CN104558191B - Construction and application of bispecific antibody CD20 xCD 3 - Google Patents

Abstract

The invention provides a bispecific antibody, which consists of a single-chain unit and a monovalent unit, wherein the single-chain unit has specific binding capacity for a surface antigen CD3 of an immune cell, and the monovalent unit has specific binding capacity for a tumor cell surface antigen CD 20; the single chain unit comprises a single chain variable fragment (ScFv) fused to an Fc fragment, and the monovalent unit comprises a light chain and a heavy chain pair. The present application also provides methods for the preparation of bispecific antibodies, pharmaceutical uses of these antibodies.

Description

Construction and application of bispecific antibody CD20 xCD 3

Technical Field

The present invention relates to the field of immunology. In particular to a construction and preparation method of a bispecific antibody and a method for detecting the function and the property of the bispecific antibody.

Background

Bispecific antibodies (biabs) are artificial antibodies that contain two specific antigen binding sites and can bridge between target cells and functional molecules (cells) to produce a targeted effector function. BiAb has wide application prospect in biomedicine, especially in the immunotherapy of tumors. The method is characterized in that the BiAb can be simultaneously combined with tumor-associated antigens and target molecules on immune response cells to directly trigger the specific killing of immune effector cells on tumor cells. The following is a description of some background art directed to the immune cell antigens and tumor cell antigens of interest, and related technology developments.

1.CD3

The CD3 molecule consists of 4 subunits: gamma and zeta have molecular masses of 18.9k Da, 23.1k Da, 20.5kDa and 18.7k Da respectively and lengths of 171, 207, 182 and 164 amino acid residues respectively. Together, they form 6 peptides, often tightly associated with a T Cell Receptor (TCR) to form a TCR-CD3 complex containing 8 peptides, the structure of which is schematically shown in fig. 1. This complex has the function of T cell activation signaling, stabilizing the TCR structure. The CD3 cytoplasmic segment contains immunoreceptor tyrosine-based activation motif (ITAM), and TCR recognizes and binds to antigenic peptides presented by MHC (major histocompatibility complex) molecules, so that tyrosine residues of conserved sequences of ITAM of CD3 are phosphorylated by tyrosine protein kinase p56lck in T cells, and then other tyrosine protein kinases (such as ZAP-70) containing SH2(Scr homology 2) structural domains can be recruited. Phosphorylation of ITAM and binding to ZAP-70 are one of the important biochemical reactions in the early stages of the T cell activation signaling process. Thus, the function of the CD3 molecule is to transduce an activation signal generated by the TCR recognition antigen.

2.CD20

CD20 is a specific marker molecule on the surface of B lymphocytes, expressed on more than 95% of normal or malignant B lymphocytes, and is devoid of CD20 antigen expression in hematopoietic stem cells, progenitor cells, and other normal tissues. The protein consists of 297 amino acids, has a molecular weight of 33kD to 36kD, belongs to non-glycosylated phosphoprotein, and has an epitope which is a loop region consisting of 43 amino acids in a third and fourth transmembrane region. After being combined with corresponding antibodies, the CD20 has no obvious internalization phenomenon and no obvious cell surface shedding phenomenon, and is an ideal target for treating B lymphocyte related diseases. The research shows that CD20 has the function of calcium ion channel, influences cell cycle, regulates cell proliferation and differentiation and even leads to apoptosis by regulating the concentration of calcium ions in cells. At present, two monoclonal antibody medicaments aiming at CD20 targets, namely rituximab and Ordovician, are available, the action mechanisms of the medicaments mainly comprise complement-dependent cytotoxicity reaction, antibody-dependent cell-mediated cytotoxicity and improvement of sensitivity of cells to the cytotoxicity, and in addition, the immunization action and the like also cause apoptosis of B cells. Since the original normal B cells are not affected by the anti-CD 20 mab, the anti-CD 20 mab is increasingly used to treat B cell related diseases, even after the anti-CD 20 mab kills all lymphoma cells expressing CD20 molecules and normal B cells, and the B cell population can be reconstituted.

3. Bispecific antibody technology development

Bispecific antibodies, antibodies in which two antigen-binding sites in one antibody molecule can bind to two different epitopes, respectively.

