CN104558191A - Construction and application of bispecific antibody CD20*CD3 - Google Patents
Construction and application of bispecific antibody CD20*CD3 Download PDFInfo
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Abstract
The invention provides a bispecific antibody. The bispecific antibody comprises a single-chain unit and a monovalent unit, wherein the single-chain unit has a specific binding capability against the surface antigen CD3 of an immune cell, and the monovalent unit has a specific binding capability against the surface antigen CD20 of a tumor cell; and the single-chain unit comprises a single-chain variable fragment (ScFv) which is fused with an Fc fragment, and the monovalent unit comprises light chain and heavy chain pairs. The invention further provides a method for preparing the bispecific antibody and the medicinal application of the antibody.
Description
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 (bispecificantibody, BiAb) is the artificial antibody containing two species-specific antigen binding sites, can erect bridge between target cell and functional molecular (cell), produces the effector function of guidance quality.BiAb, in biomedicine, particularly has broad application prospects in the immunotherapy of tumour.The focus that tumour cell is current immunotherapy applied research is killed by BiAb mediated cell toxic action, its principal feature is BiAb can answer on cell simultaneously target molecule in conjunction with tumor associated antigen and immunity, 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 18.9k Da, 23.1kDa, 20.5k Da, 18.7k Da, 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 (immunoreceptortyrosine-based activation motif, ITAM), TCR identifies and combines the antigen peptide of being offered by MHC (majorhisto-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 (Scrhomology 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.CD20
CD20 is the specific significant molecule on bone-marrow-derived lymphocyte surface, be expressed in more than 95% normal or malignant B surperficial, without the expression of CD20 antigen in hemopoietic stem cell, progenitor cell and other healthy tissuess.It is made up of 297 amino acid, and molecular weight is 33kD, and 34 ~ 36kD belongs to non-glycosylated phosphoprotein, and its epitope is the ring district be made up of 43 amino acid in third and fourth cross-film interval.Obvious internalization phenomenon not occurring after CD20 is combined with corresponding antibodies, obvious cell surface obscission does not occur yet, is the promising target for the treatment of bone-marrow-derived lymphocyte relative disease.Research shows, CD20 has the function of calcium channel, by regulating the concentration of intracellular calcium, affects the cell cycle, regulates cell proliferation and differentiation, even causes apoptotic generation.There are two kinds of monoclonal antibody drug Mabtheras for CD20 target spot and Ao Fa wood at present, its mechanism of action mainly contains complement-dependent cytotoxicity, Antibody-dependent cell cytotoxicity effect and improves the susceptibility of cell by cell toxic action, and Immunization etc. also all cause the apoptosis of B cell in addition.Because original normal B cells is not by the effect of IDEC-C2B8, even if after the lymphoma cell having killed and wounded all expression CD20 molecules at IDEC-C2B8 and normal B cells, still can rebuild B cell group, therefore anti-CD-20 monoclonal antibody is also more and more is applied in the treatment of B cell relative disease.
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, described antibody 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 CD20, EPCAM and CD30, and more preferably this TCSA is CD20; (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 CD20; The heavy chain constant domain CH1 of described heavy chain and heavy-chain variable domains also target in tumour antigen epi-position CD20; 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 antibody anti-CD 20 for CD20.
In one embodiment, the aminoacid sequence of the heavy chain of antibody anti-CD 20 is the aminoacid sequence shown in sequence number 1, the aminoacid sequence of the light chain of antibody anti-CD 20 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-CD 20 heavy chain on 224 sites is connected with the form of disulfide linkage with the halfcystine on light chain 213 site of anti-CD 20, described anti-CD 20 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 233 sites respectively 230, described anti-CD 20 heavy chain on 396 with 413 sites with 428 and 397 sites of anti-CD3 ScFv-Fc form salt bridge be connected, described anti-CD 20 heavy chain on 370 sites with 436 sites of anti-CD3 ScFv-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-CD 20 antibodies, its light chain the primer that increases is Kozak (EcoR V) F, MK-leader sequence (EcoRV) F, Rituxan-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, Rituxan-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, the expression vector of acquisition loading anti-CD 20 light chain; Then carry out homologous recombination with HC again after cutting with AvrI I and BstZl7I enzyme, obtain the pCHO1.0 expression vector of anti-CD 20, plasmid called after pCHO1.0-anti-CD 20-HL-LDY;
Described strand unit is anti-CD3 ScFv-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, 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-KKW.
