CN104774268A

CN104774268A - Construction and application of bispecific antibody EGFR*CD3

Info

Publication number: CN104774268A
Application number: CN201510030519.9A
Authority: CN
Inventors: 王涛; 方丽娟; 杨锦霞; 戴晴; 刘勇; 周鹏飞
Original assignee: YZY BIOPHARMA CO Ltd
Current assignee: Wuhan youzhiyou biopharmaceutical Co.,Ltd.
Priority date: 2015-01-21
Filing date: 2015-01-21
Publication date: 2015-07-15
Anticipated expiration: 2035-01-21
Also published as: CN104774268B

Abstract

The invention provides a bispecific antibody. The bispecific antibody is composed of a single brand unit and a univalent unit, wherein the single brand unit has specific binding ability on surface antigen CD3 of immune cells, and the univalent unit has specific binding ability on surface antigen EGFR of tumor cells; and the single brand unit includes a single brand variable fragment (ScFv) fused with an Fc fragment, and the univalent unit includes a light strand and heavy strand pair. The invention also provides a preparation method and a pharmaceutical use of the bispecific antibody.

Description

The construction and application of a kind of bi-specific antibody EGFR × CD3

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-based activation 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.EGFR

EGF-R ELISA (EGFR, Epidermal Growth Factor Receptor) is the acceptor of Urogastron (EGF) cell proliferation and intracellular signaling.EGFR belongs to the one of ErbB receptor family, and this family comprises EGFR (ErbB-1), HER2/c-neu (ErbB-2), Her 3 (ErbB-3) and Her 4 (ErbB-4).EGFR is also referred to as HER1, ErbB1, and sudden change or process LAN generally can cause tumour.EGFR is a kind of glycoprotein, and belong to Tyrosylprotein kinase receptor, cytolemma is through, molecular weight 170KDa.EGFR is positioned at surface of cell membrane, by activating with ligand binding, comprises EGF and TGF α (transforming growth factor α).After activation, EGFR is dimer by conversion of monomer, although also evidence suggests, also there is dimer before activation.EGFR also may be polymerized with other members of ErbB receptor family and activates, such as ErbB2/Her2/neu.

After part is combined with EGFR, acceptor there occurs dimerisation, dimerisation had both comprised the combination (homology dimerisation) of two cognate receptor molecules, also comprised the combination (heterology dimerisation) of the different members in mankind EGF related receptor (HER) family tyrosine kinase.It is the autophosphorylation effect of tyrosine residues after dimerisation.The residue of these phosphorylations is the binding sites raising adaptor protein and extra tyrosine kinase substrate.Protein interacts in the receptor complex activated stimulates ras albumen, the generation causing phosphorylation cascade to react and mitogen-activated protein (MAP) kinase whose activation.Or transcription signal conduction and activation, phosphatidyl inositol kinase-3 (PI3K)-Akt and stress activated protein kinase (SAPK) signal transduction pathway will be activated.These signal paths trigger genetic transcription successively, control hyperplasia simultaneously, the path of differentiation and existence is activated.

Specificity and the intensity of EGFR mediation signal path depend on the character of activator and the level of four kinds of EGFR family members.The part be combined with HER2 is not quite clear, but when HER2 and EGFR coexpression, the former often with the latter of ligand activation in conjunction with formation dimer.This heterology dimer, compared with EGFR homology dimer, often has the ability of higher reuse ratio, stability and conducted signal.Also can there is dimerisation with HER3 and HER4 in EGFR, its product has higher persistence and stronger PI3K is active.EGFR signal transduction pathway is once the EGFR of ligand binding is swallowed cell by interior, and signal will stop, and acceptor will be degraded or be recycled to surface of cell membrane, and this depends on the character of part.Such as, the acceptor that EGF combines will be degraded, and the acceptor that TGF-α combines then enters recirculation.Different somatomedins can affect quantity and the time length of EGFR signal path.

