CN104829726A - Construction and application of bispecific antibody CD19*CD3 - Google Patents
Construction and application of bispecific antibody CD19*CD3 Download PDFInfo
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Abstract
The invention provides a bispecific antibody which is composed of a single-chain unit A and a single-chain unit B, wherein the single-chain unit A, aiming to surface antigen CD19 of immune cells, has a specifically combining capability while the single-chain unit B, aiming to surface antigen CD3 of tumor cells, has a specifically combining capability. The single-chain unit A and the single-chain unit B both include a single-chain variable fragment (ScFv) fused with an Fc fragment. The application also provides a preparation of the bispecific antibody and medicinal applications of these antibodies.
Description
Technical field
The present invention relates to immunologic technical field.Specifically, the application relates to structure and the application of bi-specific antibody.
Background technology
Bi-specific antibody (bispecific antibody, BiAb) is the artificial antibody containing two species-specific antigen binding sites, can erect bridge between target cell and functional molecular (cell), produces the effector function of guidance quality.BiAb, in biomedicine, particularly has broad application prospects in the immunotherapy of tumour.The focus that tumour cell is current immunotherapy applied research is killed by BiAb mediated cell toxic action, its principal feature is that BiAb can simultaneously in conjunction with the target molecule on tumor associated antigen and immune effector cell, and direct triggering immune effector cell is to the specific killing of tumour cell.Be below for studied Immune Cell Antigens and tumor-cell antigen, and some background technologies of correlation technique development are introduced.
1.CD3
CD3 molecule is made up of 4 subunits: δ, ε, γ, ζ, and its molecular mass is respectively 18.9kDa, 23.1kDa, 20.5kDa, 18.7kDa, and its length has 171,207,182,164 amino-acid residues respectively.They form 6 peptide chains together, and the normal TCR-CD3 complex body being formed and contain 8 peptide chains of combining closely with φt cell receptor (T cell receptor, TCR), structural representation is shown in Fig. 1.This complex body has T cell activation signal transduction, stablizes the function of TCR structure.CD3 kytoplasm section is containing immunoreceptor tyrosine-based activation motif (immunoreceptor tyrosine-based activation motif, ITAM), TCR identifies and combines the antigen peptide of being offered by MHC (major histo-compatibility complex) molecule, cause the tyrosine residues of the conserved sequence of the ITAM of CD3 by the tyrosine protein kinase p56lck phosphorylation in T cell, then can raise the tyrosine protein kinase (as ZAP-70) that other contain SH2 (Scr homology 2) structural domain.The phosphorylation of ITAM and be one of important biochemical reaction of T cell activation intracellular signaling process commitment with the combination of ZAP-70.Therefore, the function of CD3 molecule is that transduction TCR identifies the activation signals that antigen produces.
2.CD19
CD19 all has expression in normal and malignant B, to be regarded as in B cell growth course one and to contain stage longer surface marker the most reliably.In normal lymphoid tissue, CD19 is expressed in the dendron shape maxicell in T cell district between the B cell of germinal center and dendritic cells,follicular, amphicyte, folliculus, substantially identical with CD20 with CD22 staining pattern, but compare with CD20, CD19 also expresses in pre B cell.In addition, by flow cyctometry detection method, CD19 is separated in tissue in the plasmocyte obtained and can detects.As a rule CD19 expresses in bone-marrow-derived lymphocyte knurl, comprising B lymphocytic lymphoma, small lymphocyte lymphoma, lymphoma mantle cell, follicular lymphoma, Burkitt lymphoma, marginarium lymph.
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 genetic engineering double specific antibody, and be mainly divided into dual specific miniantibody, double-chain antibody, strand bivalent antibody, multivalence bi-specific antibody four class.At present, existing Several gene engineering bispecific antibody drug enters clinical experimental stage in the world, and shows good application prospect.