The antibody medicine is a biological macromolecular medicine prepared by an antibody engineering technology taking a cell engineering technology and a genetic engineering technology as main bodies, and has the advantages of high specificity, uniform property, capability of being directionally prepared aiming at a specific target spot and the like. The monoclonal antibody is mainly applied to the following three aspects in clinic: tumor treatment, immune disease treatment, and anti-infection treatment. The tumor therapy is the most widely applied field of the monoclonal antibody at present, and the quantity of the monoclonal antibody used for tumor therapy is about 50 percent in the monoclonal antibody products which are clinically tested and marketed at present. Monoclonal antibody for treating tumor is an immunotherapy of killing target cells by stimulating immune system to target specific target of pathological cells, and in order to enhance the effect function of antibody, especially the effect of killing tumor cells, people try to modify antibody molecules by various methods, and bispecific antibody is one of the development directions of improving the therapeutic effect of antibody, and is now a hot spot in the research field of antibody engineering.

The bispecific antibody for immunotherapy is an artificial antibody containing 2 specific antigen binding sites, can bridge between target cells and functional molecules (cells), stimulates targeted immune response, and has wide application prospect in tumor immunotherapy.

4. Bispecific antibody preparation

Bispecific antibodies are available in a variety of ways, and are prepared mainly by: chemical coupling method, hybrid-hybridoma method and gene engineering antibody preparation method. The first concept of bispecific monoclonal antibodies was prepared by chemically coupling 2 different monoclonal antibodies together. The hybrid-hybridoma method is to produce bispecific monoclonal antibodies by means of cell hybridization or ternary hybridoma, which is obtained by fusion of established hybridomas or established hybridomas with lymphocytes obtained from mice, and which can only produce bispecific antibodies of murine origin, and its application is greatly limited. With the rapid development of molecular biology technology, various construction modes of genetically engineered humanized bispecific antibodies appear, and the antibodies are mainly classified into four classes of bispecific miniantibodies, diabodies, single-chain diabodies and multivalent bispecific antibodies. At present, several genetic engineering bispecific antibody medicaments enter a clinical test stage internationally and show good application prospects.

5. Adoptive immunotherapy of tumors

The adoptive immunotherapy of tumor is to directly kill tumor cells and regulate and enhance the immune function of the body by inputting autologous or allogeneic immunocompetent cells into a patient after in vitro amplification, and mainly comprises immunotherapy of LAK cells, TIL cells, activated T lymphocytes and CIK cells. Immunotherapy can only eliminate a small amount of scattered tumor cells, and has limited efficacy for advanced solid tumors. Therefore, it is often used as an adjuvant therapy in combination with conventional methods such as surgery, chemotherapy, and radiotherapy. After a large amount of tumor cells are cleaned by a conventional method, residual tumor cells are cleaned by an immunotherapy, so that the comprehensive treatment effect of the tumor can be improved. The adoptive immunotherapy is a new method in the comprehensive treatment of tumors, has been widely matched with conventional operative treatment, radiotherapy, chemotherapy and other cell and molecular therapies, and has shown wide application prospects in the treatment of various tumors. However, a more desirable way would be that one end of the bispecific antibody could bind to the surface antigen CD3 of cultured immune cells and be delivered into the body along with it, while the other end of the bispecific antibody binds well to the surface antigen of tumor cells; thus, the bispecific antibody can bridge between tumor cells and immune cells in vivo, so that the immune cells are concentrated around the tumor cells, and further kill the tumor cells. The method can effectively solve the problem of tumor cell metastasis and diffusion, and overcomes the defects of incomplete treatment, easy metastasis, great side effect and the like after three traditional treatment modes of operation, radiotherapy and chemotherapy.

Disclosure of Invention

Terms and abbreviations

BiAb bispecific antibody (bispecific antibody)

TA tumor antigen (tumor antigen)

VH heavy chain variable region (heavy chain variable region).

VL light chain variable region (light chain variable region).

CL: a constant region of light chain (constant region of light chain).

CDR: the abbreviation is used for the English Complementary Determining Regions (CDRs), and refers to the antigen Complementarity determining regions of the antibody.

ScFv: single-chain variable region antibody fragments (also known as single-chain antibodies).

CLD cell line development (cell line)

FACS: fluorescence-activated cell sorting (Fluorescence-activated cell sorting), also known as flow cytometry sorting.

Aiming at the defects of the conventional monoclonal antibody, the invention creates a new molecule-bispecific antibody by a genetic engineering and antibody engineering method, increases the immunotherapy of mediated T cells on the basis that the traditional monoclonal antibody kills tumor cells mainly by CDC, ADCC and apoptosis capacity, and greatly improves the efficiency of an immune system in killing the tumor cells.