EGFR signal path has multiple biological action.Such as, ras-MAPK signal transduction pathway irritation cell division and migrate.EGFR is also the important amboceptor of multiple receptor pathway, plays the effect of signal convergent point, can by signal whole and with variation.Such as, stress, in the reaction of film unzipping and some non-physiologic stimulator (comprising oxygenant, radioactive rays and alkylating agent), Reverse Activity can be induced the phosphorylation of EGFR Tyrosylprotein kinase and the transduction of signal is occurred subsequently.The member of EGFR family plays important effect in normal development, but frequent overexpression out of hand in human tumor.

Research shows in many noumenal tumours, there is EGFR high expression level or unconventionality expression.The propagation of EGFR and tumour cell, vasculogenesis, tumor invasion, transfer and apoptotic suppression are relevant.Its mechanism has: the enhancing that the high expression level of EGFR causes downstream signal to conduct; The increase of mutant egf R acceptor or ligand expression causes the continuous activation of EGFR; The effect of autocrine loop strengthens; The destruction of receptor down-regulated mechanism; The activation etc. of abnormal signal conduction path.The process LAN of EGFR plays an important role in the evolution of malignant tumour, has the process LAN of EGFR in the tissue such as spongiocyte, kidney, lung cancer, prostate cancer, carcinoma of the pancreas, mammary cancer.The research of glioma is found that the high expression level of EGFR is main relevant with its gene amplification.But after the dysregulation of EGFR expression level is also present in translation and translation sometimes.The high expression level of EGFR in tumour also may reduce relevant with degrade after activation, and some are studied and point out that c-Src is by suppression acceptor ubiquitination and endocytosis and raise EGFR level.There is mutant egf R to exist in many tumours, have now found that many kinds of EGFR saltant types.The effect of mutant egf R may comprise: the cell continuous activation with part independent form acceptor; The destruction, the activation of abnormal signal conduction path, apoptotic suppression etc. of receptor down-regulated mechanism is caused due to some structural domain disappearance of EGFR.The generation of mutant is due to the disappearance of EGFR gene, sudden change and rearrangement.The part of EGFR has a significant impact Cellular Signaling Transduction Mediated.The part of EGFR activates EGFR by autocrine form and promotes cell proliferation, their coexpression often indicates that tumor prognosis is bad, such as, finds in the research of infiltration ductal carcinomas of breast, TGF α and EGFR coexpression, and the survival rate significant correlation of this coexpression and patient.The research of the people such as Kopp to the knot/rectum cancer shows that the autocrine growth of tumour is process LAN and the coefficient result of ligand expression thereof of EGFR.

In addition, the research of EGFR and the vasculogenesis of tumour, high aggressive and transfer relationship is found that EGFR can affect tumor-blood-vessel growth by the adjustment of the factor levels such as Ang-1 and VEGF.

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, a kind of bi-specific antibody is provided, it is characterized in that, this antibody described comprises: (a) unit price unit, for light-heavy chain pair, this light-heavy chain has specific binding capacity to for TCSA, and preferably this TCSA is EGFR, CD20, CD30 and CD133, and more preferably this TCSA is EGFR; (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.

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, 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-CD3 of antibody for people source CD3, and unit price unit comprises the antibody anti-EGFR for EGFR, preferably, the aminoacid sequence of described anti-EGFR heavy chain is the aminoacid sequence shown in sequence number 1, the aminoacid sequence of the light chain of anti-EGFR 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 9, and the halfcystine of anti-EGFR heavy chain on 222 sites is connected with the form of disulfide linkage with the halfcystine on light chain 215 site of anti-EGFR, described anti-EGFR 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 231 sites respectively 228, described anti-EGFR heavy chain on 394 with 411 sites with 428 and 397 sites of anti-CD3ScFv-Fc form salt bridge be connected, described anti-EGFR heavy chain on 368 sites with 436 sites of anti-CD3ScFv-Fc are formed knuckle-enter-cave and are connected,