5. the adoptive immunotherapy of tumour
The adoptive immunotherapy of tumour is inputted after amplification in vitro in patient body by immunologically competent cell that is autologous or allosome, direct killing tumour cell, the immunologic function of adjustment and enhancing body, mainly comprises LAK cell, til cell, the T lymphocyte of activation and the immunotherapy of CIK cell.And immunotherapy can only remove a small amount of, scattered tumour cell, the entity tumor curative effect for late period is limited.Therefore often it can be used as the ordinary method combined utilization such as a kind of adjuvant therapy and operation, chemotherapy, radiotherapy.After first cleaning a large amount of tumour cells by ordinary method, then remove remaining tumour cell by immunotherapy, the effect of combined therapy of tumour can be improved.Wherein, adoptive immunotherapy, as the novel method of in combined therapy of tumour, is treated with routine operation, radiotherapy, chemotherapy and other cells and molecular therapy is extensively coordinated, illustrate application prospect widely in the treatment of kinds of tumors.But one more preferably mode should be, bi-specific antibody one end in conjunction with the surface antigen CD3 of cultured immunocyte, and can input in body thereupon together, and the other end of bi-specific antibody can well in conjunction with the surface antigen of tumour cell; Like this, bi-specific antibody just can erect the bridge between tumour cell and immunocyte in vivo, makes immunocyte concentrate near tumor cells, and then kills and wounds tumour cell.Effectively can solve the metastasis and extension of tumour cell by this method, overcome drawbacks such as " not thoroughly, easily transfers, side effect large " after operation, chemicotherapy three great tradition therapeutic modality.
Summary of the invention
Term and shortenings
BiAb: bi-specific antibody (bispecific antibody)
TA: tumour antigen (tumor antigen)
VH: variable region of heavy chain (heavy chain variable region).
VL: variable region of light chain (light chain variable region).
CL: constant region of light chain (constant region of light chain).
CDR: the abbreviation being English Complementarity determining regions (CDRs), refers to the antigen complementary determining region of antibody.
ScFv: Single chain antibody fragment (single-chain variable fragment), is also called single-chain antibody.
CLD: clone exploitation (cell line development)
FACS: fluorescence-activated cell sorting (Fluorescence-activated cell sorting), also referred to as Flow cytometry.
The present invention is directed to the weak point of conventional monoclonal antibody, the initiative of the recruit-bi-specific antibody undertaken by the method for genetically engineered and antibody engineering, at conventional monoclonal antibody mainly through CDC, ADCC and apoptosis capacity are come on the basis of killing tumor cell, add the immunotherapy of mediate T cell, substantially increase effect of immunity system killing tumor cell.
Particularly, the invention provides following technical scheme:
In one embodiment, a kind of bi-specific antibody is provided, it is characterized in that, this antibody comprises: (a) strand unit A, for fusogenic peptide, this fusogenic peptide comprises single chain variable fragment ScFv and has the Fc fragment of hinge area, CH2 structural domain and CH3 structural domain, and wherein this fusogenic peptide has specific binding capacity for TCSA, preferably this TCSA is CD19, CD20, CD30 and CD133, and more preferably this TCSA is CD19; (b) strand unit B 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 described strand unit A and described strand unit B is between ScFv fragment and CH3 structural domain.
In one embodiment, the single chain variable fragment of described strand unit A and described strand unit B is made up of variable region of light chain and heavy chain variable domain, their respectively all target in epitope CD19 and epitope CD3; The variable region of light chain of strand unit A single chain variable fragment and variable region of heavy chain are by (GGGGS)
3small peptide connect, as VH-(GGGGS)
3-VL; The variable region of light chain of strand unit B single chain variable fragment and variable region of heavy chain (GGGGS)
3small peptide connect, as VH-(GGGGS)
3-VL.
In one embodiment, the hinge area of described strand unit A and the hinge area of described strand unit B are by being connected by one or more disulfide linkage.
In one embodiment, strand unit A comprises the anti-CD19 of antibody for people source CD19; Strand unit B comprises the anti-CD3 of antibody for people source CD3;
Preferably, the aminoacid sequence of described anti-CD19ScFv-Fc is the aminoacid sequence shown in sequence number 1, and the aminoacid sequence of described anti-CD3ScFv-Fc is the aminoacid sequence shown in sequence number 3; Described anti-CD19 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 268 sites respectively 265, anti-CD19ScFv-Fc is 438, with 421 of described anti-CD3ScFv-Fc on 446 sites, 395 sites are formed salt bridge to connect, described anti-CD19ScFv-Fc on 407 sites with 438 sites of anti-CD3ScFv-Fc are formed knuckle-enter-cave is connected.