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 CD20 specific antigen express caused by tumour or relative disease, or express CD20 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 expressing the medicine screening in CD20 tumor cell line and be used for the treatment of the tumour cell relative disease of expressing CD20 specific antigen, or evaluates the drug effect of the medicine being used for the treatment of the tumour cell relative disease of expressing CD20 specific antigen.
Present invention also offers following technical scheme:
The invention provides a kind of novel method and prepare bi-specific antibody MSBODY (ScFv and monomerbispecific 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 CD20 and CD3, be named as CD20 × CD3, as Fig. 2, anti-CD 20 be here IgG form, comprises anti-CD 20 heavy chain and light chain, and anti-CD3 this side is ScFv-Fc form, comprises anti-CD3 VH, 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.
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 immunocyte killing tumor cell that a kind of novel bispecific antibodies MSBODY mediates.The present invention includes that the immunocyte mediated in bispecific antibody drug research process kills and wounds, the preparation of bi-specific antibody, set up the detection of pharmacophore model and bi-specific antibody drug effect with the tumor cell line of expressing h CD20 albumen.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 CD20, 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 people 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), excites the immune response with guidance quality, has 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 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 ".
Surprisingly, the application proves that this asymmetrical antibody is stable and has high antigen joint efficiency.This makes us feeling surprised, even because the homodimer of verified single-chain antibody is in physiological conditions all unstable.Such as, Ahmad etc. " ScFv Antibody:Principles and ClinicalApplication; " Clinical and Developmental Immunology, 2012:980250 (2012), the 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 .CD20 × CD3 bi-specific antibody molecule schematic diagram.
Fig. 3. electrophoresis detection PCR primer figure, M:DL10000 labeled nucleic acid molecule; 1. anti-CD20 antibodies heavy chain; 2. anti-CD20 antibodies light chain.
Fig. 4. electrophoresis detection PCR primer figure, M:DL2000 labeled nucleic acid molecule; 1. anti-cd 3 antibodies ScFv-Fc.
Fig. 5. the double antibody electrophoresis of purifying and purity detecting figure, (A) CD20 × CD3 double antibody SDS-PAGE electrophoresis, M: protein markers; 1: non-reduced SDS-PAGE electrophoresis detection; 2: reduction SDS-PAGE electrophoresis detection; (B) the HPLC-SEC purity peak shape figure of CD20 × CD3.
Fig. 6. the CD20 × CD3 double antibody measured based on flow cytometric analysis and the affine of Raji cell are tried hard to; (●) Rituxan contrasts monoclonal antibody, (■) CD20 × CD3 bi-specific antibody.
Fig. 7. the CD20 × CD3 double antibody measured based on flow cytometric analysis and the affine of Jurkat cell are tried hard to; (●) L2K contrasts monoclonal antibody, (■) CD20 × CD3 bi-specific antibody.
Fig. 8. flow cytometer detection CD20 × CD3 double antibody simultaneously in conjunction with Raji and Jurkat cell, and promotes that 2 kinds of cells combine altogether; (▲) M903:CD20 × CD3 double antibody.
Fig. 9. the Tm value result figure of differential scanning calorimeter sweep measurement CD20 × CD3 double antibody.
Figure 10 .CD20 × CD3 double antibody after Overheating Treatment with Raji cell surface CD20 binding activities detected result figure, (●) CD20 × CD3 double antibody; (■) Rituxan monoclonal antibody.
Figure 11 .CD20 × CD3 double antibody detects with Jurkat cell surface C D3 binding activities after Overheating Treatment schemes, (▲) CD20 × CD3 double antibody; (▼) Anti-CD3 monoclonal antibody L2K.
Figure 12 .CIK Phenotypic examination result figure, the two positive NK class cell of the CD3 in the upper right corner, CD56.
Figure 13. under flow cytometer detection different concns antibody existence condition, effector cell CIK is to the lethal effect result figure of target cell Raji; (■) M903:CD20 × CD3 (all forms should be consistent) double antibody, (▲) Mco101: contrast 4420 X × CD3 double antibodies, (▼) Rituxan:CD20 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 Raji; (■) M903:CD20 × CD3 double antibody, (▲) Rituxan:CD20 monoclonal antibody, (▼) Mco101: contrast 4420 X × CD3 double antibodies, (●) hIgG: human IgG.
Figure 15. effect experiment result figure in double antibody body, after (A) represents inoculated tumour cell, only tail vein is to substratum, and (B) tail vein is to T cell, and (C) tail vein injection bag is by the T cell of CD20x × CD3 double antibody.