Or the aminoacid sequence of described anti-EGFR heavy chain is the aminoacid sequence shown in sequence number 5, the aminoacid sequence of the light chain of anti-EGFR is the aminoacid sequence shown in sequence number 7, and the aminoacid sequence of described anti-CD3ScFv-Fc is the aminoacid sequence shown in sequence number 9, and the halfcystine of anti-EGFR heavy chain on 222 sites is connected with the form of disulfide linkage with the halfcystine on light chain 214 site of anti-EGFR, described anti-EGFR 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 231 sites respectively 228, described anti-EGFR heavy chain on 394 with 411 sites with 428 and 397 sites of anti-CD3ScFv-Fc form salt bridge be connected, described anti-EGFR heavy chain on 368 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 IgG 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 IgG Fc fragment.

In one embodiment, the human IgG Fc section of described unit price unit and the IgG Fc 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-EGFR-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 hIgG1 (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 anti-EGFR light chain; Then homologous recombination is carried out with HC again after cutting with AvrII and BstZl7I enzyme, obtain the pCHO1.0 expression vector of anti-EGFR, with the plasmid called after pCHO1.0-ERB-HL-KKW of Erbitux antibody gene, or the plasmid called after pCHO1.0-VEC-HL-KKW of band Vectibix antibody gene;

Described strand unit is anti-CD3ScFv-Fc antibody, its the primer that increases is Kozak (Avr II) F, L2K-VH (MK) F1 and hIgG1 (BstZ17I) R, by pcr amplification AntiCD3 McAb ScFv-Fc structural domain, 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 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 EGFR specific antigen express caused by tumour or relative disease, or express EGFR 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 EGFR specific antigen or evaluation be used for the treatment of the medicine of the tumour cell relative disease of expressing EGFR specific antigen.Present invention also offers following technical scheme:

The invention provides a kind of novel method and prepare bi-specific antibody MSBODY (monomer and ScFv bispecific 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 single aggressiveness Ab, another group is 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 single aggressiveness Ab 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 EGFR and people source CD3, be named as EGFRX CD3, as Fig. 2, anti-EGFR is here IgG form, comprise anti-EGFR 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, single aggressiveness Ab heavy chain of bi-specific antibody MSBODY and single aggressiveness Ab light chain binary 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 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 will to express the plasmid co-transfection of unit price unit heavy chain, unit price unit light chain and strand unit respectively in mammalian cell, regather the expression that supernatant carries out SDS-PAGE and western blotting detection 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 foundation and the application thereof of a kind of outer effect experiment method of immunocyte contact element that 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 bi-specific antibody pharmacy in vitro model and detection.Bi-specific antibody MSBODY comprises one group of unit price unit (heavy light chain combination), another group is then strand unit (ScFv connects Fc 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 EGFR, 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), excites the immune response with guidance quality, 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; " Clinical 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, those of ordinary skill in the art are come, 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 .EGFR X CD3 bi-specific antibody molecule schematic diagram.

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

The two antigen ELISA of Fig. 4 .EGFR and CD3 detects the two target capability result figure of antibody.

Fig. 5. flow cytometer detection heat challenge experiment process after double antibody to HCT116 cell in conjunction with situation map.

Fig. 6. flow cytometer detection heat challenge experiment process after double antibody to human PBMC's cell in conjunction with situation map.

Fig. 7 .EGFR × CD3MSBODY and hPBMC is to the cell in vitro poison experimental result picture of HCT116 cell.

Fig. 8 .EGFR × CD3MSBODY and hPBMC is to the cell in vitro poison experimental result picture of MDA-MB-453 cell.

Fig. 9 .EGFR × CD3MSBODY and hPBMC is to the cell in vitro poison experimental result picture of HEK293 cell.