Specifically, the invention provides the following technical scheme:

in one embodiment, there is provided a bispecific antibody, wherein the antibody comprises: (a) a monovalent unit that is a light-heavy chain pair having specific binding capacity for a tumor cell surface antigen, preferably the tumor cell surface antigen is CD20, EPCAM and CD30, more preferably the tumor cell surface antigen is CD 20; and (b) a single chain unit which is a fusion peptide comprising a single chain variable fragment ScFv and an Fc fragment having a hinge region, a CH2 domain and a CH3 domain, wherein the immune cell against which the fusion peptide is directed is selected from a T cell, an NKT cell or a CIK cell; preferably, the fusion peptide has specific binding capacity to the immune cell surface antigen CD 3.

In one embodiment, the CH2 domain of the single chain unit of the bispecific antibody is located between the ScFv fragment and the CH3 domain; the single-stranded unit does not comprise a CH1 domain.

In one embodiment, the single chain variable fragment of the bispecific antibody consists of a light chain variable region and a heavy chain variable region domain, both of which are targeted to epitope CD 3.

In one embodiment, in a monovalent unit, both the light chain constant region domain and the light chain variable region domain of the light chain are targeted to the tumor epitope CD 20; the heavy chain constant domain CH1 and the heavy chain variable domain of the heavy chain also target the tumor epitope CD 20; the light chain is bound to the heavy chain by disulfide bonds; the heavy chain is bound to the fusion peptide by one or more disulfide bonds.

In one embodiment, the single chain unit comprises the antibody anti-CD3 directed to CD3 and the monovalent unit comprises the antibody anti-CD 20 directed to CD 20.

In one embodiment, the amino acid sequence of the heavy chain of antibody anti-CD 20 is the amino acid sequence shown in sequence No. 1, the amino acid sequence of the light chain of antibody anti-CD 20 is the amino acid sequence shown in sequence No. 3, and the amino acid sequence of the anti-CD 3ScFv-Fc is the amino acid sequence shown in sequence No. 5; and the cysteine at position 224 of the anti-CD 20 heavy chain is disulfide-linked to the cysteine at position 213 of the light chain of anti-CD 20, the cysteine at positions 230 and 233 of the anti-CD 20 heavy chain is disulfide-linked to the cysteine at positions 255 and 258 of anti-CD 3ScFv-Fc, respectively, the anti-CD 20 heavy chain is salt-bridged at positions 396 and 413 to the positions 428 and 397 of anti-CD 3ScFv-Fc, and the anti-CD 20 heavy chain is protuberance-into-hole linked at position 370 to the position 436 of anti-CD3 ScFv-Fc.

In one embodiment, the heavy chain in the monovalent unit comprises a human or humanized Fc fragment, preferably the Fc fragment of the heavy chain comprises a human IgG Fc fragment; the Fc fragment of the fusion peptide comprises a human or humanized Fc fragment, preferably the Fc fragment of the fusion peptide comprises a human IgG Fc fragment.

In one embodiment, the human IgG Fc fragment of the monovalent unit and the IgG Fc fragment of the single chain unit are linked by a salt bridge and knob-hole-in structure.

In one embodiment, a method of making a bispecific antibody is provided, the method comprising:

(1) respectively constructing heavy chains and light chains of the monovalent units on a first expression vector and constructing the single chain units on a second expression vector;

(2) co-transfecting the first and second expression vectors into a cell together, culturing and taking the supernatant;

(3) separating the expression supernatant to obtain a purified bispecific antibody; preferably, the cells are CHO-S cells; or preferably, the separating step comprises: protein A affinity chromatographic column captures all antibodies with Fc structural domains from the expression supernatant, realizes the separation of the target bispecific antibody and byproducts by SP cation exchange chromatography, passes through a Q column, and finally concentrates and replaces buffer PBS.

In one embodiment, the first expression vector is pcho 1.0; the second expression vector is pCHO1.0-hygromycin.