Embodiment
Embodiment 1: the expression vector establishment (CD20 × CD3, M903) of bi-specific antibody
1. bi-specific antibody sequences Design
Be named as M903 with the bi-specific antibody that CD20 and CD3 is target spot, as Fig. 2, anti-CD20 is here IgG form, comprises anti-CD 20 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 LDY transformation, ScFv-Fc on one side Fc carries out KKW 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. CD20 × 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 SEQ ID NO:1-8.
Anti-CD 20 heavy chain amino acid sequence (sequence number 1)
QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCRVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLASKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
Anti-CD 20 heavy chain nucleic acid sequence (sequence number 2)
caggtacaactgcagcagcctggggctgagctggtgaagcctggggcctcagtgaagatgtcctgcaaggcttctggctacacatttaccagttacaatatgcactgggtaaaacagacacctggtcggggcctggaatggattggagctatttatcccggaaatggtgatacttcctacaatcagaagttcaaaggcaaggccacattgactgcagacaaatcctccagcacagcctacatgcagctcagcagcctgacatctgaggactctgcggtctattactgtgcaagatcgacttactacggcggtgactggtacttcaatgtctggggcgcagggaccacggtcaccgtctctgcagcctccaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaaagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgccgggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctgaagtccgacggctccttcttcctcgccagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaa
Anti-CD 20 light-chain amino acid sequence (sequence number 3)
QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
Anti-CD 20 light chain nucleic acid sequence (sequence number 4)
caaattgttctctcccagtctccagcaatcctgtctgcatctccaggggagaaggtcacaatgacttgcagggccagctcaagtgtaagttacatccactggttccagcagaagccaggatcctcccccaaaccctggatttatgccacatccaacctggcttctggagtccctgttcgcttcagtggcagtgggtctgggacttcttactctctcacaatcagcagagtggaggctgaagatgctgccacttattactgccagcagtggactagtaacccacccacgttcggaggggggaccaagctggaaatcaaacgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgt
AntiCD3 McAb ScFv-Fc aminoacid sequence (sequence number 5)
DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSGGGGSGGGGSGGGGSDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKGAAAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
AntiCD3 McAb ScFv-Fc nucleotide sequence (sequence number 6)
gatatcaaactgcagcagtcaggggctgaactggcaagacctggggcctcagtgaagatgtcctgcaagacttctggctacacctttactaggtacacgatgcactgggtaaaacagaggcctggacagggtctggaatggattggatacattaatcctagccgtggttatactaattacaatcagaagttcaaggacaaggccacattgactacagacaaatcctccagcacagcctacatgcaactgagcagcctgacatctgaggactctgcagtctattactgtgcaagatattatgatgatcattactgccttgactactggggccaaggcaccactctcacagtctcctcaggaggcggcggttcaggcggaggtggaagtggtggaggaggttctgacattcagctgacccagtctccagcaatcatgtctgcatctccaggggagaaggtcaccatgacctgcagagccagttcaagtgtaagttacatgaactggtaccagcagaagtcaggcacctcccccaaaagatggatttatgacacatccaaagtggcttctggagtcccttatcgcttcagtggcagtgggtctgggacctcatactctctcacaatcagcagcatggaggctgaagatgctgccacttattactgccaacagtggagtagtaacccgctcacgttcggtgctgggaccaagctggagctgaaaggtgcggccgcagagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgtggtgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacgataccacgcctcccgtgctggactccgacggctccttcttcctctacagcgatctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga
The leader peptide sequences aminoacid sequence (sequence number 7) of mouse kappa
METDTLLLWVLLLWVPGSTG
The leader peptide sequences nucleotide sequence (sequence number 8) of mouse kappa
atggagacagacacactcctgctatgggtactgctgctctgggttccaggttccactggt
2. bi-specific antibody gene clone
PCHO1.0 is selected to remove heavy chain and the light chain gene of the anti-CD20 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 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-CD 20 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; 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 (Fig. 3); And Kozak sequence, leader sequence and restriction enzyme site AvrII and BstZl7I are introduced heavy chain (see 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, the expression vector of acquisition loading anti-CD 20 light chain; Then carry out homologous recombination with HC again after cutting with AvrII and BstZl7I enzyme, obtain the pCHO1.0 expression vector of anti-CD 20, plasmid called after pCHO1.0-anti-CD 20-HL-LDY.
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 (see Fig. 4) 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-KKW.
