and unit price unit comprises the anti-HER2 of antibody for HER2.
In one embodiment, the aminoacid sequence of the heavy chain of the anti-HER2 of antibody is the aminoacid sequence shown in sequence number 1, the aminoacid sequence of the light chain of the anti-HER2 of antibody is the aminoacid sequence shown in sequence number 3, and the aminoacid sequence of described anti-CD3ScFv-Fc is the aminoacid sequence shown in sequence number 5, and the halfcystine of anti-HER2 heavy chain on 223 sites is connected with the form of disulfide linkage with the halfcystine on light chain 214 site of anti-HER2, described anti-HER2 heavy chain is connected with the form of disulfide linkage with the halfcystine on 255 and 258 sites of anti-CD3ScFv-Fc with the halfcystine on 232 sites respectively 229, described anti-HER2 heavy chain on 395 with 412 sites with 428 and 397 sites of anti-CD3ScFv-Fc form salt bridge be connected, described anti-HER2 heavy chain on 369 sites with 436 sites of anti-CD3ScFv-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-HER2 antibody, its light chain the primer that increases is Kozak (EcoRV) F, MK-leader (EcoRV) F and hIgK (PacI) R, increased by over-lap PCR, Kozak sequence, MK-leader and restriction enzyme site EcoRV and PacI are introduced light chain; Its heavy chain the primer that increases is Kozak (AvrII) F, MK-leader (AvrI I) F and hIgG1 (BstZ17I) R, increased by over-lap PCR, Kozak sequence, MK-leader and restriction enzyme site AvrI I and BstZl7I are introduced heavy chain and sees; The LC gene fragment increased with carry out homologous recombination with the pCHO1.0 expression vector that EcoRV and PacI enzyme cuts through, acquisition loading anti-HER2 light chain expression vector; Then carry out homologous recombination with HC again after cutting with AvrI I and BstZl7I enzyme, obtain the pCHO1.0 expression vector of anti-HER2, the anti-HER2-HL-KKW of plasmid called after pCHO1.0-;
Described strand unit is anti-CD3ScFv-Fc antibody, its the primer that increases is Kozak (AvrII) F, MK-leader (AvrI I) F, L2K-VH (MK) F1 and hIgG1 (BstZ17I) R, by over-lap PCR amplification AntiCD3 McAb ScFv-Fc structural domain, and by Kozak sequence, MK-leader and restriction enzyme site AvrI I and BstZl7I introduces ScFv-Fc, the pCHO1.0-Totomycin expression vector gene fragment increased and enzyme cut through carries out homologous recombination, obtain the expression vector loading AntiCD3 McAb ScFv-Fc, plasmid called after pCHO1.0-Totomycin-L2K-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 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 antibody being called as bi-specific antibody, and set up and a kind ofly utilize the immunity system of human body to carry out immunotherapy and carry out the method for the drug efficacy study of bi-specific antibody.This bi-specific antibody, as a kind of novel antibody and for pharmacophore model, introduces T cell to specific cytotoxic effect of the tumour antigens such as HER2.
The invention provides a kind of novel method and prepare bi-specific antibody MSBODY (monomer and ScFv-Fcbispecific antibody) (as shown in Figure 2), this bi-specific antibody comprises two groups heavy light chain combination, wherein one group of a kind of antigen of specific combination, and some transformations are carried out in its heavy chain Fc district, make its versus wild type, not easily self forms dimer; And the another kind of antigen of another group 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 heavy and light chains 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 MSBODY simultaneously, and each domain arrangement order of MSBODY 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 CD3, be named as HER2XCD3, as Fig. 2, anti-HER2 is here unit price unit form, comprise anti-HER2 heavy chain and light chain, anti-CD3 is here ScFv-Fc form, comprises anti-CD3VH, 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 MSBODY and unit price unit light chain double promoter expression vector, and ScFv-Fc expression vector.According to unit price unit light chain (LC), unit price unit heavy chain (HC), the multiple clone site design primer in ScFv, Fc gene order and carrier.Wherein LC, HC, ScFv and Fc carry out pcr amplification respectively, obtain 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 MSBODY 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 MSBODY.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 the foundation of effect experiment method and application thereof in immunocyte contact element ectosome that a kind of novel bispecific antibodies MSBODY 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 pharmacophore model and detection in the external body of bi-specific antibody.Bi-specific antibody MSBODY 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 unit price 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 strand 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 Clinical Application; " (Cl inical and Developmental Immunology, 2012:980250 (2012)), the IgG 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 .HER2 × CD3 bi-specific antibody molecule schematic diagram.