In one embodiment, the Fc fragment of described strand unit A comprises people or humanized Fc fragment, and preferably, the Fc fragment of this fusogenic peptide comprises human IgG Fc fragment; The Fc fragment of described strand unit B 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 strand unit A and the human IgG Fc of described strand unit B are by salt bridge and knuckle-enter-cave anatomical connectivity.
In one embodiment, the invention provides a kind of method preparing bi-specific antibody, described method comprises step:
(1) respectively strand unit A is building up on the first expression vector, strand unit B is building up on the second expression vector;
(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, in the step (1) of aforesaid method:
Described strand unit A is anti-CD19ScFv-Fc antibody, its the primer that increases is Kozak (Avr II) F, MK-Leader (AvrI I) F, AC19-VH F1, hIgG1 (sbfI) R, increased by over-lap PCR, Kozak sequence, leader sequence and restriction enzyme site EcoR V and PacI are introduced ScFv-Fc; By the ScFv-Fc gene fragment increased with carry out homologous recombination with the pCHO1.0 expression vector that EcoR V and PacI enzyme cut through, obtain the pCHO1.0 expression vector of anti-CD19ScFv-Fc, the anti-CD19-ScFv-Fc-KKW of plasmid called after pCHO1.0-;
Described strand unit B is anti-CD3ScFv-Fc antibody, its the primer that increases is Kozak (Avr II) F, MK-leader (AvrI I) F, L2K-VH (MK) F1, hIgG1 (sbfI) R, expanded by over-lap PCR, Kozak sequence, leader sequence and restriction enzyme site AvrII and BstZl7I are introduced ScFv-Fc, the pCHO1.0-Totomycin expression vector gene fragment increased and enzyme cut through carries out homologous recombination, obtain the expression vector loading anti-CD3ScFv-Fc, plasmid called after pCHO1.0-Totomycin-L2K-ScFv-Fc-LDY.
In one embodiment, the invention provides bi-specific antibody and preparing the purposes in medicine, described medicine be used for the treatment of CD19 specific antigen express caused by tumour or relative disease, or express CD19 cell for killing.
In one embodiment, the invention provides bi-specific antibody and preparing the purposes in medicine, described medicine is used for the treatment of the drug effect of the medicine of the tumour cell relative disease of expressing CD19 specific antigen for the medicine or evaluation screening the tumour cell relative disease being used for the treatment of expression CD19 specific antigen in tumor cell line.Present invention also offers following technical scheme:
The invention provides a kind of novel antibody being called as bi-specific antibody, and set up a kind of immunity system of human body that utilizes and carry out immunotherapy, carry out putting of the drug efficacy study of bi-specific antibody.These bi-specific antibodies, as a kind of novel antibody and for pharmacophore model, introduce T cell to specific cytotoxic effect of the tumour antigens such as CD19.
The invention provides a kind of novel method and prepare bi-specific antibody SSBODY (ScFv and ScFv bispecific antibody) (as shown in Figure 2), this bi-specific antibody comprises the combination of two groups of ScFv-Fc, wherein one group is the ScFv-Fc of a kind of antigen of energy 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 another group is the ScFv-Fc of the another kind of antigen of energy specific combination, carry out other transformation equally in its heavy chain Fc district, also not easily self forms dimer, and is easy between these two groups of single chain variable fragments form heterozygosis dimer.After Fc transformation, two groups of ScFv-Fc single-chain antibody nature different dimerizations, form each domain arrangement order of SSBODY, SSBODY and structural representation is 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 people source CD19 and people source CD3, be named as M904, as Fig. 2, anti-CD19 is here ScFv-Fc form, comprise anti-CD19VH, VL, Fc structural domain, anti-CD3 is also here ScFv-Fc form, comprises anti-CD3VH, VL, Fc structural domain.Above bi-specific antibody is built by antibody genetic engineering method, and the ScFv-Fc of the anti-CD19 of bi-specific antibody SSBODY is expressed by the first expression vector, and the ScFv-Fc of AntiCD3 McAb is expressed by the second expression vector.According to the multiple clone site design primer in respective ScFv, Fc gene order and carrier.Obtain gene fragment by PCR or Overlap extension PCR method, then cloned by homologous recombination method.Enzyme cuts pCHO1.0 or pCHO1.0-hygromycin vector, then purifying reclaim PCR primer and enzyme cut after carrier, be cloned on pCHO1.0 carrier by anti-CD19ScFv-Fc homologous recombination, the ScFv-Fc fragment homologous recombination of AntiCD3 McAb is cloned on pCHO1.0-hygromycin vector, and checks order.The expression of recombinant protein SSBODY in mammalian cell, detection, use transfection reagent will to express two kinds of plasmid co-transfections of the strand (Single chain) of anti-CD19 and CD3 respectively in mammalian cell, regather the expression that supernatant carries out SDS-PAGE and western blotting detection SSBODY.By centrifugal for the nutrient solution supernatant after transfection expression, filter, with the dilution of binding buffer liquid, cross affinity column, elution buffer wash-out, SDS-PAGE detects protein purification.