Embodiment

Embodiment 1: the expression vector establishment (EGFR × CD3, M1001, M1003) of bi-specific antibody

1. bi-specific antibody sequences Design

EGFR × CD3MSBODY is named as with the bi-specific antibody that EGFR and CD3 is target spot, wherein unit price unit is the heavy chain light chain pair of anti-EGFR, the sequence (PDB database serial number 1YY8) of variable region amino acid sequence reference monoclonal antibody Erbitux and the sequence (source is the sequence number 38 and 49 in patent US6235883) of Vectibix, comprise anti-EGFR 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 EGFR × CD3MSBODY, EGFR × CD3MSBODY that unit price unit derives from Erbitux is numbered M1001, and EGFR × CD3MSBODY that unit price unit derives from Vectibix is numbered M1003., in order to EGFR × CD3MSBODY 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: 1-12.Signal peptide is directly connected in the N end of antibody variable region.

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

QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

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

caggtgcagctgaaacagagcggcccgggcctggtgcagccgagccagagcctgagcattacctgcaccgtgagcggctttagcctgaccaactatggcgtgcattgggtgcgccagagcccgggcaaaggcctggaatggctgggcgtgatttggagcggcggcaacaccgattataacaccccgtttaccagccgcctgagcattaacaaagataacagcaaaagccaggtgttttttaaaatgaacagcctgcagagcaacgataccgcgatttattattgcgcgcgcgcgctgacctattatgattatgaatttgcgtattggggccagggcaccctggtgaccgtgagcgcggcgtcgaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaaagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctg gggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtctacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgtggtgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacgataccacgcctcccgtgctggactccgacggctccttcttcctctacagcgatctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga

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

DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

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

gatattctgctgacccagagcccggtgattctgagcgtgagcccgggcgaacgcgtgagctttagctgccgcgcgagccagagcattggcaccaacattcattggtatcagcagcgcaccaacggcagcccgcgcctgctgattaaatatgcgagcgaaagcattagcggcattccgagccgctttagcggcagcggcagcggcaccgattttaccctgagcattaacagcgtggaaagcgaagatattgcggattattattgccagcagaacaacaactggccgaccacctttggcgcgggcaccaaactggaactgaaacgccgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgttag

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

QVQLQESGPGLVKPSETLSLTCTVSGGSVSSGDYYWTWIRQSPGKGLEWIGHIYYSGNTNYNPSLKSRLTISIDTSKTQ FSLKLSSVTAADTAIYYCVRDRVTGAFDIWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

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

caggtgcagctgcaggagtcgggcccaggactggtgaagccttcggagaccctgtccctcacctgcactgtctctggtggctccgtcagcagtggtgattactactggacctggatccggcagtccccagggaagggactggagtggattggacacatctattacagtgggaacaccaattataacccctccctcaagagtcgactcaccatatcaattgacacgtccaagactcagttctccctgaagctgagttctgtgaccgctgcggacacggccatttattactgtgtgcgagatcgagtgactggtgcttttgatatctggggccaagggacaatggtcaccgtctcttcagcgtcgaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaaagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtctacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgtggtgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacgataccacgcctcccgtgctggactccgacggctccttcttcctctacagcgatctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga

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

DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYFCQHFDHLPLAFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

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

gacatccagatgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttgccaggcgagtcaggacatcagcaactatttaaattggtatcagcagaaaccagggaaagcccctaaactcctgatctacgatgcatccaatttggaaacaggggtcccatcaaggttcagtggaagtggatctgggacagattttactttcaccatcagcagcctgcagcctgaagatattgcaacatatttctgtcaacactttgatcatctcccgctcgctttcggcggagggaccaaggtggagatcaaacgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgttag

Strand unit aminoacid sequence (L2K, sequence number 9)

DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSGGGGSGGGGSGGGGSDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKGAAAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCRVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLASKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Strand unit nucleotide sequence (L2K, sequence number 10)