Fig. 3. the double antibody electrophoresis of purifying and purity detecting result figure, (A) non-reduced SDS-PAGE electrophoresis, M: protein markers; 1:M802; (B) the HPLC-SEC purity peak shape figure of M802.
Fig. 4. the HER2 × CD3 double antibody measured based on flow cytometric analysis and the avidity situation map of SK-BR-3 cell.
Fig. 5. the HER2 × CD3 double antibody measured based on flow cytometric analysis and the avidity situation map of Jurkat cell.
Fig. 6 .HER2 positive cell NCI-N87 (CFSE dyeing) and Jurkat cell (PKH26 dyeing) are without antibody streaming scatter diagram.
Fig. 7 .HER2 positive cell NCI-N87 (CFSE dyeing) and Jurkat cell (PKH26 dyeing) have M802 antibody to hatch streaming scatter diagram altogether.
Fig. 8. flow cytometer detection heat challenge experiment process after double antibody to SK-BR-3 cell in conjunction with situation map.
Fig. 9. flow cytometer detection heat challenge experiment process after double antibody to human PBMC's cell in conjunction with situation map.
Figure 10 .M802 and hPBMC is to the cell in vitro poison experimental result picture of SK-BR-3 cell.
Figure 11 .M802 and hPBMC is to the cell in vitro poison experimental result picture of NCI-N87 cell.
Figure 12 .M802 and hPBMC is to the cell in vitro poison experimental result picture of MDA-MB-231 cell.
Figure 13 .M802 and hPBMC is to the cell in vitro poison experimental result picture of HEK-293 cell.
The interior animal experiment result figure of Figure 14 .M802.
Embodiment
Embodiment 1: the expression vector establishment (HER2 × CD3, M802) of bi-specific antibody
1. bi-specific antibody sequences Design
HER2 × CD3MSBODY 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 anti-HER2, the sequence (PDB database No.1N8Z) of variable region amino acid sequence reference monoclonal antibody Herceptin, comprise anti-HER2 heavy chain and light chain, containing Fab and Fc structural domain; Strand unit is the ScFv-Fc form of AntiCD3 McAb, and variable region amino acid, with reference to the sequence (with reference to US20070123479 sequence number 2) of monoclonal antibody L2K, comprises AntiCD3 McAb VH, VL, Fc structural domain.Wherein the heavy chain Fc of unit price unit and the Fc (the heavy chain Fc with human IgG1) 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 × CD3MSBODY, is numbered M802.Meanwhile, in order to M802 can at Chinese hamster ovary cell (CHO (Cricetulusgriseus, hamster, Chinese, ovary) cells, and can be secreted in substratum, 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 MSBODY of a kind of anti-HER2 × CD3, unit price unit variable region is from Trastuzumab, and strand unit variable region is from humanization OKT3, and this MSBODY is numbered M801 in the present invention, as a comparison antibody.
Unit price unit heavy chain amino acid sequence (Trastuzumab, sequence number 1)
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK-
Unit price unit heavy chain nucleic acid sequence (Trastuzumab, sequence number 2)
GAAGTGCAGCTGGTGGAAAGCGGCGGCGGCCTGGTGCAGCCGGGCGGATCCCTGCGCCTGAGCTGCGCGGCGAGCGGCTTTAACATTAAAGATACCTATATTCATTGGGTGCGCCAGGCGCCGGGCAAAGGCCTGGAATGGGTGGCGCGCATTTATCCGACCAACGGCTATACCCGCTATGCGGATAGCGTGAAAGGCCGCTTTACCATTAGCGCGGATACCAGCAAAAACACCGCGTATCTGCAGATGAACAGCCTGCGCGCGGAAGATACCGCGGTGTATTATTGCAGCCGCTGGGGCGGCGATGGCTTTTATGCGATGGATTATTGGGGCCAGGGCACCCTGGTGACCGTGAGCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACGATACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCGATCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA
Unit price unit light-chain amino acid sequence (Trastuzumab, sequence number 3)
DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC-