The useful technique effect of technical scheme of the present invention has:
1. this application provides a kind of heterodimeric antibodies, this antibody comprises two different antigen-binding polypeptides unit.This heterodimer is different from its corresponding homodimer molecular size range, the size of molecular weight can be utilized to distinguish heterodimer and homodimer, thus more conveniently determine the purity of bi-specific antibody.One of these two antigen-binding polypeptides unit comprise the light-heavy chain pair being similar to wild-type antibodies, and in whole the application, this unit is also referred to as " unit price unit ".Another antigen-binding polypeptides unit comprises single chain variable fragment (ScFv).Such ScFv can merge the constant fragment (Fc) to antibody.In the application's full text, this fusogenic peptide is also referred to as " strand unit ".
2. the invention discloses foundation and the application thereof of the animal model of the immunocyte killing tumor cell that a kind of novel bispecific antibodies SSBODY mediates.The present invention includes that the immunocyte mediated in bispecific antibody drug research process kills and wounds, the preparation of bi-specific antibody, and the foundation of bi-specific antibody pharmacophore model and detection.Bi-specific antibody SSBODY comprises two groups of ScFv and connects Fc combination, wherein the tumor-cell antigen of one group of a kind of people of specific combination, comprises a series of tumour cell film surface antigens such as CD19, and carries out some transformations 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.And utilize the Mediated by Bi-specific Antibodies built, it can simultaneously in conjunction with the immune accessory cell that tumour cell, the T cell expressing CD3 and energy Fc combine, a bridge block is set up between immunocyte and tumour cell, form an immunocomplex, there is strong immune response in immunocyte, secretion cytokine profiles, kills and wounds tumour cell, and then the growth of Tumor suppression.Like this, bi-specific antibody can erect bridge between target cell and functional molecular (cell), excites the immune response with guidance quality, has broad application prospects in the immunotherapy of tumour.
Surprisingly, the application proves that this antibody is also stable, has high antigen joint efficiency.This makes us feeling surprised, even because the homodimer of verified single-chain antibody is in physiological conditions all unstable.Such as, Ahmad etc. " ScFv Antibody:Principles and Clinical Appl ication; " Clinical and Developmental Immunology, 2012:980250 (2012), the IgG antibody-like shown based on ScFv is unstable, and needs transformation further assemble to reduce and improve stability.
In addition, because have asymmetry, heterodimer has and the homodimer difference iso-electric point be made up of wherein arbitrary antigen-binding polypeptides unit.Based on the iso-electric point difference between heterodimer and homodimer, easily the heterodimer of needs can be separated with homodimer, greatly reduce the difficulty that the ubiquitous downstream process exploitation of bi-specific antibody exists.
Accompanying drawing explanation
In order to be illustrated more clearly in the technical scheme in the embodiment of the present application, be briefly described to the accompanying drawing used required in embodiment below, apparently, the accompanying drawing that the following describes is only some embodiments recorded in the application, those of ordinary skill in the art are come, under the prerequisite not paying creative work, other accompanying drawing can also be obtained according to these accompanying drawings, wherein:
Fig. 1 .CD3 schematic arrangement.