GACATCAAACTGCAGCAGTCAGGGGCTGAACTGGCAAGACCTGGGGCCTCAGTGAAGATGTCCTGCAAGACTTCTGGCTACACCTTTACTAGGTACACGATGCACTGGGTAAAACAGAGGCCTGGACAGGGTCTGGAATGGATTGGATACATTAATCCTAGCCGTGGTTATACTAATTACAATCAGAAGTTCAAGGACAAGGCCACATTGACTACAGACAAATCCTCC AGCACAGCCTACATGCAACTGAGCAGCCTGACATCTGAGGACTCTGCAGTCTATTACTGTGCAAGATATTATGATGATCATTACTGCCTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCAGGAGGCGGCGGTTCAGGCGGAGGTGGAAGTGGTGGAGGAGGTTCTGACATTCAGCTGACCCAGTCTCCAGCAATCATGTCTGCATCTCCAGGGGAGAAGGTCACCATGACCTGCAGAGCCAGTTCAAGTGTAAGTTACATGAACTGGTACCAGCAGAAGTCAGGCACCTCCCCCAAAAGATGGATTTATGACACATCCAAAGTGGCTTCTGGAGTCCCTTATCGCTTCAGTGGCAGTGGGTCTGGGACCTCATACTCTCTCACAATCAGCAGCATGGAGGCTGAAGATGCTGCCACTTATTACTGCCAACAGTGGAGTAGTAACCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAAGGTGCGGCCGCAGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCGGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGAAGTCCGACGGCTCCTTCTTCCTCGCCAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA

The leader peptide sequences aminoacid sequence (sequence number 11) of mouse kappa

METDTLLLWVLLLWVPGSTG

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

atggagacagacacactcctgctatgggtactgctgctctgggttccaggttccactggt

2. bi-specific antibody gene clone

PCHO1.0 is selected to remove heavy chain and the light chain gene of the anti-EGFR of cloning and expressing 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 the ScFv-Fc fusion gene of cloning and expressing AntiCD3 McAb.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 by gold only intelligence gene chemical synthesis the gene plasmid be cloned on pUC57, comprise the variable region of Erbitux and Vectibix, then anti-EGFR is heavy, light chain is building up on the expression vector of pCHO1.0 respectively respectively, is building up to by AntiCD3 McAb ScFv-Fc on the expression vector of pCHO1.0-Totomycin.

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

Initial PCR amplification template DNA: M1001 and M1003 uses identical primer; Its light chain the primer that increases is Kozak (EcoR V) F, MK-Leader (EcoRV) F, with 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 hIgG1 (BstZ17I) R, increased by over-lap PCR, Kozak sequence, leader sequence and restriction enzyme site AvrII and BstZl7I are introduced heavy chain; Described strand unit is anti-CD3ScFv-Fc antibody, its the primer that increases is Kozak (Avr II) F, L2K-VH (MK) F1 and hIgG1 (BstZ17I) R, by pcr amplification AntiCD3 McAb ScFv-Fc structural domain, and Kozak sequence, leader sequence and restriction enzyme site AvrII and BstZl7I are introduced ScFv-Fc; The consumption of template DNA is 35ng/ pipe, e.g., and 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 microfuge 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 anti-EGFR light chain; Then homologous recombination is carried out with HC again after cutting with AvrII and BstZl7I enzyme, obtain the pCHO1.0 expression vector of anti-EGFR, with the plasmid called after pCHO1.0-ERB-HL-KKW of Erbitux antibody gene, the plasmid called after pCHO1.0-VEC-HL-KKW of band Vectibix antibody gene.