Unit price unit light chain nucleic acid sequence (Trastuzumab, sequence number 4)
GATATTCAGATGACCCAGAGCCCGTCAAGCTTAAGCGCGAGCGTGGGCGATCGCGTGACCATTACCTGCCGCGCGAGCCAGGATGTGAACACCGCGGTGGCGTGGTATCAGCAGAAACCGGGCAAAGCGCCGAAACTGCTGATTTATAGCGCGAGCTTTCTGTATAGCGGCGTGCCGAGCCGCTTTAGCGGCAGCCGCAGCGGCACCGATTTTACCCTGACCATTAGCAGCCTGCAGCCGGAAGATTTTGCGACCTATTATTGCCAGCAGCATTATACCACCCCGCCGACCTTTGGCCAGGGTACCAAAGTGGAAATTAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG
Strand strand unit aminoacid sequence (L2K, sequence number 5)
DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSGGGGSGGGGSGGGGSDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKGAAAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCRVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLASKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK-
Strand strand unit nucleotide sequence (L2K, sequence number 6)
GACATCAAACTGCAGCAGTCAGGGGCTGAACTGGCAAGACCTGGGGCCTCAGTGAAGATGTCCTGCAAGACTTCTGGCTACACCTTTACTAGGTACACGATGCACTGGGTAAAACAGAGGCCTGGACAGGGTCTGGAATGGATTGGATACATTAATCCTAGCCGTGGTTATACTAATTACAATCAGAAGTTCAAGGACAAGGCCACATTGACTACAGACAAATCCTCCAGCACAGCCTACATGCAACTGAGCAGCCTGACATCTGAGGACTCTGCAGTCTATTACTGTGCAAGATATTATGATGATCATTACTGCCTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCAGGAGGCGGCGGTTCAGGCGGAGGTGGAAGTGGTGGAGGAGGTTCTGACATTCAGCTGACCCAGTCTCCAGCAATCATGTCTGCATCTCCAGGGGAGAAGGTCACCATGACCTGCAGAGCCAGTTCAAGTGTAAGTTACATGAACTGGTACCAGCAGAAGTCAGGCACCTCCCCCAAAAGATGGATTTATGACACATCCAAAGTGGCTTCTGGAGTCCCTTATCGCTTCAGTGGCAGTGGGTCTGGGACCTCATACTCTCTCACAATCAGCAGCATGGAGGCTGAAGATGCTGCCACTTATTACTGCCAACAGTGGAGTAGTAACCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAAGGTGCGGCCGCAGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCGGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGAAGTCCGACGGCTCCTTCTTCCTCGCCAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA
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
Select0ExpressionVector (is called for short pCHO1.0, purchased from the test kit of Lifetechnologiesarticle 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 10xPCRBuffer damping fluid of 2.5 μ l; The 10mMdNTP of 1 μ l; 2.5 units/μ lPyrobestDNA polysaccharase (Takara, R005A) of 1 μ l; Softly mix in 200 μ LPCR pipes 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.Gene Amp PCR System9700 (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, MK-leader and restriction enzyme site EcoRV and PacI are introduced light chain; And Kozak sequence, MK-leader 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 EcoRV and PacI enzyme cuts through, acquisition loading anti-HER2 light chain expression vector; Then carry out homologous recombination with HC again after cutting with AvrII and BstZl7I enzyme, obtain the pCHO1.0 expression vector of anti-HER2, plasmid called after pCHO1.0--Trastuzumab-HL-KKW.
By over-lap PCR amplification AntiCD3 McAb ScFv-Fc structural domain, and Kozak sequence, MK-leader 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 AntiCD3 McAb Scfv-Fc, plasmid called after pCHO1.0-Totomycin-L2K-ScFv-Fc-LDY.
Embodiment 2: bi-specific antibody expression and purification
1. the expression of bi-specific antibody
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 CDFortiCHO substratum (Invitrogen, article No. A11483-1), is placed in 37 DEG C, 5%CO2cultivate in cell culture incubator, after getting out cell, according to the specification sheets (Maxcyte) of manufacturers, use MaxcyteSTX electroporation that plasmid pCHO1.0-Trastuzumab-HL-KKW cotransfection together with pCHO1.0-Totomycin-L2K-ScFv-Fc-LDY, in CHO-S cell, is expressed the bi-specific antibody M802 of anti-HER2 × CD3.Cultivate after 14 days, 800Xg harvested by centrifugation expresses supernatant.