Fig. 2 .CD19 × CD3SSBODY bi-specific antibody molecule schematic diagram.
Fig. 3. electrophoresis detection PCR primer, Fig. 3 A:M, DL10000 labeled nucleic acid molecule; 1. anti-CD 19 antibodies ScFv-Fc; Fig. 3 B:M, DL10000 labeled nucleic acid molecule; 2. anti-CD 3 antibodies ScFv-Fc.
Fig. 4 .CD19 × CD3SSBODY double antibody SDS-PAGE electrophorogram, M: protein markers; 2: non-reduced SDS-PAGE electrophoresis detection; 3: reduction SDS-PAGE electrophoresis detection.
The HPLC-SEC purity peak shape figure of Fig. 5 .CD19 × CD3SSBODY.
Fig. 6. the CD19 × CD3SSBODY double antibody measured based on flow cytometric analysis and the avidity result figure of Raj i cell, (▼) M904; The anti-CD19 of (▲) control antibodies.
Fig. 7. the CD19 × CD3SSBODY double antibody measured based on flow cytometric analysis and the avidity result figure of Jurkat cell, (▼) M904; (▲) control antibodies L2K.
Fig. 8. the dual anti-physical efficiency of flow cytometer detection CD19 × CD3SSBODY promotes the common combination of Raji and Jurkat two kinds of cells, (●) M904; (▼) control antibodies MT103.
Fig. 9. differential scanning calorimeter measures the Tm value figure of M904 double antibody.
Figure 10 .CD19 × CD3SSBODY double antibody Activity determination figure, Figure 10 A. and CD19 binding activities after Overheating Treatment detect, (●) M904; (■) Anti-CD19 monoclonal antibody; Figure 10 B. and CD3 binding activities detect, (▲) M904; (▼) Anti-CD3 monoclonal antibody L2K.
Figure 11 .CIK Phenotypic examination result figure, the two positive NK class cell of the CD3 in the upper right corner, CD56.
Figure 12. double antibody effectively mediates the result figure that CIK cell kills and wounds CD19 positive tumor cell.Figure 12 A: double antibody mediation CIK cell kills and wounds Daudi cell (▲) M904, (▼) Anti-CD19 monoclonal antibody (■) 4420 × CD3 double antibody, Mco101, (●) hIgG negative control; Figure 12 B: double antibody mediation CIK cell kills and wounds Raji cell (●) M904, (▲) Anti-CD19 monoclonal antibody (■) 4420 × CD3 double antibody, Mco101, (△) hIgG negative control.
Figure 13. double antibody effectively mediates the result figure of PBMC cell killing Raji tumour cell, (■) M904, (▼) Anti-CD19 monoclonal antibody (▲) 4420 × CD3 double antibody, Mco101, (●) hIgG negative control.
Embodiment
Embodiment 1: the expression vector establishment (CD19 × CD3, M904) of bi-specific antibody
1. bi-specific antibody sequences Design
Be named as CD19 × CD3 with the bi-specific antibody that CD19 and CD3 is target spot, M904, as figure (Fig. 2), anti-CD19 and anti-CD3 is ScFv-Fc form, comprises the fusogenic peptide of anti-CD3, containing VH, VL, Fc structural domain.Wherein anti-CD19 one side Fc carries out KKW transformation, antibody CD3 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. CD19 × CD3 bi-specific antibody.Meanwhile, in order to dual anti-physical efficiency is expressed in Chinese hamster ovary cell (CHO), and can be secreted in substratum, 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 1-6.
