CN104592392A

CN104592392A - Construction method and application of bispecific antibody EpCAM*CD3

Info

Publication number: CN104592392A
Application number: CN201510031516.7A
Authority: CN
Inventors: 王涛; 方丽娟; 胡柳; 刘杨; 张雨; 范克索; 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: 2015-05-06
Anticipated expiration: 2035-01-21
Also published as: CN104592392B

Abstract

The invention provides a bispecific antibody. The bispecific antibody is composed of a single-chain unit and a univalent unit, wherein the single-chain unit has specific binding capacity for a surface antigen CD3 of an immune cell; the univalent unit has specific binding capacity for a surface antigen EpCAM of a tumor cell; the single-chain unit contains a single-chain variable fragment (scFv) fused with a Fc fragment; and the univalent unit contains a light and heavy chain pair. The invention also provides a preparation method of the bispecific antibody and pharmaceutical application of the bispecific antibody.

Description

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

Technical field

The present invention relates to immunologic technical field.Specifically, relate to structure and the preparation method of bi-specific antibody, and double antibody function and nature examination method.

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.But in bispecific antibody drug R&D process, ubiquity and is expressed difficulty, yield poorly, many obstacles such as purifying difficulty, poor stability, therefore, build new bi-specific antibody, above-mentioned obstacle can be overcome, and set up corresponding immunologic cytotoxicity animal model, be necessary.The invention provides a kind of structure of novel pair of characteristic antibody, and the research method described its pharmacodynamics and result.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 kinds of 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-compatibi lity 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.EpCAM

EpCAM (CD326) is I type transmembrane glycoprotein, as epithelial specific cell adhesion molecule.It also relates to some other process, comprises cell migration, propagation, differentiation etc.EpCAM is one of tumor associated antigen of identifying of using monoclonal antibody technology the earliest, and it is with polymeric form wide expression in epithelium surface, and mediation calcium independence cell syndio form adhesive function, can be included into adhesion molecule family accordingly.EpCAM also possesses other characteristics of adhesion molecule family, participates in the various procedures such as interaction, migration, cytodifferentiation, form, Cycle Regulation, intracellular signaling, metabolism comprising cell and matrix.Meanwhile, EpCAM in process LAN, points out itself and tumour closely related in the tumour of multiple epithelial origin.Under pathologic condition, EpCAM in various degree be expressed in gland cancer, comprise colorectal cancer, adenocarcinoma of stomach, mammary cancer, ovarian cancer, adenocarcinoma of lung, prostate cancer, carcinoma of the pancreas and hepatocellular carcinoma and retinoblastoma.Multinomial research has confirmed the propagation of the expression of EpCAM and mammary cancer and colon cancer cell, period profile, transfer relevant (see table 1).Monospecific anti-EpCAM monoclonal antibody (MAB), such as 17-1A MAB (glaxowellcome, Centocor), it is the assisting therapy of first German EpCAM targeted therapy colorectal cancer of approval, but, a large amount of clinical application data presentation, this Mono-specific antibodies does not have significantly more useful effect compared with chemotherapy.At present, some other EpCAM targeted therapy, comprise bi-specific antibody, developing into the direction of cancer therapy, bi-specific antibody MT110 and Catumaxomab is the therapeutic double antibody medicine for tumour antigen EpCAM, wherein Catumaxomab was ratified to be used for the treatment of pernicious cancer ascites in 2009 by European Union, and MT110 in clinical studies.Obviously, EpCAM has become one of focus target of current oncotherapy research.

Table 1 EpCAM tumour distribution widely

Tumour	EpCAM positive rate
		Ovarian cancer	88-100％
Cancer of the stomach	98％
		Colorectal carcinoma	99％
Carcinoma of the pancreas	96％
		Mammary cancer	90％
Carcinoma of endometrium	91-96％
		Lung cancer	87％
Prostate cancer	98％

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 is combined to the special target spot of sick cell by it, thus stimulating immune system kills and wounds 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, prepares bispecific monoclonal antibody, and this is bispecific monoclonal antibody concept the earliest, and the shortcoming of this sample preparation method is apparent.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, to improve the effect of combined therapy of tumour.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 EpCAM, CD20, CD30 and CD133, and more preferably this TCSA is EpCAM; (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 EpCAM; The heavy chain constant domain CH1 of described heavy chain and heavy-chain variable domains also target in tumour antigen epi-position EpCAM; 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 CD3, and unit price unit comprises the anti-EpCAM of antibody for EpCAM.