In one embodiment, the monovalent unit is an anti-CD 20 antibody, the primers used to amplify its light chain are Kozak (EcoR V) F, MK-leader (EcoRV) F, Rituxan-VL F1 and higk (PacI) R, and the Kozak sequence, leader and cleavage sites EcoR V and PacI are introduced into the light chain by overlapping PCR amplification; primers for amplifying the heavy chain are Kozak (Avr II) F, MK-leader sequence (AvrII) F, Rituxan-VH F1 and hIgG1(sbfI) R, and Kozak sequence, leader sequence and cleavage site AvrI and BstZL7I are introduced into the heavy chain by overlapping PCR amplification; carrying out homologous recombination on the amplified LC gene fragment and a pCHO1.0 expression vector cut by EcoR V and PacI enzyme to obtain an expression vector filled with an anti-CD 20 light chain; then carrying out enzyme digestion on AvrI and BstZL7I, and then carrying out homologous recombination on the AvrI and BstZL7I and HC to obtain a pCHO1.0 expression vector of anti-CD 20, wherein the plasmid is named as pCHO1.0-anti-CD 20-HL-LDY;

the single-chain unit is an anti-CD 3ScFv-Fc antibody, primers used for amplifying the anti-CD 3ScFv-Fc antibody are Kozak (avr II) F, MK-leader sequence (avrI) F, L2K-VH (MK) F1 and hIgG1(sbfI) R, an anti-CD 3ScFv-Fc domain is amplified through overlapping PCR, the Kozak sequence, the leader sequence, the enzyme digestion site AvrII and BstZL7I are introduced into ScFv-Fc, the amplified gene fragment and the enzyme digested pCHO1.0-hygromycin expression vector are subjected to homologous recombination to obtain the expression vector filled with the anti-CD 3ScFv-Fc, and the plasmid is named pCHO1.0-hygromycin-L2K-ScFv-Fc-KKW.

In one embodiment, the use of a bispecific antibody of any one of the above or a bispecific antibody prepared according to any one of the above methods in the manufacture of a medicament for the treatment of a tumor or related disease caused by the expression of a CD 20-specific antigen or for killing a cell expressing CD 20.

In one embodiment, the use of a bispecific antibody of any one of the above or a bispecific antibody prepared according to any one of the above methods for the manufacture of a medicament for screening for a medicament for the treatment of a tumor cell associated disease expressing a CD20 specific antigen in a CD20 tumor cell line or for evaluating the efficacy of a medicament for the treatment of a tumor cell associated disease expressing a CD20 specific antigen.

The invention also provides the following technical scheme:

the invention provides a new method for preparing a bispecific antibody MSBODY (ScFv and monomerbispecific antibody) (as shown in figure 2), the bispecific antibody comprises two groups of heavy-light chain combinations, one group of the heavy-light chain combinations specifically binds to an antigen, and the heavy-chain Fc region is modified for some times, so that the bispecific antibody is not easy to form a dimer by itself compared with a wild type antibody; while the other group specifically binds to the other antigen, and is also less prone to form dimers themselves by additional modifications in its heavy chain Fc region, whereas hybrid dimers are readily formed between the two heavy and light chains. And the antibody structure of one group is a monomeric Ab, and the other group is ScFv-Fc, so that the possibility of mismatching of the respective light chain and the heavy chain of the other party is avoided, and the bispecific antibody protein molecule with 125KD is formed. After Fc transformation, heavy chain and single chain of the monomeric Ab are naturally heterodimerized, and CL and CH1 are naturally dimerized at the same time, finally MSBODY is formed, and the arrangement sequence and the structural schematic diagram of each domain of the MSBODY are shown in FIG. 2.

In the present invention, the bispecific antibody is prepared by the above method for preparing a bispecific antibody. Among them, bispecific antibodies targeting CD20 and CD3 were named CD20 × CD3, as shown in fig. 2, with anti-CD 20 being in the form of IgG comprising heavy and light chains of anti-CD 20 and anti-CD3 being in the form of ScFv-Fc comprising VH, VL, Fc domains of anti-CD 3. The bispecific antibody is constructed by an antibody genetic engineering method, and a binary expression vector of a monomer Ab heavy chain and a monomer Ab light chain of the bispecific antibody MSBODY and an ScFv-Fc expression vector. Primers were designed based on LC, HC, ScFv, Fc gene sequences and the multiple cloning sites in the vector. Wherein LC, HC, ScFv and Fc are respectively subjected to PCR amplification, gene fragments are obtained by a PCR method or an overlap extension PCR method, and then cloning is carried out by a homologous recombination method. The pCHO1.0 or pCHO1.0-hygromycin vector is enzyme-cut, then the PCR product and the enzyme-cut vector are purified and recovered, the LC fragment and the HC fragment are cloned on the pCHO1.0 vector through homologous recombination and the ScFv-Fc fragment is cloned on the pCHO1.0-hygromycin vector through homologous recombination in two steps, and the sequencing is carried out. The expression and detection of recombinant protein MSBODY in mammalian cell, co-transfecting two kinds of plasmid expressing monomer Ab heavy chain, monomer Ab light chain and Single chain with transfection reagent, collecting supernatant and SDS-PAGE and Western blotting to detect the expression of MSBODY. Centrifuging the supernatant of the culture solution after transfection expression, filtering, diluting with a binding buffer solution, passing through an affinity chromatography column, eluting with an elution buffer solution, and detecting and purifying the protein by SDS-PAGE.