2. the purifying of bi-specific antibody
Express supernatant 0.22uM membrane filtration, utilize MabselectSuRe 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 NaH2pO4+ 40.5mM Na2hPO4, 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 NaH2pO4+ 6.2mM Na2hPO4, pH6.0) balance chromatography column after, sample, with between two pure water dilution conductance to 3.0-3.5ms, is crossed after SP pillar combines, with elution buffer B (43.8mM NaH2pO4+ 6.2mM Na2hPO4+ 1M NaCl, pH6.0) 20 column volume linear elutions; Finally concentrated displacement BufferPBS.Bi-specific antibody after purifying carries out SDS-PAGE, SEC and detects, and purity, more than 95%, is shown in Fig. 3.
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 × 106individual cell/ml, plating cells in 96 orifice plates, every hole 50ul (2 × 105individual cell), add the bi-specific antibody that 50ul has diluted, incubated at room 1 hour; Centrifugally remove supernatant, cell is washed twice with PBS, again with the anti-human igg FC antibody (Biolegend of the PE mark diluted, 409304) re-suspended cell, room temperature lucifuge hatches 30 minutes, and PBS washes twice, use 100ulPBS 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 GraphPadPrism5.0.The SK-BR-3 cell of result display HER2 × CD3 double antibody and the HER2 positive has good binding activities, sees Fig. 4.With HER2 positive cell SK-BR-3 in conjunction with situation: the KD value of M801 is the KD value of 14.84nM, M802 is 10.61nM, and the KD value of Trastuzumab is 3.772nM.
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 100ulPBS, 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 Prism 5.0.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 M801 is the KD value of 7.25nM, M802 is 6.61nM, and the KD value of Trastuzumab is 0.39nM.
3. the immunocyte of double antibody mediation and the common Binding experiment of tumour cell
By cultured NCI-N87 (HER2 positive gastric carcinoma cell, purchased from China typical culture collection center) and Jurkat cell, collected by centrifugation also washes 2 times with PBS, respectively with CFSE and PKH-26 dyeing.Dilute M802 to 160nM simultaneously.By the NCI-N87 dyeed with Jurkat cell is centrifugal removes supernatant, wash twice with PBS+1%FBS, then add PBS+1%FBS re-suspended cell to 4 × 106individual cell/ml, mixes by 1:1, by plating cells in 96 orifice plates, and every hole 50ul (2 × 105individual cell), add the bi-specific antibody that 50ul has diluted, incubated at room 1 hour; Centrifugally remove supernatant, wash cell twice, finally use 100ulPBS resuspended with PBS, upper machine (FC500, Beckman) is detected, and analyzes the ratio of two positive cell, by carrying out analytical calculation with software GraphPadPrism5.0.Result when show there is no a M802, the ratio very low (Fig. 6) of the two fluorescence of flow cytometer detection; When adding HER2 × CD3 double antibody M802, the ratio of the two fluorescence of flow cytometer detection reaches 26.3%, show that M802 simultaneously in conjunction with the NCI-N87 cell of the HER2 positive and the Jurkat cell of the CD3 positive, can promote the common combination (see Fig. 7) of two kinds of cells.
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 (ABIPCRsystem9700).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 sigmoidal dose response with variable slope) model with GraphPad Prism 5 software with variable slope is analyzed.The temperature mid point value of thermal denaturation curve is T50.
Single chain antibody fragments (ScFv) is by a connection peptides (Gly4ser)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 (MichaelsonJS of antibody drug, etc., Farrington GK, LugovskoyA, Joseph I, Bailly V, Wang X, Garber E, BrowningJ, Glaser SM.Anti-tumoractivity of stability-engineered IgG-like bispecificantibodies targeting TRAIL-R2and LTbetaR.MAbs.2009Mar-Apr; 1 (2): 128-41.).The unit price unit of M802 and M801 completely the same, the T that both are combined with SK-BR-350also closely (Fig. 8), the T of M80250=60.60 DEG C, the T of M80150=57.97 DEG C; But the strand unit of M802 uses the variable region of L2K, the strand unit of M801 to use the variable region of humanization OKT3, with the T that T cell is combined50value difference not comparatively large (Fig. 9), the T of M80250=59.98 DEG C, the T of M80150=48.79 DEG C, M802 thermostability is obviously better than M801.