In one embodiment, the aminoacid sequence of the heavy chain of the anti-EpCAM of antibody is the aminoacid sequence shown in sequence number 1, the aminoacid sequence of the light chain of the anti-EpCAM 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-EpCAM heavy chain on 223 sites is connected with the form of disulfide linkage with the halfcystine on light chain 220 site of anti-EpCAM, described anti-EpCAM 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-EpCAM heavy chain on 395 with 412 sites with 428 and 397 sites of anti-CD3ScFv-Fc form salt bridge be connected, described anti-EpCAM 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 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-EpCAM antibody, its light chain the primer that increases is Kozak (EcoR V) F, MK-leader sequence (EcoRV) F, M701-VL F1 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 sequence (AvrII) F, M701-VH F1 and hIgG1 (sbfI) R, increased by over-lap PCR, Kozak sequence, leader sequence and restriction enzyme site AvrI I and BstZl7I are introduced heavy chain; The LC gene fragment increased with carry out homologous recombination with the pCHO1.0 expression vector that EcoR V and PacI enzyme cut through, acquisition loading anti-EpCAM 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-EpCAM, the anti-EpCAM-HL-KKW of plasmid called after pCHO1.0-;

Described strand unit is anti-CD3ScFv-Fc antibody, its the primer that increases is Kozak (Avr II) F, MK-leader sequence (AvrI I) F, L2K-VH (MK) F1 and hIgG1 (sbfI) R, by over-lap PCR amplification AntiCD3 McAb ScFv-Fc structural domain, and by Kozak sequence, leader sequence and restriction enzyme site AvrI I and BstZl7I introduce ScFv-Fc, the pCHO1.0-Totomycin expression vector gene fragment increased and enzyme cut through carries out homologous recombination, obtain the expression vector loading 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 EpCAM specific antigen express caused by tumour or relative disease, or express EpCAM 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 double antibody medicine of the tumour cell relative disease of expressing EpCAM specific antigen or evaluation be used for the treatment of the double antibody medicine of the tumour cell relative disease of expressing EpCAM 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 EpCAM.

The invention provides a kind of novel method and prepare bi-specific antibody MSBODY (monomer and ScFvbispecific 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 EpCAM and CD3, be named as EpCAMX CD3, as Fig. 2, anti-EpCAM is here IgG form, comprise anti-EpCAM 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 two kinds of plasmid co-transfections of single aggressiveness Ab heavy chain, single aggressiveness Ab light chain and strand (Single chain) 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.

Present invention also offers the method for tumor model foundation and Composition analyzed.The foundation of pharmacophore model and Composition analyzed, main is exactly the people's CIK cell mixing utilizing people source tumor stabilisation clone and the separation and Culture expressing people source EpCAM, tumour is formed efficiently in the Mice Body of immune deficiency, and utilize the Mediated by Bi-specific Antibodies built, it can simultaneously in conjunction with tumour cell, express the T cell of CD3 and the immune accessory cell of energy and Fc combination, a bridge block is set up between immunocyte and tumour cell, form an immunocomplex, there is strong immune response in immunocyte, secretion cytokine profiles, tumour cell is killed and wounded, thus the growth of Tumor suppression.This model carries out in the immunity system of a simulation, immunocyte is utilized to kill and wound tumour, and the drug effect of the effective reaction double specific antibody mediated immunity cell killing tumour cell of energy, for the bispecific antibody drug exploitation of target immunocyte and tumour cell provides a fine pharmacodynamic assessment method.

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

1. the invention discloses foundation and the application thereof of the animal model of the structure of a kind of novel bispecific antibodies MSBODY and the immunocyte killing tumor cell of its mediation.The present invention includes the preparation of institute's mediated immunity cell killing, bi-specific antibody in bispecific antibody drug research process, and the foundation of bi-specific antibody pharmacophore model and detection.Bi-specific antibody MSBODY comprises one group heavy light chain combination, another group is then for ScFv connects Fc combination, the wherein tumor-cell antigen of one group of a kind of people of specific combination, comprise a series of tumour cell film surface antigens such as EpCAM, 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 the another kind of mouse 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.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, therefore, have broad application prospects in the immunotherapy of tumour.