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, cotransfection is in CHO-S cell together with pCHO1.0-Totomycin-L2K-ScFv-Fc-KKW by plasmid pCHO1.0-anti-CD 20-HL-LDY to use Maxcyte STX electroporation, and these two kinds of plasmids of design cotransfection are to express the bi-specific antibody M903 to CD20 × 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, pH6.0) balance chromatography column after, sample, with between two pure water dilution conductance to 3.0-3.5ms, is crossed after SP pillar combines, with elution buffer B (43.8mM NaH
2pO
4+ 6.2mM Na
2hPO
4+ 1M NaCl, pH6.0) 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. 5.
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 Raji (purchased from ATCC, CCL-86) as the cell of the CD20 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 Raji cell
Cultivate enough Raji cells, centrifugal collecting cell.Dilute bi-specific antibody, concentration is from 1000nmol, and 3 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 Raji with software GraphPadPrism5.0.The Raji cell of result display CD20 × CD3 double antibody and the CD20 positive has good binding activities, and see Fig. 6, its KD value is 274.5nM.
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 CD20 × CD3 double antibody and the CD3 positive has good binding activities, and see Fig. 7, its KD value is 571.3nM.
3. the common binding activities of double antibody mediation detects
By cultured Raji 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 Raji 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.The dual anti-physical efficiency of result display CD20 × CD3, simultaneously in conjunction with the Raji cell of the CD20 positive and the Jurkat cell of the CD3 positive, forms the cell conjugate of two fluorescence, sees Fig. 8, and result display relies on antibody concentration and changes, and ceiling rate reaches about 15%.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 calorimeter (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 display (Fig. 9), the Tm value of bi-specific antibody, all more than 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 the quality (MichaelsonJS1 of antibody drug, etc., Anti-tumor activity of stability-engineered IgG-like bispecificantibodies targeting TRAIL-R2 and LTbetaR.MAbs.2009 Mar-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 10 and 11, result display CD20 × CD3 double antibody T that CD20 is combined on Raji cell
50value is 60.69; The T that CD3 is combined in Jurkat cell
50value is 58.99, all show 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 (serum-free X-Vivo cell culture fluid+75ng/ml Anti-human CD3 ε, 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 12, and phenotypic results display CIK cell has the two positive of CD3 and CD56 of 18.1%, and cultured cells has the ratio of good NK T cell.
2. double antibody effectively mediates the detection of CIK cell killing tumor cell
Collect Raji single cell suspension, with CFSE dyeing (staining procedure is shown in that protocol-1 CFSE 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.Take out 96 orifice plates after 48h, 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 Raji.Detected result is shown in Figure 13, and cell killing result display CD20 × 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 Mabthera monoclonal antibody.
3. double antibody effectively mediates the detection of PBMC cell killing tumour cell
Preparation Raji single cell suspension.With CFSE dyeing (staining procedure is shown in that protocol-1 CFSE 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 PBMC cell, and 50ul/ hole, arranges 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.Take out 96 orifice plates after 48h, 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 Raji.Detected result is shown in Figure 14, and cell killing result display CD20 × CD3 MSBODY Mediated by Bi-specific Antibodies PBMC cell killing tumour cell shows good fragmentation effect, and its maximum killing-efficiency and EC50 are obviously better than Mabthera monoclonal antibody.
Embodiment 6: bi-specific antibody kills and wounds the Composition analyzed of subcutaneous transplantation knurl
CD20 × CD3 MSBODY pharmacodynamic evaluation is expressed on the blood diffusion knurl model of positive raji cell foundation based on CD20 and is completed.Method for establishing model: 1x10
6raji cell by tail vein injection in female NOD/SCID Mice Body.Evaluating drug effect: wherein antagonist treatment group fed back the human T-cell's cell 1.6x10 being coated with CD20 × CD3MSBODY to inoculation raji cell to mouse random packet in the 3rd day
7/ only (induce human PBMC to cultivate acquisition by anti-CD3antibody and IL-2), two contrasts feed back human T-cell's (being only all added with IL-22000U/ in all adoptive therapy mouse) of PBS and non-coated antibody respectively, carry out adoptive therapy in an identical manner first after adoptive therapy at the 6th, 9,12,14 day to mouse.To mouse state and body weight Real-Time Monitoring in the middle of whole therapeutic process, after about 15 days, every day observes mouse, adds up each group of survival rate.