Anti-CD19ScFv-Fc aminoacid sequence (sequence number 1)
QVQLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIWPGDGDTNYNGKFKGKATLTADESSSTAYMQLSSLASEDSAVYFCARRETTTVGRYYYAMDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIQLTQSPASLAVSLGQRATISCKASQSVDYDGDSYLNWYQQIPGQPPKLLIYDASNLVSGIPPRFSGSGSGTDFTLNIHPVEKVDAATYHCQQSTEDPWTFGGGTKLEIKGAAAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
Anti-CD19ScFv-Fc nucleotide sequence (sequence number 2)
caggtgcagctgcagcagtctggggctgagctggtgaggcctgggtcctcagtgaagatttcctgcaaggcttctggctatgcattcagtagctactggatgaactgggtgaagcagaggcctggacagggtcttgagtggattggacagatttggcctggagatggtgatactaactacaatggaaagttcaagggtaaagccactctgactgcagacgaatcctccagcacagcctacatgcaactcagcagcctagcatctgaggactctgcggtctatttctgtgcaagacgggagactacgacggtaggccgttattactatgctatggactactggggccaagggaccacggtcaccgtctccagcggaggcggcggttcaggcggaggtggaagtggtggaggaggttctgatatccagctgacccagtctccagcttctttggctgtgtctctagggcagagggccaccatctcctgcaaggccagccaaagtgttgattatgatggtgatagttatttgaactggtaccaacagattccaggacagccacccaaactcctcatctatgatgcatccaatctagtttctgggatcccacccaggtttagtggcagtgggtctgggacagacttcaccctcaacatccatcctgtggagaaggtggatgctgcaacctatcactgtcagcaaagtactgaggatccgtggacgttcggtggagggaccaagctcgagatcaaaggtgcggccgcagagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgtggtgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacgataccacgcctcccgtgctggactccgacggctccttcttcctctacagcga tctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga
Anti-CD3ScFv-Fc aminoacid sequence (sequence number 3)
DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSGGGGSGGGGSGGGGSDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKGAAAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCRVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLASKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK-
Anti-CD3ScFv-Fc nucleotide sequence (sequence number 4)
GACATCAAACTGCAGCAGTCAGGGGCTGAACTGGCAAGACCTGGGGCCTCAGTGAAGATGTCCTGCAAGACTTCTGGCTACACCTTTACTAGGTACACGATGCACTGGGTAAAACAGAGGCCTGGACAGGGTCTGGAATGGATTGGATACATTAATCCTAGCCGTGGTTATACTAATTACAATCAGAAGTTCAAGGACAAGGCCACATTGACTACAGACAAATCCTCCAGCACAGCCTACATGCAACTGAGCAGCCTGACATCTGAGGACTCTGCAGTCTATTACTGTGCAAGATATTATGATGATCATTACTGCCTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCAGGAGGCGGCGGTTCAGGCGGAGGTGGAAGTGGTGGAGGAGGTTCTGACATTCAGCTGACCCAGTCTCCAGCAATCATGTCTGCATCTCCAGGGGAGAAGGTCACCATGACCTGCAGAGCCAGTTCAAGTGTAAGTTACATGAACTGGTACCAGCAGAAGTCAGGCACCTCCCCCAAAAGATGGATTTATGACACATCCAAAGTGGCTTCTGGAGTCCCTTATCGCTTCAGTGGCAGTGGGTCTGGGACCTCATACTCTCTCACAATCAGCAGCATGGAGGCTGAAGATGCTGCCACTTATTACTGCCAACAGTGGAGTAGTAACCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAAGGTGCGGCCGCAGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAA AGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCGGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGAAGTCCGACGGCTCCTTCTTCCTCGCCAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA
The leader peptide sequences aminoacid sequence (sequence number 5) of mouse kappa
METDTLLLWVLLLWVPGSTG
The leader peptide sequences nucleotide sequence (sequence number 6) of mouse kappa
atggagacagacacactcctgctatgggtactgctgctctgggttccaggttccactggt
2. bi-specific antibody gene clone
PCHO1.0 is selected to remove the ScFv-Fc gene of the anti-CD19 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 the anti-CD3 of cloning and expressing.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 respectively the ScFv-Fc of anti-CD19 is building up on the expression vector of pCHO1.0, anti-CD3ScFv-Fc is building up 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 ScFv-Fc, sees Fig. 3 A.First by the ScFv-Fc gene fragment increased with carry out homologous recombination with the pCHO1.0 expression vector that EcoR V and PacI enzyme cut through, obtain the pCHO1.0 expression vector of anti-CD19, the anti-CD19-ScFv-Fc-KKW of plasmid called after pCHO1.0-.