2. 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 effectively 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 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 ".

Surprisingly, the application proves that this asymmetrical antibody is stable and has high antigen joint efficiency.This is unexpected, 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, apparent, 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 .EpCAM × CD3 bi-specific antibody molecule schematic diagram.

Fig. 3. electrophoresis detection PCR primer figure; M:DL10000 labeled nucleic acid molecule; 1. anti-cd 3 antibodies ScFv-Fc; 2. anti-EpCAM heavy chain of antibody; 3. anti-EpCAM light chain of antibody.

Fig. 4. the double antibody electrophoresis of purifying and purity detecting result figure; (4A) sex change SDS-PAGE electrophoresis, M: protein markers; 1:EpCAM × CD3 double antibody; (4B) non denatured SDS-PAGE electrophoresis, M: protein markers; 1:EpCAM × CD3 double antibody; (4C) the HPLC-SEC purity peak shape figure of EpCAM × CD3.

Fig. 5. the EpCAM × CD3 double antibody measured based on flow cytometric analysis and the avidity situation map of HCT116 cell, (■) EpCAM × CD3 MSBODY; (●) Anti-EpCAM monoclonal antibody.

Fig. 6. the EpCAM × CD3 double antibody measured based on flow cytometric analysis and the avidity situation map of Jurkat cell, (■) EpCAM × CD3 MSBODY; (●) Anti-CD3 monoclonal antibody L2K.

Fig. 7. flow cytometer detection EpCAM × CD3 double antibody to move to Jurkat cell and 2 kinds of cells in conjunction with HCT116 simultaneously together with result figure; (●) is EpCAM × CD3 double antibody; (■) EpCAM monoclonal antibody body; (▲) anti-CD49d McAb body L2K; (▼) control antibodies MCO101.

Fig. 8. the Tm value result figure of differential scanning calorimeter sweep measurement EpCAM × CD3 MSBODY double antibody.

Fig. 9. antibody is Activity determination result after Overheating Treatment, and 9A. and EpCAM binding activities detects, (●) anti-EpCAM monoclonal antibody; (▲) EpCAM × CD3 MSBODY double antibody; 9B. and CD3 binding activities detects, (●) anti-CD3 monoclonal antibody L2K; (■) EpCAM × CD3 MSBODY double antibody.

Figure 10 .CIK Phenotypic examination result figure, the two positive NK class cell of the CD3 in the upper right corner, CD56.

Figure 11. under flow cytometer detection different concns antibody existence condition, effector cell CIK is to the lethal effect result figure of target cell HCT116; (■) EpCAM × CD3 MSBODY double antibody, (▲) Mco101: contrast 4420 × CD3 double antibody, (▼) Anti-EpCAM: anti-EpCAM monoclonal antibody, (●) hIgG: human IgG.EpCAM×CD3

Figure 12. under flow cytometer detection different concns antibody existence condition, effector cell CIK is to the lethal effect result figure of target cell NCI-N87; (■) EpCAM × CD3 MSBODY double antibody, (▲) Mco101: contrast 4420 × CD3 double antibody, (▼) Anti-EpCAM: anti-EpCAM monoclonal antibody, (●) hIgG: human IgG.

Figure 13. under flow cytometer detection different concns antibody existence condition, effector cell PBMC is to the lethal effect result figure of target cell HCT116; (■) EpCAM × CD3 MSBODY double antibody, (▲) Mco101: contrast 4420 × CD3 double antibody, (▼) EpCAM:EpCAM monoclonal antibody, (●) hIgG: human IgG.

Figure 14. under flow cytometer detection different concns antibody existence condition, effector cell PBMC is to the lethal effect result figure of target cell NCI-N87; (■) EpCAM × CD3 MSBODY double antibody, (▲) Mco101: contrast 4420 × CD3 double antibody, (▼) EpCAM:EpCAM monoclonal antibody, (●) hIgG: human IgG.