The technical scheme of the invention has the beneficial technical effects that:

1. the invention discloses establishment and application of a novel bispecific antibody MSBODY mediated animal model for killing tumor cells by immune cells. The invention comprises mediated immune cell killing in the process of bispecific antibody drug research, preparation of bispecific antibody, establishment of drug effect model by tumor cell line expressing human CD20 protein, and detection of drug effect of bispecific antibody. The bispecific antibody MSBODY comprises a group of heavy-light chain combinations, and the other group is ScFv connection Fc combinations, wherein one group is specifically combined with a human tumor cell antigen, including a series of tumor cell membrane surface antigens such as CD20, and the heavy chain Fc region is modified to ensure that the heavy chain Fc region is not easy to form a dimer by itself relative to a wild type; while the other group specifically binds to the other human T cell antigen CD3, and some additional modifications to the Fc region of its heavy chain are also less prone to form dimers themselves, whereas hybrid dimers are readily formed between the two heavy and light chains. Meanwhile, the bispecific antibody can bridge between a target cell and a functional molecule (cell), stimulate a guided immune response, and has a wide application prospect in the immunotherapy of tumors.

2. The present application provides a heterodimeric antibody comprising two distinct antigen-binding polypeptide units. The heterodimer has different molecular weight from the corresponding homodimer, and the purity of the bispecific antibody can be conveniently determined by using the molecular weight to distinguish the heterodimer from the homodimer. One of these two antigen-binding polypeptide units comprises a light chain-heavy chain pair similar to a wild-type antibody, which unit is also referred to as a "monovalent unit" throughout this application. Another antigen-binding polypeptide unit comprises a single chain variable fragment (ScFv). Such ScFv can be fused to the constant fragment (Fc) of an antibody. This fusion peptide is also referred to as a "single-chain unit" throughout the application.

Surprisingly, the present application demonstrates that such asymmetric antibodies are stable and have high antigen binding efficiency. This is surprising, since it has been demonstrated that homodimers of even single chain antibodies are unstable under physiological conditions. For example, the "ScFv Antibody: Principles and Clinical Application," Clinical and Developmental Immunology,2012:980250(2012) by Ahmad et al, shows that ScFv-based IgG class antibodies are unstable and need further modification to reduce aggregation and improve stability.

In addition, because of the asymmetry, heterodimers have different isoelectric points than homodimers, which are composed of any of the antigen-binding polypeptide units. Based on the isoelectric point difference between heterodimers and homodimers, the desired heterodimers can be easily separated from homodimers, greatly reducing difficulties in downstream process development that are prevalent with bispecific antibodies.

Drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed to be used in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments described in the present application, and other drawings can be obtained by those skilled in the art without inventive efforts, wherein:

FIG. 1 is a molecular structure diagram of CD 3.

FIG. 2 is a schematic representation of a CD20 × CD3 bispecific antibody molecule.

FIG. 3 is a diagram of the detection of PCR products by electrophoresis, M: DL10000 nucleic acid molecular marker; 1. an anti-CD 20 antibody heavy chain; 2. anti-CD 20 antibody light chain.

FIG. 4 is a diagram of the detection of PCR products by electrophoresis, M: a DL2000 nucleic acid molecular marker; 1. the anti-CD3 antibody ScFv-Fc.

FIG. 5 purified diabody electrophoresis and purity detection scheme, (A) CD20 × CD3 diabody SDS-PAGE, M: protein molecular weight markers; 1: non-reducing SDS-PAGE electrophoresis detection; 2: reducing SDS-PAGE electrophoresis detection; (B) HPLC-SEC purity peak profile of CD20 × CD 3.

FIG. 6 is an affinity diagram of the CD20 × CD3 diabody with Raji cells, determined based on flow cytometry; (●) Rituxan control monoclonal antibody, (■) CD20 × CD3 bispecific antibody.

FIG. 7 is an affinity diagram of CD20 × CD3 diabody and Jurkat cells determined based on flow cytometry; (●) L2K control monoclonal antibody, (■) CD20 × CD3 bispecific antibody.