To be increased anti-CD3ScFv-Fc structural domain by over-lap PCR, and Kozak sequence, leader sequence and restriction enzyme site AvrII and BstZl7I are introduced ScFv-Fc, by the gene fragment increased, see Fig. 3 B, the pCHO1.0-Totomycin expression vector cut through with enzyme carries out homologous recombination, obtain the expression vector loading anti-CD3ScFv-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 is at CD CHO substratum (Gibco, 10743-029), be placed in 37 DEG C, cultivate in 5%CO2 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-CD19-SCFV-FC-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 M904 to CD19 × CD3.
Difference the 2nd day after transfection, culture temperature is lowered to 32 DEG C, and every day adds 3.5%FeedA, cultivates 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 damping fluid PBS.Bi-specific antibody after purifying carries out SDS-PAGE, SEC and detects, and purity is (see Fig. 4 and Fig. 5) more than 95%.
Embodiment 3: the binding activities of bi-specific antibody and cell measures (FACS)
The target antigen of bi-specific antibody of the present invention on corresponding cell is combined.The present invention is using Raji (purchased from ATCC, CCL-86) as the cell of the CD19 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 Daji 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 GraphPad Prism5.0.The Raji cell of result display CD19 × CD3 double antibody and the CD19 positive has good binding activities, sees Fig. 6.
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 CD19 × CD3 double antibody and the CD3 positive has good binding activities, sees Fig. 7.
3. the immunocyte of double antibody mediation and the common Binding experiment of tumour cell
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 160nmol, and 4 times of gradient dilutions, obtain 6 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 100ul PBS 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 CD19 × CD3 simultaneously in conjunction with the Raji cell of the CD19 positive and the Jurkat cell of the CD3 positive, and promotes the common combination of 2 kinds of cells, sees Fig. 8.
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 shows, and see Fig. 9, the Tm value of bi-specific antibody, all more than 70 DEG C, shows except 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 antibodies targeting 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 10, and result shows, and the Tm value of bi-specific antibody, all more than 50 DEG C, 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 in 75cm2 culturing bottle, is 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 11, 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 CIK cell killing tumor cell
Collect Raji single cell suspension, with CFSE dyeing (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 Raji.The results are shown in Figure 12, cell killing result display CD19 × CD3SSBODY 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-CD19 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-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 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.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 Raji.The results are shown in Figure 13, cell killing result display CD19 × CD3SSBODY 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-CD19 monoclonal antibody.
Example 6: bi-specific antibody kills and wounds the Composition analyzed of subcutaneous transplantation knurl
CD19 × CD3SSBODY pharmacodynamic evaluation is expressed on the blood diffusion knurl model of positive raji cell foundation based on CD19 and is completed.Method for establishing model: 1x106raji cell by tail vein injection in female NOD/SCID Mice Body.Evaluating drug effect: inoculation raji cell the 3rd day is to mouse random packet, wherein antagonist treatment group feeds back the human T-cell's cell 1.6x107/ being coated with CD19 × CD3SSBODY and only (cultivates acquisition by anti-CD3antibody and IL-2 induction human PBMC), two contrasts feed back people's (being only all added with LI-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.
Following result is obtained after pharmacodynamic evaluation being carried out to CD19 × CD3 SSBODY based on above experimental model, feed back PBS and do not wrap and started death in inoculation after 25 days by the T cell control group of CD19 × CD3 SSBODY, after 28 days, PBS group is all dead, after 35 days, T cell group is all dead, Continuous Observation 40 days CD19 × CD3 SSBODY drug treatment group mouse survival rates 100% and health states is normal.Illustrate thus, T cell is combined and is gathered in around CD19 positive tumor cell after being mediated by CD19 × CD3 SSBODY, 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 CD19 × CD3 SSBODY to mediate does not obtain killing and wounding thoroughly and removing in time.This result conforms to Design Theory, and the T cell mediated by CD19 × CD3 SSBODY can also play the lethal effect being more greater than simple T cell by the specific CD19 of killing and wounding positive tumor cell.