Figure 15. effect experiment result in double antibody body, mouse: NOD-SCID; Inoculation (i.h): SW480 (5X10 ⁶) and people CIK (5X10 ⁶) combined inoculation; Administration process (i.v): EpCAM mAb:4mg/kg; Day (0,2,4), 4420 × CD3:4mg/kg; Day (0,2,4), EpCAM × CD3 (MSBODY)-1:4mg/kg; Day (0,2,4), EpCAM × CD3 (MSBODY)-2:2mg/kg; Day (0,2,4).(●) represents PBS, only tail vein is to PBS, () Anti-EpCAM monoclonal antibody, (△) 4420 × CD3 unrelated control double antibody, (▼) M701-1:EpCAM × CD3SMBODY double antibody 4mg/kg concentration group (zero) M701-2:EpCAM × CD3SMBODY double antibody 2mg/kg concentration group.

Embodiment

Below in conjunction with specific embodiment, and in further detail the present invention is described with reference to accompanying drawing.Should be understood that the embodiment in present disclosure is in order to demonstrate the invention, but not limit the scope of the invention by any way.

Embodiment 1: the expression vector establishment (EpCAM × CD3, M701) of bi-specific antibody

1. bi-specific antibody sequences Design

Be named as M701 with the bi-specific antibody that EpCAM and CD3 is target spot, as Fig. 2, anti-EpCAM is here IgG form, comprises anti-EpCAM heavy chain and light chain, containing Fab and Fc structural domain; AntiCD3 McAb is here ScFv-Fc form, comprises AntiCD3 McAb VH, VL, Fc structural domain.Wherein IgG form one side Fc carries out KKW transformation, ScFv-Fc on one side Fc carries out LDY transformation, and concrete Fc transformation process, see PCT/CN2012/084982, makes it not easily form homodimer separately, and be easy to be formed heterozygosis dimer, i.e. EpCAM × CD3 bi-specific antibody., in order to dual anti-physical efficiency is expressed in Chinese hamster ovary celI, and can be secreted in substratum meanwhile, have selected the leader peptide sequences of mouse 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.

Anti-EpCAM heavy chain (aminoacid sequence sequence number 1)

EVQLLEQSGAELVRPGTSVKISCKASGYAFTNYWLGWVKQRPGHGLEWIGDIFPGSGNIHYNEKFKGKATLTADKSSSTAYMQLSSLTFEDSAVYFCARLRNWDEPMDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Anti-EpCAM heavy chain (nucleic acid sequences number 2)

GAGGTGCAGCTGCTCGAGCAGTCTGGAGCTGAGCTGGTAAGGCCTGGGACTTCAGTGAAGATATCCTGCAAGGCTTCTGGATACGCCTTCACTAACTACTGGCTAGGTTGGGTAAAGCAGAGGCCTGGACATGGACTTGAGTGGATTGGAGATATTTTCCCTGGAAGTGGTAATATCCACTACAATGAGAAGTTCAAGGGCAAAGCCACACTGACTGCAGACAAATCTTCGAGCACAGCCTATATGCAGCTCAGTAGCCTGACATTTGAGGACTCTGCTGTCTATTTCTGTGCAAGACTGAGGAACTGGGACGAGCCTATGGACTACTGGGGCCAAGGGACCACGGTCACCGTCTCCTCCGCGTCGACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTCTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACGATACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCGATCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA

Anti-EpCAM light chain (aminoacid sequence sequence number 3)

ELVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDYSYPLTFGAGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Anti-EpCAM light chain (nucleic acid sequences number 4)

GAGCTCGTGATGACACAGTCTCCATCCTCCCTGACTGTGACAGCAGGAGAGAAGGTCACTATGAGCTGCAAGTCCAGTCAGAGTCTGTTAAACAGTGGAAATCAAAAGAACTACTTGACCTGGTACCAGCAGAAACCAGGGCAGCCTCCTAAACTGTTGATCTACTGGGCATCCACTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGAACAGATTTCACTCTCACCATCAGCAGTGTGCAGGCTGAAGACCTGGCAGTTTATTACTGTCAGAATGATTATAGTTATCCGCTCACGTTCGGTGCTGGGACCAAGCTTGAGATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG

AntiCD3 McAb ScFv-Fc (aminoacid sequence sequence number 5)

QVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSGGGGSGGGGSGGGGSQIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYDTSKLASGVPAHFRGSGSGTSYSLTISGMEAEDAATYYCQQWSSNPFTFGSGTKLEINRGAAAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

AntiCD3 McAb ScFv-Fc (nucleic acid sequences number 6)

CAGGTGCAGCTGGTGCAGAGCGGCGGCGGCGTCGTGCAGCCGGGCAGGTCCCTGAGACTGTCTTGTAAGGCTTCTGGATACACCTTCACTAGATACACAATGCACTGGGTCAGACAGGCTCCTGGAAAGGGACTCGAGTGGATTGGATACATTAATCCTAGCAGAGGTTATACTAACTACAATCAGAAGTTTAAGGACAGATTCACAATTTCTACTGACAAATCTAAGAGTACAGCCTTCCTGCAGATGGACTCACTCAGACCTGAGGATACCGGAGTCTATTTTTGTGCTAGATATTACGATGACCACTACTGTCTGGACTACTGGGGCCAAGGTACCCCGGTCACCGTGAGCTCAGGAGGCGGCGGTTCAGGCGGAGGTGGAAGTGGTGGAGGAGGTTCTGATATTCAGATGACCCAGAGCCCGTCAAGCTTATCTGCTTCTGTCGGAGACAGAGTCACAATCACATGTTCTGCTTCTAGCTCTGTCTCTTACATGAACTGGTATCAGCAGACACCTGGAAAGGCTCCTAAGCGGTGGATCTACGACACATCTAAGCTCGCTTCTGGAGTCCCTTCTAGATTCTCTGGTTCTGGCTCTGGAACAGACTACACATTCACAATCTCTTCTCTCCAACCTGAGGACATCGCTACATACTACTGCCAACAGTGGTCTAGCAATCCTTTCACATTCGGACAGGGTACCAAACTGCAGATCACAAGAGGTGCGGCCGCAGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA

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

METDTLLLWVLLLWVPGSTG

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

atggagacagacacactcctgctatgggtactgctgctctgggttccaggttccactggt

2. bi-specific antibody gene clone

PCHO1.0 is selected to remove heavy chain and the light chain gene of cloning and expressing anti-EpCAM 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 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 anti-EpCAM 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

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; 10 × 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, sees Fig. 3; And Kozak sequence, leader sequence and restriction enzyme site AvrII and BstZl7I introducing heavy chain is shown in Fig. 3 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, acquisition loading anti-EpCAM 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-EpCAM, the anti-EpCAM-HL-KKW of plasmid called after pCHO1.0-.

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 that the gene fragment increased (Fig. 3) and enzyme cut through is carried 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 CHO substratum (Gibco, 10743-029), 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 by anti-for plasmid pCHO1.0-EpCAM-HL-KKW and pCHO1.0-Totomycin-L2K-ScFv-Fc-LDY together cotransfection in CHO-S cell, these two kinds of plasmids of design cotransfection are to express the bi-specific antibody M701 to EpCAM × CD3.

The 2nd day after transfection, culture temperature was lowered to 32 DEG C, and every day adds 3.5%FeedA, cultivated after 14 days, and 800*g 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, 18-1153-45,17-5438-01) 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 (18-1153-44,17-1087-01), 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 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 HCT116 (purchased from ATCC, CCL-247) as the cell of the EpCAM positive, and Jurkat (Jurkat, 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 HCT116 cell

Cultivate enough HCT116 cells, with 0.25% trysinization, centrifugal collecting cell.Dilute bi-specific antibody, concentration is from 10ug/ml, and 10 times of gradient dilutions, obtain 12 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 HCT116 with software GraphPad Prism5.0.The HCT116 cell of result display EpCAM × CD3 double antibody and the EpCAM positive has good binding activities, and see Fig. 5, its KD value is the KD detected result of 9.602nM, Anti-EpCAM is 1.661nM.

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, by cell resuspended for 100ul PBS, and upper machine testing, then 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 EpCAM × CD3 double antibody and the CD3 positive has good binding activities, and see Fig. 6, its KD value is 14.27nM, shows good avidity.