By over-lap PCR amplification AntiCD3 McAb ScFv-Fc structural domain, 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 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 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-Erbitux-HL-KKW cotransfection together with pCHO1.0-Totomycin-L2K-ScFv-Fc-LDY, in CHO-S cell, is expressed the bi-specific antibody M1001 of anti-EGFR × CD3; Cotransfection is in CHO-S cell together with pCHO1.0-Totomycin-L2K-ScFv-Fc-LDY by plasmid pCHO1.0-Vectibix-HL-KKW to use Maxcyte STX electroporation, and method is the same, expresses the bi-specific antibody M1003 of anti-EGFR × 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 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 (43.8mM NaH ₂pO ₄+ 6.2mM Na ₂hPO ₄+ 1M NaCl, pH 6.0) 20 column volume linear elutions; Finally concentrated displacement Buffer PBS.Bi-specific antibody after purifying carries out SDS-PAGE, SEC and detects, and two kinds of MSBODY purity, all about 80%, are 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 HT29 (purchased from China typical culture collection center) as the cell of the EGFR positive, and Jurkat (ATCC, TIB-152) as the cell of the CD3 positive, and measures its cell-bound activity with double antibody prepared by the present invention.

1. utilize flow cytometer showed method to detect the binding activities of bi-specific antibody and HT29 cell

Cultivate enough HT29 cells, with 0.25% trysinization, centrifugal collecting cell.Dilute bi-specific antibody, concentration is from 160nM, 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 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 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 HT29 with software GraphPad Prism5.0.The HT29 cell of result display EGFR × CD3 double antibody and the EGFR positive has good binding activities (see table 2).With EGFR positive cell HT29 in conjunction with situation: the KD value of the KD value of M1001 to be the KD value of 0.65 ± 0.27nM, M1003 be 0.65 ± 0.18nM, Erbitux is 0.12 ± 0.03nM.The avidity of two kinds of EGFR × CD3MSBODY is close, and is more or less the same with the avidity of monoclonal antibody Erbitux.

Table 2 EGFR × CD3MSBODY is to the avidity of HT29 cell

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 EGFR × CD3 double antibody and the CD3 positive has good binding activities (see table 3).With CD3 positive cell Jurkat in conjunction with situation: the KD value of M1001 is the KD value of 8.73 ± 5.29nM, M1003 is 14.36 ± 5.79nM, and avidity is all lower than monoclonal antibody L2K (KD value is 0.42 ± 0.12nM).The structure of instruction book chain unit can reduce the avidity of antibody.

Table 3 EGFR × CD3MSBODY is to the avidity of Jurkat cell

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

1) antigen preparation: EGFR antigen design of primers is with reference to Genbank SeqNo.NM_005228.3, and primer sequence is in EGFR-F and EGFR-R of table 2; MRNA extracts from HT29 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 EGFR-R, with primer EGFR-F and EGFR-R, the ectodomain of this antigen is increased out again, then primer EGFR-F and Histag-R is used to add histidine-tagged (7 × His) with PCR method, build up in expression vector pcDNA3.1/Hygro (+) (Invitrogen), transfection expression method is with the transfection expression (see embodiment 2) of M1001, 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 EGFR (see step 1).

3) envelope antigen: EGFR 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 4, M1001 and M1003 all can simultaneously in conjunction with EGFR 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 PCRsystem 9700).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 HCT116 cell (purchased from China typical culture collection center) 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 Prism5 software with variable slope is analyzed.The temperature mid point value of thermomechanical curve is T ₅₀.

Single chain antibody fragments (ScFv) is coupled together variable region of heavy chain and variable region of light chain by a connection peptides (Gly4Ser) 3 and is formed.But there is the unstable of report ScFv inherence may affect quality (the Michaelson JS1 of antibody drug, etc., Farrington GK, Lugovskoy A, Joseph I, Bailly V, Wang X, Garber E, Browning J, Glaser SM.Anti-tumor activity of stability-engineered IgG-like bispecific antibodies targeting TRAIL-R2and LTbetaR.MAbs.2009Mar-Apr; 1 (2): 128-41).The strand unit of M1001 and M1003 completely the same, the T that both are combined with Jurkat ₅₀also closely (Fig. 5), the T of M1001 ₅₀=60.26 ± 0.24 DEG C, the T of M1003 ₅₀=59.71 ± 0.64 DEG C; The unit price unit of M1001 uses light chain and the heavy chain of Erbitux, the unit price unit of M1003 to use light chain and the heavy chain of Vectibix, the T be combined with HCT116 ₅₀value difference very not little (Fig. 6), the T of M1001 ₅₀=59.66 ± 0.50 DEG C, the T of M1003 ₅₀=59.20 ± 0.70 DEG C.The thermostability difference of two kinds of MSBODY is little.