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) strand unit A, for fusogenic peptide, this fusogenic peptide comprises single chain variable fragment ScFv and has the Fc fragment of hinge area, CH2 structural domain and CH3 structural domain, wherein this fusogenic peptide has specific binding capacity to TCSA, and preferably this TCSA is CD19, CD20, CD30 and CD133, and more preferably this TCSA is CD19; (b) strand unit B 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 A and described strand unit B is all between ScFv fragment and CH3 structural domain.
3. described bi-specific antibody according to claim 1, it is characterized in that: the single chain variable fragment of described strand unit A and described strand unit B is made up of variable region of light chain and heavy chain variable domain, their respectively all target in epitope CD19 and epitope CD3; The variable region of light chain of strand unit A single chain variable fragment and variable region of heavy chain are by (GGGGS)
3small peptide connect, as VH-(GGGGS)
3-VL; The variable region of light chain of strand unit B single chain variable fragment and variable region of heavy chain are by (GGGGS)
3small peptide connect, as VH-(GGGGS)
3-VL.
4. described bi-specific antibody according to claim 1, is characterized in that: the hinge area of described strand unit A and the hinge area of described strand unit B are by being connected by one or more disulfide linkage.
5. the described bi-specific antibody of claim 1, is characterized in that: strand unit A comprises the anti-CD19 of antibody for people source CD19; Strand unit B comprises the anti-CD3 of antibody for people source CD3;
Preferably, the aminoacid sequence of described anti-CD19ScFv-Fc is the aminoacid sequence shown in sequence number 1, and the aminoacid sequence of described anti-CD3ScFv-Fc is the aminoacid sequence shown in sequence number 3; Described anti-CD19 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 268 sites respectively 265, anti-CD19ScFv-Fc is 438, with 421 of described anti-CD3ScFv-Fc on 446 sites, 395 sites are formed salt bridge to connect, described anti-CD19ScFv-Fc on 407 sites with 438 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 Fc fragment of described strand unit A comprises people or humanized Fc fragment, and preferably, the Fc fragment of this fusogenic peptide comprises human IgG Fc fragment; The Fc fragment of described strand unit B 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 strand unit A and the human IgG Fc of described strand unit B 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 strand unit A is building up on the first expression vector, strand unit B is built on the second expression vector;
(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, the first described expression vector is pCHO1.0; The second described expression vector is pCHO1.0-Totomycin.
10. method according to 8 of claim, is characterized in that, in the step (1) of described method:
Described strand unit A is anti-CD19ScFv-Fc antibody, its the primer that increases is Kozak (Avr II) F, MK-Leader (AvrII) F, AC19-VH F1, hIgG1 (sbfI) R, increased by over-lap PCR, Kozak sequence, leader sequence and restriction enzyme site EcoR V and PacI are introduced ScFv-Fc; By the ScFv-Fc gene fragment increased with carry out homologous recombination with the pCHO1.0 expression vector that EcoR V and PacI enzyme cut through, obtain the pCHO1.0 expression vector of anti-CD19ScFv-Fc, the anti-CD19-ScFv-Fc-KKW of plasmid called after pCHO1.0-;
Described strand unit B is anti-CD3ScFv-Fc antibody, its the primer that increases is Kozak (Avr II) F, MK-leader (AvrII) F, L2K-VH (MK) F1, hIgG1 (sbfI) R, expanded by over-lap PCR, Kozak sequence, leader sequence and restriction enzyme site AvrII and BstZl7I are introduced ScFv-Fc, the pCHO1.0-Totomycin expression vector gene fragment increased and enzyme cut through carries out homologous recombination, obtain the expression vector loading anti-CD3ScFv-Fc, plasmid called after pCHO1.0-Totomycin-L2K-ScFv-Fc-LDY.
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 CD19 specific antigen express caused by tumour or relative disease, or express CD19 cell for killing.
12. bi-specific antibody prepared by bi-specific antibody according to any one of claim 1-7 or the method according to the bi-specific antibody prepared any one of claim 8-10 is preparing the purposes in medicine, described medicine is being used for the treatment of the drug effect of the medicine of the tumour cell relative disease of expressing CD19 specific antigen for the medicine or evaluation screening the tumour cell relative disease being used for the treatment of expression CD19 specific antigen in tumor cell line.
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