Obtain result such as Figure 15 after carrying out pharmacodynamic evaluation based on above experimental model to CD20 × CD3 MSBODY to show, feed back PBS and do not wrap and started death in inoculation after 25 days by the T cell control group of CD20 × CD3 MSBODY, after 28 days, PBS group is all dead, after 35 days, T cell group is all dead, Continuous Observation 40 days CD20 × CD3 MSBODY drug treatment group mouse survival rates 100% and health states is normal.Illustrate thus, T cell combines and is gathered in around CD20 positive tumor cell after by CD20 × CD3MSBODY mediation, by the cell toxicant killing tumor cell that CD3 stimulates T cell to produce, and control group tumour cell when not having T cell or have T cell not have CD20 × CD3 MSBODY to mediate does not obtain killing and wounding thoroughly and removing in time.This result conforms to Design Theory, can also play the lethal effect being more greater than simple T cell by the specific CD20 of killing and wounding positive tumor cell by the T cell of CD20 × CD3MSBODY mediation.
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 (12)
1. bi-specific antibody, it is characterized in that, described antibody comprises: (a) unit price unit, for light-heavy chain pair, this light-heavy chain has specific binding capacity to TCSA, preferably this TCSA is CD20, CD30 and EPCAM, and more preferably this TCSA is CD20; (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. described bi-specific antibody according to claim 1, is characterized in that: the CH2 structural domain of described strand unit is between ScFv fragment and CH3 structural domain; Do not comprise CH1 structural domain.
3. described 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. described 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 CD20; The heavy chain constant domain CH1 of described heavy chain and heavy-chain variable domains also target in tumour antigen epi-position CD20; 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. the described bi-specific antibody of claim 1, is characterized in that: described strand unit comprises the anti-CD3 of antibody for CD3, and unit price unit comprises the antibody anti-CD 20 for CD20;
Preferably, the aminoacid sequence of described anti-CD 20 heavy chain is the aminoacid sequence shown in sequence number 1, the aminoacid sequence of the light chain of anti-CD 20 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-CD 20 heavy chain on 224 sites is connected with the form of disulfide linkage with the halfcystine on light chain 213 site of anti-CD 20, described anti-CD 20 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 233 sites respectively 230, described anti-CD 20 heavy chain on 396 with 413 sites with 428 and 397 sites of anti-CD3ScFv-Fc form salt bridge be connected, described anti-CD 20 heavy chain on 370 sites with 436 sites of anti-CD3ScFv-Fc are formed knuckle-enter-cave is connected.
6. described 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. described 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 described bi-specific antibody of preparation any one of claim 1-7, it is characterized in that, described method comprises step:
(1) respectively heavy, the light chain of unit price unit are building up on the first expression vector, by strand cell formation on the second expression vector respectively;
(2) by the first and second expression vectors together cotransfection in cell, cultivate and get supernatant;
(3) bi-specific antibody after obtaining purifying is separated by expressing supernatant; Preferably, described cell is CHO-S cell; Or preferably, described separating step comprises: the antibody of all band Fc structural domains caught by protein A affinity chromatography post from expressing supernatant, by being separated of SP cation-exchange chromatography realize target bi-specific antibody and by product, after Q post, finally concentrated displacement damping fluid PBS.
9. described method according to claim 8, described first expression vector is pCHO1.0; Described second expression vector is pCHO1.0-Totomycin.
10. described method according to claim 8, is characterized in that, in the step (1) of described method:
Described unit price unit is anti-CD 20 antibodies, its light chain the primer that increases is Kozak (EcoR V) F, MK-leader sequence (EcoRV) F, Rituxan-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, Rituxan-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, the expression vector of acquisition loading anti-CD 20 light chain; Then carry out homologous recombination with HC again after cutting with AvrII and BstZl7I enzyme, obtain the pCHO1.0 expression vector of anti-CD 20, plasmid called after pCHO1.0-anti-CD 20-HL-LDY;
Described strand unit is anti-CD3ScFv-Fc antibody, its the primer that increases is Kozak (Avr II) 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, 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-KKW.
Described bi-specific antibody 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 CD20 specific antigen express caused by tumour or relative disease, or express CD20 cell for killing.
Described bi-specific antibody any one of 12. 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 is used for the treatment of the drug effect of the medicine of the tumour cell relative disease of expressing CD20 specific antigen for screening the medicine that is used for the treatment of the tumour cell relative disease of expressing CD20 specific antigen or evaluation.