3. the immunocyte of double antibody mediation and the common Binding experiment of tumour cell

By cultured HCT116 and Jurkat cell, collected by centrifugation also washes 2 times with PBS, respectively with CFSE and PKH-26 dyeing.Dilute bi-specific antibody, concentration is from 10ug/ml, and 10 times of gradient dilutions, obtain 12 concentration gradients, for subsequent use simultaneously.By the HCT116 dyeed with Jurkat cell is centrifugal removes supernatant, wash twice with PBS+1%FBS, then add PBS+1%FBS re-suspended cell to 4 × 10 ⁶individual cell/ml, mixes by 1:1, by plating cells in 96 orifice plates, and every hole 50ul (2 × 10 ⁵individual cell), add the bi-specific antibody that 50ul has diluted, incubated at room 1 hour; Centrifugally removing supernatant, wash cell twice with PBS, finally use 100ulPBS resuspended, upper machine testing, analyzing the ratio of two positive cell, by carrying out analytical calculation with software GraphPad Prism5.0.Result when show there is no a M701, the ratio very low (Fig. 7) of the two fluorescence of flow cytometer detection; When adding EpCAM × CD3 double antibody M701, the ratio of the two fluorescence of flow cytometer detection reaches 27.5%, show that M701 simultaneously in conjunction with the HCT116 cell of the EpCAM positive and the Jurkat cell of the CD3 positive, can promote the aggregation of two kinds of cells, form an immunologic cytotoxicity complex body.

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

1. the Tm pH-value determination pH of bi-specific antibody

The thermostability of bi-specific antibody is by Differential Scanning Calorimetry instrument (MicroCal VP-DSC, GE company) measure, double antibody Sample Purification on Single rear substitution is in PBS damping fluid, with PBS damping fluid in contrast, calorimetric scan data are carried out scanning with the heating rate of 60 DEG C/h from 10 DEG C to 100 DEG C and are obtained.Scanning result (Fig. 8) shows, and the Tm value of bi-specific antibody, all at about 70 DEG C, shows good thermostability.

2. the hot challenge experiment of bi-specific antibody

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., Anti-tumor activity of stability-engineered IgG-like bispecific antibodiestargeting TRAIL-R2and LTbetaR.MAbs.2009Mar-Apr; 1 (2): 128-41.].Therefore, antibody dilution to 0.4mg/ml, is distinguished 4 DEG C, 37 DEG C, 42 DEG C, 47 DEG C, 52 DEG C, 57 DEG C, 62 DEG C, 67 DEG C, 72 DEG C, 77 DEG C, 82 DEG C, PCR instrument process 1h, often pipe 15ul by us.Centrifuging and taking supernatant, carries out flow cytometer detection according to following steps, collects single cell suspension and adds 96 orifice plates, 3 × 10 ⁵/ hole, adds various process antibody, and adds fluorescence two and resist, and machine testing in streaming, the results are shown in Figure 9, Fig. 9 A and determine Anti-EpCAM and EpCAM × CD3 MSBODY bi-specific antibody in the thermostability in conjunction with EpCAM, its T ₅₀value be respectively 73.28 and 61.01, Fig. 9 B determine the thermostability of L2K and EpCAM × CD3 MSBODY bi-specific antibody at the antibody in conjunction with CD3, its T ₅₀value is respectively 69.33 and 60.30, all shows good thermostability.

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

The separation of 1.PBMC cell and CIK cell are cultivated

Get fresh anticoagulation, 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 can carry out the subsequent experimental such as counting with after suitable 4 DEG C of PBS re-suspended cells precipitation.

The cultivation of CIK cell, fills 30ml with CIK cell Primary culture liquid (serum-free X-Vivo cell culture fluid+750IU/ml IFN-γ ± 2% autologous plasma) by every part of cell, is added to 75cm ²in culturing bottle, be placed in saturated humidity, 37 DEG C, 5.0%CO ₂incubator is cultivated.Cultivate after 24 hours, add CIK cell stimulating factor mixed solution 1ml (the anti-human CD3 ε of serum-free X-Vivo cell culture fluid+75ng/ml, 750IU/ml IL-2,0.6ng/ml IL-1 α), continue to be placed in saturated humidity, 37 DEG C, 5.0%CO ₂cultivate in incubator.Following step determines the thing of fluid infusion (serum-free X-Vivo nutrient solution+750IU/ml IL-2 ± 2% autologous plasma), sub-bottle according to the growing state of CIK cell, substantially will maintain cell and grow about the concentration of 2*10^6.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.Detected result is shown in Figure 10, and phenotypic results display CIK cell has the two positive of CD3 and CD56 of more than 35%, and cultured cells has the ratio of good NK T cell.