FIG. 8 flow assay CD20 × CD3 diabody binds to both Raji and Jurkat cells simultaneously and promotes co-binding of 2 cells; (. tangle-solidup.) M903: CD20 × CD3 diabodies.

FIG. 9 is a graph showing the results of measuring the Tm of the CD20X CD3 diabody by differential scanning calorimeter.

FIG. 10 shows the result of the detection of the binding activity of CD20 × CD3 diabody to Raji cell surface CD20 after heat treatment, (●) CD20 × CD3 diabody; (■) Rituxan monoclonal antibody.

FIG. 11 is a graph of CD20 CD3 diabody heat-treated for binding activity to Jurkat cell surface CD3, (. tangle-solidup.) CD20 CD3 diabody; the t.tag) Anti-CD3 monoclonal antibody L2K.

FIG. 12 is a CIK phenotype test result chart showing NK-like cells double positive for CD3 and CD56 in the upper right corner.

FIG. 13 is a diagram showing the result of the killing effect of effector cell CIK on target cell Raji in the presence of antibodies of different concentrations by flow detection; (■) M903: CD20 × CD3 (all formats should be kept consistent) diabodies, (. tangle-solidup) Mco 101: control 4420 × CD3 diabody, (xxx) Rituxan: CD20 mab, (●) hIgG: human IgG.

FIG. 14 is a graph showing the results of flow detection of the killing effect of effector cell PBMC on target cells Raji in the presence of antibodies of different concentrations; (■) M903: CD20 × CD3 diabody, (. tangle-solidup) Rituxan: CD20 mab, (tχ) Mco 101: control 4420X CD3 diabody, (●) hIgG: human IgG.

FIG. 15 is a graph showing the results of in vivo potency test of diabodies, wherein (A) shows that only the tail vein is administered with culture medium after tumor cell inoculation, (B) the tail vein is administered with T cells, and (C) the tail vein is injected with T cells coated with CD20x XCD 3 diabodies.

Detailed Description

Example 1: construction of expression vector for bispecific antibody (CD20 XCD 3, M903)

1. Bispecific antibody sequence design

Bispecific antibodies targeting CD20 and CD3 were designated M903, as in fig. 2, anti-CD 20 is here in the form of an IgG comprising an anti-CD 20 heavy and light chain, containing Fab and Fc domains; anti-CD3 is here in the form of an ScFv-Fc comprising an anti-CD 3VH, VL, Fc domain. Wherein the IgG form is subjected to LDY modification on one side of Fc, ScFv-Fc is subjected to KKW modification on one side of Fc, and the specific Fc modification process is shown in PCT/CN2012/084982, so that the IgG form is not easy to form a homodimer and is easy to form a hybrid dimer, namely the CD20 multiplied by CD3 bispecific antibody. Meanwhile, in order that the diabody can be expressed in CHO cells and secreted into the medium, a leader peptide sequence of the murine kappa chain was selected as a secretion signal peptide. The amino acid sequence and nucleic acid sequence of each domain and signal peptide are shown in SEQ ID NO 1-8.

anti-CD 20 heavy chain amino acid sequence (SEQ ID NO: 1)

QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCRVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFFLASKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

anti-CD 20 heavy chain nucleic acid sequence (SEQ ID NO: 2)

caggtacaactgcagcagcctggggctgagctggtgaagcctggggcctcagtgaagatgtcctgcaaggcttctggctacacatttaccagttacaatatgcactgggtaaaacagacacctggtcggggcctggaatggattggagctatttatcccggaaatggtgatacttcctacaatcagaagttcaaaggcaaggccacattgactgcagacaaatcctccagcacagcctacatgcagctcagcagcctgacatctgaggactctgcggtctattactgtgcaagatcgacttactacggcggtgactggtacttcaatgtctggggcgcagggaccacggtcaccgtctctgcagcctccaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaaagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgccgggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctgaagtccgacggctccttcttcctcgccagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaa

anti-CD 20 light chain amino acid sequence (SEQ ID NO: 3)

QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

anti-CD 20 light chain nucleic acid sequence (SEQ ID NO. 4)

caaattgttctctcccagtctccagcaatcctgtctgcatctccaggggagaaggtcacaatgacttgcagggccagctcaagtgtaagttacatccactggttccagcagaagccaggatcctcccccaaaccctggatttatgccacatccaacctggcttctggagtccctgttcgcttcagtggcagtgggtctgggacttcttactctctcacaatcagcagagtggaggctgaagatgctgccacttattactgccagcagtggactagtaacccacccacgttcggaggggggaccaagctggaaatcaaacgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgt

anti-CD 3ScFv-Fc amino acid sequence (SEQ ID NO: 5)

DIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSGGGGSGGGGSGGGGSDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELKGAAAEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

anti-CD 3ScFv-Fc nucleic acid sequence (SEQ ID NO: 6)

gatatcaaactgcagcagtcaggggctgaactggcaagacctggggcctcagtgaagatgtcctgcaagacttctggctacacctttactaggtacacgatgcactgggtaaaacagaggcctggacagggtctggaatggattggatacattaatcctagccgtggttatactaattacaatcagaagttcaaggacaaggccacattgactacagacaaatcctccagcacagcctacatgcaactgagcagcctgacatctgaggactctgcagtctattactgtgcaagatattatgatgatcattactgccttgactactggggccaaggcaccactctcacagtctcctcaggaggcggcggttcaggcggaggtggaagtggtggaggaggttctgacattcagctgacccagtctccagcaatcatgtctgcatctccaggggagaaggtcaccatgacctgcagagccagttcaagtgtaagttacatgaactggtaccagcagaagtcaggcacctcccccaaaagatggatttatgacacatccaaagtggcttctggagtcccttatcgcttcagtggcagtgggtctgggacctcatactctctcacaatcagcagcatggaggctgaagatgctgccacttattactgccaacagtggagtagtaacccgctcacgttcggtgctgggaccaagctggagctgaaaggtgcggccgcagagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgtggtgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacgataccacgcctcccgtgctggactccgacggctccttcttcctctacagcgatctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga

Leader peptide sequence of mouse kappa chain amino acid sequence (SEQ ID NO: 7)

METDTLLLWVLLLWVPGSTG

Leader peptide sequence of mouse kappa chain nucleic acid sequence (SEQ ID NO: 8)

atggagacagacacactcctgctatgggtactgctgctctgggttccaggttccactggt

2. Bispecific antibody gene cloning

pCHO1.0 was selected as an expression vector for cloning and expressing the anti-CD 20 heavy and light chain genes, and pCHO1.0-hygromycin expression vector was modified by replacing the puromycin gene in pCHO1.0 with the hygromycin resistance gene, and was selected for cloning and expressing the ScFv-Fc fusion gene of anti-CD 3. The primers in Table 1 were designed according to the cloning protocol and sent to Sovium Kirgiz Biotechnology Inc. for synthesis. PCR amplification is carried out by using the primers in the table 1, the template is a gene plasmid synthesized by genes in an early experiment or subcloned to pCDNA3.1 or pUC57, the PCT/CN2012/084982 patent is described in detail, then anti-CD 20 heavy and light chains are respectively constructed on an expression vector of pCHO1.0, and anti-CD 3ScFv-Fc is constructed on an expression vector of pCHO1.0-hygromycin.

TABLE 1 primers used in bispecific antibody gene cloning

Initial PCR amplification of template DNA: 35ng of template DNA, e.g., the light and heavy chains of the antibody of interest; 1 μ l of 10 μ M forward and reverse primers; 2.5. mu.l of 10 XPCR Buffer; 1 μ l of 10mM dNTP; 1 μ l of 2.5 units/. mu.l Pyrobest DNA polymerase (Takara, R005A); and distilled water to a total volume of 25 μ l were gently mixed in a microfuge tube and spun rapidly in a microfuge to collect the reaction mixture to the bottom of the tube. The PCR reaction was performed using GeneAmp PCR System 9700(Applied Biosystem) and the following settings: 5 minutes at 95 ℃; the following 25 cycles: at 95 ℃ for 30 seconds each time; 56 ℃ for 30 seconds; and 72 ℃ for 1 minute.

The Kozak sequence, leader sequence and cleavage sites EcoR V and PacI were introduced into the light chain by several rounds of overlapping PCR amplification (fig. 3); and corresponding primers introduce the Kozak sequence, leader sequence and cleavage sites AvrII and BstZL7I into the heavy chain (see FIG. 3). Firstly, carrying out homologous recombination on the amplified LC gene fragment and a pCHO1.0 expression vector which is cut by EcoR V and PacI enzyme to obtain an expression vector loaded with an anti-CD 20 light chain; then, the plasmid was cleaved with AvrII and BstZL7I, and then subjected to homologous recombination with HC to obtain an anti-CD 20 pCHO1.0 expression vector, which was named pCHO1.0-anti-CD 20-HL-LDY.