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| CN107759694A (en) * | 2016-08-19 | 2018-03-06 | 安源医药科技（上海）有限公司 | Bispecific antibody and preparation method thereof and purposes |
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| CN110964114A (en) * | 2018-09-29 | 2020-04-07 | 上海博槿生物科技有限公司 | Double-target antigen binding molecule |
| WO2020088437A1 (en) * | 2018-11-01 | 2020-05-07 | 安源医药科技（上海）有限公司 | Bispecific antibody binding to cd20 and cd3 and uses thereof |
| CN111394387A (en) * | 2020-03-13 | 2020-07-10 | 苏州智享众创孵化管理有限公司 | Construction and screening method of bispecific antibody cell strain |
| CN112300283A (en) * | 2019-07-29 | 2021-02-02 | 深圳华大生命科学研究院 | Monoclonal antibody for resisting human pancreatic cancer and preparation method and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102030826A (en) * | 2009-09-25 | 2011-04-27 | 上海抗体药物国家工程研究中心有限公司 | High-affinity CD20-resistance monoclonal antibody |
| WO2014028776A1 (en) * | 2012-08-15 | 2014-02-20 | Zyngenia, Inc. | Monovalent and multivalent multispecific complexes and uses thereof |
| CN104271602A (en) * | 2012-11-21 | 2015-01-07 | 武汉友芝友生物制药有限公司 | Bispecific antibody |
-
2015
- 2015-01-21 CN CN201510029908.XA patent/CN104558191B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102030826A (en) * | 2009-09-25 | 2011-04-27 | 上海抗体药物国家工程研究中心有限公司 | High-affinity CD20-resistance monoclonal antibody |
| WO2014028776A1 (en) * | 2012-08-15 | 2014-02-20 | Zyngenia, Inc. | Monovalent and multivalent multispecific complexes and uses thereof |
| CN104271602A (en) * | 2012-11-21 | 2015-01-07 | 武汉友芝友生物制药有限公司 | Bispecific antibody |
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| CN105907789A (en) * | 2016-03-14 | 2016-08-31 | 紫程瑞生会(北京)生物技术发展有限公司 | Preparation method and kit of cytokine-induced killing cell for inducing antibody-dependent cellular cytotoxicity |
| CN110603266A (en) * | 2016-06-02 | 2019-12-20 | 豪夫迈·罗氏有限公司 | Type II anti-CD 20 and anti-CD 20/CD3 bispecific antibodies for the treatment of cancer |
| CN107759694A (en) * | 2016-08-19 | 2018-03-06 | 安源医药科技（上海）有限公司 | Bispecific antibody and preparation method thereof and purposes |
| WO2018177371A1 (en) * | 2017-03-29 | 2018-10-04 | Taipei Medical University | Antigen-specific t cells and uses thereof |
| CN108690138A (en) * | 2017-04-12 | 2018-10-23 | 鸿运华宁（杭州）生物医药有限公司 | It is a kind of can be with people CD19 or CD20 and people the CD3 bispecific antibody combined and its application |
| CN108059680A (en) * | 2017-12-26 | 2018-05-22 | 北京东方百泰生物科技有限公司 | A kind of bispecific antibody for CD20 and CD3 |
| CN108059680B (en) * | 2017-12-26 | 2020-07-24 | 北京东方百泰生物科技有限公司 | Bispecific antibody aiming at CD20 and CD3 |
| CN110964114B (en) * | 2018-09-29 | 2023-07-04 | 上海博槿生物科技有限公司 | Double-target antigen binding molecule |
| CN110964114A (en) * | 2018-09-29 | 2020-04-07 | 上海博槿生物科技有限公司 | Double-target antigen binding molecule |
| WO2020088437A1 (en) * | 2018-11-01 | 2020-05-07 | 安源医药科技（上海）有限公司 | Bispecific antibody binding to cd20 and cd3 and uses thereof |
| CN112300283B (en) * | 2019-07-29 | 2024-01-02 | 深圳华大生命科学研究院 | Anti-human pancreatic cancer monoclonal antibody and preparation method and application thereof |
| CN112300283A (en) * | 2019-07-29 | 2021-02-02 | 深圳华大生命科学研究院 | Monoclonal antibody for resisting human pancreatic cancer and preparation method and application thereof |
| CN110669137A (en) * | 2019-10-24 | 2020-01-10 | 北京免疫方舟医药科技有限公司 | Multi-specificity antibody and preparation method and application thereof |
| CN111394387A (en) * | 2020-03-13 | 2020-07-10 | 苏州智享众创孵化管理有限公司 | Construction and screening method of bispecific antibody cell strain |
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