2. double antibody effectively mediates the detection of PBMC cell killing tumour cell

With trysinization HCT116 or NCI-N87 cell, prepare single cell suspension.With final concentration be 5uM CFSE dye HCT116 or NCI-N87 (staining procedure is shown in that protocol-1CFSE dyes), 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 100ul/ hole adds 96 orifice plate overnight incubation.Experimental design adds the CIK cell of cultivation, and 50ul/ hole, arranges control wells, and the substratum without the need to the Kong Zeyong same volume adding CIK cell fills into.Empirically design while adding CIK 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 EP pipe, 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 1ug/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 HCT116 or NCI-N87, the results are shown in Figure 11 and 12, cell killing result display EpCAM × CD3 MSBODY Mediated by Bi-specific Antibodies CIK cell killing tumor cell shows good fragmentation effect, and its maximum killing-efficiency and EC50 are obviously better than Anti-EpCAM monoclonal antibody.

3. double antibody effectively mediates the detection of PBMC cell killing tumour cell

With trysinization HCT116 or NCI-N87 cell, prepare single cell suspension.With CFSE dyeing HCT116 or NCI-N87 (staining procedure is shown in that protocol-1CFSE dyes) that final concentration is 5uM, 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 100ul/ hole adds 96 orifice plate overnight incubation.Experimental design adds the CIK cell of cultivation, and 50ul/ hole, arranges control wells, and the substratum without the need to the Kong Zeyong same volume adding CIK cell fills into.Empirically design while adding CIK 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 EP pipe, 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 1ug/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 HCT116 or NCI-N87.The results are shown in Figure 13 and 14, cell killing result display EpCAM × CD3 MSBODY Mediated by Bi-specific Antibodies PBMC killing tumor cell shows good fragmentation effect, and its maximum killing-efficiency and EC50 are obviously better than Anti-EpCAM monoclonal antibody.

Embodiment 6: bi-specific antibody kills and wounds the Composition analyzed of subcutaneous transplantation knurl

The foundation of Tumor Xenograft Models is by by 5 × 10 ⁶sW480 and 5 × 10 ⁶cIK cell mixes, and forms (N=8 group) in female NOD/SCID mouse right dorsal subcutaneous inoculation growth.In 2 hours, mouse random packet, Antybody therapy group, starts with 2, and 1 and 0.5mg/kg EpCAM × CD3 MSBODY, carry out intravenous injection respectively by tail vein.Control group comprises one group of anti-EpCAM monoclonal antibody with 2mg/kg and one group of nothing to do with control MSBODY (4420 × CD3).Contrast MSBODY is that a kind of anti-fluorescein antibody (4-4-20) sequence construct forms (Kranz DM, Voss EW Jr., Partialelucidation of an anti-hapten repertoire in BALB/c mice:comparativecharacterization of several monoclonal antifluorescyl antibodies.Mol Immunol.1981; 18 (10): 889-898).And continue to continue the identical dosage of administration at the 2nd day with the 4th day.At corresponding control animals intravenous injection PBS.Within every three days, result calculates and adopts following formula to carry out: 1/2 × length x width x width (mm with digital vernier kind of calliper gross tumor volume ³).

Antitumous effect assessment in EpCAM × CD3 MSBODY body is completed by adoptive transfer Transplanted tumor model.Gastric cancer tumor clone SW480 cell is used to set up Transplanted tumor model on immunodeficient mouse NOD/SCID, and people's CIK cell, by after separating peripheral blood mononuclear cells, stimulates to cultivate and gets, 1:1 ratio combined inoculation before inoculation.As Figure 15 display, PBS group, control antibodies Anti-EpCAM, 4420 × CD3 Antybody therapy all to tumor growth without obvious suppression, but EpCAM × CD3 (2mg/kg, 4mg/kg) treatment group does not find tumor growth in same experiment, therefore, the immune tumor-killing that EpCAM × CD3 MSBODY mediates can obviously Tumor suppression growth.Also as expected, even if remain with CD3 specific molecular, but lack during EpCAM specific MSBODY molecule MCO101 (4420 × CD3) tests in vivo and can not demonstrate significant anti-tumor activity.