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, the centrifugal 2-3 minute of 400 × g, abandons 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 EGFR positive tumor cell

(the HCT116 colon cancer cell of EGFR high expression level is comprised with trysinization target cell, the MDA-MB-453 breast cancer cell of the low expression of EGFR and the HEK-293 HEKC of EGFR 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, centrifugal 500g × 5min.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 HCT116 fragmentation effect of EGFR high expression level all clearly, most High Fragmentation rate reaches more than 60% to M1001 and M1003, and using dosage is all far below monoclonal antibody Erbitux and L2K (Fig. 7); Also have the tumour cell MDA-MB-453 of the low expression of EGFR and significantly kill and wound, and effect is better than Erbitux and L2K (Fig. 8) greatly.But for the cell HEK293 that EGFR is completely negative, M1001 and M1003 does not show fragmentation effect (Fig. 9).EGFR × CD3MSBODY is described in vitro in cellulotoxic experiment, in the presence of immunocyte, the tumour cell different to EGFR positive expression amount all has good fragmentation effect, and does not substantially have toxicity for the cell that EGFR 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

1. bi-specific antibody, it is characterized in that, this antibody described comprises: (a) unit price unit, for light-heavy chain pair, this light-heavy chain has specific binding capacity to for TCSA, preferably this TCSA is EGFR, CD20, CD30 and CD133, and more preferably this TCSA is EGFR; (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.

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 CD3.

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-CD3 of antibody for people source CD3, and unit price unit comprises the antibody anti-EGFR for EGFR;

Preferably, the aminoacid sequence of described anti-EGFR heavy chain is the aminoacid sequence shown in sequence number 1, the aminoacid sequence of the light chain of anti-EGFR 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 9, and the halfcystine of anti-EGFR heavy chain on 222 sites is connected with the form of disulfide linkage with the halfcystine on light chain 215 site of anti-EGFR, described anti-EGFR 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 231 sites respectively 228, described anti-EGFR heavy chain on 394 with 411 sites with 428 and 397 sites of anti-CD3ScFv-Fc form salt bridge be connected, described anti-EGFR heavy chain on 368 sites with 436 sites of anti-CD3ScFv-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:

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-EGFR-antibodies, its light chain the primer that increases is Kozak (EcoR V) F, MK-Leader (EcoRV) F, with 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 I I) F, MK-Leader (AvrI I) F and hIgG1 (BstZ17I) R, increased by over-lap PCR, Kozak sequence, leader sequence and restriction enzyme site AvrI I 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 anti-EGFR light chain; Then homologous recombination is carried out with HC again after cutting with AvrII and BstZl7I enzyme, obtain the pCHO1.0 expression vector of anti-EGFR, with the plasmid called after pCHO1.0-ERB-HL-KKW of Erbitux antibody gene, or the plasmid called after pCHO1.0-VEC-HL-KKW of band Vectibix antibody gene;

Described strand unit is anti-CD3ScFv-Fc antibody, its the primer that increases is Kozak (Avr I I) F, L2K-VH (MK) F1 and hIgG1 (BstZ17I) R, by pcr amplification AntiCD3 McAb ScFv-Fc structural domain, 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 AntiCD3 McAb ScFv-Fc, plasmid called after pCHO1.0-Totomycin-L2K-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 EGFR specific antigen express caused by tumour or relative disease, or express EGFR 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 EGFR specific antigen for the medicine or evaluation screening the tumour cell relative disease being used for the treatment of expression EGFR specific antigen in tumor cell line.