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 400g, 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 400g, 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 SK-BR-3 breast cancer cell of HER2 high expression level is comprised with trysinization target cell, the NCI-N87 stomach cancer cell of HER2 high expression level, the MDA-MB-231 breast cancer cell of the low expression of HER2 and the HEK-293 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 × 105/ ml, according to 2 × 104/ 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 min of 500 × g.Abandon supernatant, each hole adds the resuspended mixing cell of 150 μ l1%FBS-PBS.Each pipe in streaming before machine 10-15min add PI (final concentration is 1 μ g/ml) dyeing.In streaming, the two positive cell of machine testing CFSE, PI accounts for the mortality ratio that CFSE positive cell ratio is target cell.
To the tumor cytotoxicity effect of HER2 high expression level clearly, most High Fragmentation rate reaches 80% to M802, and using dosage is far below Trastuzumab and L2K (Figure 10,11); Also have the tumour cell of the low expression of HER2 and significantly kill and wound, and effect is better than Trastuzumab and L2K (Figure 12) greatly.But for the cell that HER2 is completely negative, M802 does not show fragmentation effect (Figure 13).M802 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 does not substantially have toxicity for the cell that HER2 does not express.
Embodiment 6: bi-specific antibody kills and wounds the Composition analyzed of subcutaneous transplantation knurl
The cultivation of CIK cell: with CIK cell Primary culture liquid (serum-free X-Vivo cell culture fluid+750IU/mlIFN-γ ± 2% autologous plasma), every part of cell is filled 30ml, be added to 75cm2in culturing bottle, be placed in saturated humidity, 37 DEG C, 5.0%CO2incubator is cultivated.Cultivate after 24 hours, (serum-free X-Vivo cell culture fluid+75ng/ml OKT3 monoclonal antibody (self-control), 750IU/ml interleukin-22 (IL-2), 0.6ng/ml interleukin-11 (IL-1 α) continue to be placed in saturated humidity, 37 DEG C, 5.0%CO to add CIK cell stimulating factor mixed solution 1ml2cultivate in incubator.The thing that following step determines fluid infusion (serum-free X-Vivo nutrient solution+750IU/ml IL-2 ± 2% autologous plasma) according to the growing state of CIK cell, goes down to posterity, will maintain cell substantially at 2*106the density growth of about/ml.Finally with flow cytometer FC500, Phenotypic examination is carried out to the CIK cell of collecting, comprising: CD3, CD56, CD4, CD8, detect the expression of these cell-surface antigenss in CIK cell.
Tumor inoculation and CIK injection are carried out simultaneously, and 5 × 106nCI-N87 tumour cell and 5 × 106the right dorsal part of female NOD/SCID mouse is injected in after CIK cell mixing.By mouse random packet (mouse 8/group) in two hours, by tail vein injection administration, dosage is respectively the M802 of 4,2 and 1mg/kg.Control group is: (1) dosage is the Trastuzumab of 4mg/kg, and (2) dosage is the MCO101 of 4mg/kg.MCO101 is also MSBODY, its strand unit and M802 completely the same, unit price unit variable region is 4420 (a kind of anti-fluorescein antibodies, see Kranz DM, Voss EW Jr., Partialelucidation of an anti-hapten repertoire in BALB/c mice:comparativecharacterization of several monoclonal anti fluorescyl antibodies.MolImmunol.1981; 18 (10): 889-898) variable region.(3) negative control group, only injects PBS.Administration same day is the 0th day.Within 2nd day and the 4th day, continue administration, dosage is constant.Within every 3 days, measure the volume of a tumour, calculating cubature formula is 1/2 × length × wide × wide (mm3).
In fig. 14, tumour cell NCI-N87 is subcutaneous in female NOD/SCID mouse with identical quantity co-injection with immunocyte CIK; Administration after two hours, tail vein injection; Within after administration 3 days and 5 days, be administered once respectively, dosage is constant again, and every 3 staggering amount knurls once.Figure 14 shows, the M802 administration of various dose demonstrates the curative effect of good Tumor suppression growth, wherein 2 and all mouse (the totally 16) tumour of 4mg/kg dosage treatment group is completely suppressed 53 days time even disappears, and the mouse Partial tumors of 1mg/kg dosage treatment group also suppresses (3/8) completely, remaining 5 mouse also only has less knurl block to there is (<150mm3).In control group, Trastuzumab treatment group tumors is suppressed, after 44 days, have a small amount of growth; MCO101 treatment group tumors does not suppress, and knurl block is long-pending reaches 300mm3.The tumor growth situation of negative group is normal, reaches 800mm when 53 days3left and right.
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, many Equivalents of specific embodiment of the present invention described in this article.These Equivalents are intended to comprise in the appended claims.