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.

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 EpCAM, CD20 and CD30, and more preferably this TCSA is EpCAM; (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 scFv fragment and CH3 structural domain; Do not comprise CH1 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 unit price unit, the light chain constant domain of described light chain and light chain variable domain all target in tumour antigen epi-position EpCAM; The heavy-chain constant domains CH1 of described heavy chain and heavy chain variable domain also target in tumour antigen epi-position EpCAM; Described light chain is combined with described heavy chain by disulfide linkage; Described heavy chain is combined with described fusogenic peptide by one or more disulfide linkage.

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

Preferably, the aminoacid sequence of described anti-EpCAM heavy chain is the aminoacid sequence shown in sequence number 1, the aminoacid sequence of the light chain of anti-EpCAM is the aminoacid sequence shown in sequence number 3, and the aminoacid sequence of described anti-CD3 ScFv-Fc is the aminoacid sequence shown in sequence number 5, and the halfcystine of anti-EpCAM heavy chain on 223 sites is connected with the form of disulfide linkage with the halfcystine on light chain 220 site of anti-EpCAM, described anti-EpCAM heavy chain is connected with the form of disulfide linkage with the halfcystine on 255 and 258 sites of anti-CD3 ScFv-Fc with the halfcystine on 232 sites respectively 229, described anti-EpCAM heavy chain on 395 with 412 sites with 428 and 397 sites of anti-CD3 ScFv-Fc form salt bridge be connected, described anti-EpCAM heavy chain on 369 sites with 436 sites of anti-CD3 ScFv-Fc are formed knuckle-enter-cave is connected.

6. bi-specific antibody according to claim 1, is characterized in that: the heavy chain in described 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.

7. bi-specific antibody according to claim 6, is characterized in that: 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.

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

9. method according to claim 8, the first described expression vector is pCHO1.0; The second described 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-EpCAM antibody, its light chain the primer that increases is Kozak (EcoR V) F, MK-leader sequence (EcoRV) F, M701-VL F1 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 sequence (AvrII) F, M701-VH F1 and hIgG1 (sbfI) R, increased by over-lap PCR, Kozak sequence, leader sequence and restriction enzyme site AvrII and BstZl7I are introduced heavy chain; The LC gene fragment increased with carry out homologous recombination with the pCHO1.0 expression vector that EcoR V and PacI enzyme cut through, acquisition loading anti-EpCAM 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-EpCAM, the anti-EpCAM-HL-KKW of plasmid called after pCHO1.0-;

Described strand unit is anti-CD3 ScFv-Fc antibody, its the primer that increases is Kozak (AvrII) F, MK-leader sequence (AvrII) F, L2K-VH (MK) F1 and hIgG1 (sbfI) R, by over-lap PCR amplification AntiCD3 McAb ScFv-Fc structural domain, and by Kozak sequence, leader sequence and restriction enzyme site AvrII and BstZl7I introduce ScFv-Fc, the pCHO1.0-Totomycin expression vector gene fragment increased and enzyme cut through carries out homologous recombination, obtain the expression vector loading AntiCD3 McAb ScFv-Fc, plasmid called after pCHO1.0-Totomycin-L2K-ScFv-Fc-LDY.

Bi-specific antibody according to any one of 11. claim 1-7 or preparing the purposes in medicine according to bi-specific antibody prepared by the method for the bi-specific antibody prepared any one of claim 8-10, described medicine be used for the treatment of EpCAM specific antigen express caused by tumour or relative disease, or express EpCAM 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 double antibody medicine of the tumour cell relative disease of expressing EpCAM specific antigen for the double antibody medicine or evaluation screening the tumour cell relative disease being used for the treatment of expression EpCAM specific antigen in tumor cell line.