An anti-CD 3ScFv-Fc domain is amplified through overlapping PCR, a Kozak sequence, a leader sequence, an enzyme cutting site AvrII and BstZL7I are introduced into ScFv-Fc, and the amplified gene fragment (shown in figure 4) and an enzyme-cut pCHO1.0-hygromycin expression vector are subjected to homologous recombination to obtain an expression vector filled with the anti-CD 3ScFv-Fc, wherein the plasmid is named pCHO1.0-hygromycin-L2K-ScFv-Fc-KKW.

Example 2: bispecific antibody expression and purification

1. Expression of bispecific antibodies

Plasmid bulk extraction was performed using an endotoxin free bulk extraction kit (Qiagen, 12391), the specific procedures were performed according to the instructions provided by the manufacturer. CHO-S cell culture in CD CHO Medium (Gibco, 10743-029) at 37 ℃ with 5% CO according to the instructions provided by the manufacturer₂After culture in a cell incubator and cell preparation, the plasmid pCHO1.0-anti-CD 20-HL-LDY was co-transfected into CHO-S cells using a Maxcell STX electrotransfer instrument, together with pCHO1.0-hygromycin-L2K-ScFv-Fc-KKW, both plasmids were designed to express bispecific antibody M903 against CD20 × CD 3.

On day 2 post-transfection, the culture temperature was adjusted to 32 ℃ and supplemented with 3.5% feed a daily, and after 14 days of culture, expression supernatants were harvested by centrifugation at 800 × g.

2. Purification of bispecific antibodies

The expression supernatant was filtered through a 0.22uM filter, all the Fc domain-carrying antibodies were captured from the expression supernatant using a Mabselect Sure affinity column (available from GE, 18-1153-45,17-5438-01), and equilibration buffer (9.5mM NaH)₂PO₄+40.5mM Na₂HPO₄Ph7.0) and then passed through an affinity column and eluted with elution buffer (50mM citric acid +100mM arginine, ph 3.2). Separation of the target bispecific antibody from the byproducts was achieved by SP cation exchange chromatography, using equilibration buffer A (43.8mM NaH), a cation exchange column from GE (18-1153-44,17-1087-01)₂PO₄+6.2mM Na₂HPO₄pH6.0) is balanced with the column,the sample was diluted with deionized water to a conductivity of between 3.0 and 3.5ms, bound by SP column, and eluted with elution buffer B (43.8mM NaH)₂PO₄+6.2mM Na₂HPO₄+1M NaCl, ph6.0)20 column volumes for linear elution; finally, the displacement Buffer PBS was concentrated. The purified bispecific antibody was tested by SDS-PAGE and SEC and had a purity of 95% or more, as shown in FIG. 5.

Example 3: determination of binding Activity of bispecific antibody to cells (FACS)

The bispecific antibodies of the invention bind to a target antigen on the corresponding cell. Raji (purchased from ATCC, CCL-86) was used as CD 20-positive cells and Jurkat (Jurkat, TIB-152) was used as CD 3-positive cells, and the cell binding activity was measured using the diabody prepared according to the present invention.

1. Detection of binding Activity of bispecific antibody to Raji cells Using flow assay

Sufficient Raji cells were cultured, centrifuged to collect cells, bispecific antibody was simultaneously diluted at a concentration of 1000nmol, 3-fold gradient dilutions were made to obtain 12 concentration gradients for use, the collected cells were washed twice with PBS + 1% FBS, and PBS + 1% FBS was added to resuspend the cells to 4 × 10⁶Cells/ml, cells were plated in 96-well plates at 50ul per well (2 × 10)⁵Cell), 50ul of diluted bispecific antibody was added, the cells were incubated at room temperature for 1 hour, the supernatant was centrifuged, the cells were washed twice with PBS, the cells were resuspended with diluted PE-labeled anti-human IgG FC antibody (Biolegend, 409304), incubated at room temperature for 30 minutes in the dark, washed twice with PBS, resuspended with 100ul of PBS, tested on the machine, and the binding affinity KD value between the diabody and Raji was calculated by analysis with the software GraphPadPrism 5.0. the results show that CD20 × CD3 diabody has good binding activity with CD 20-positive Raji cells, see FIG. 6, whose KD value is 274.5 nM.

2. Detection of binding Activity of bispecific antibody with Jurkat cells by flow assay

Sufficient Jurkat suspension cells were cultured and harvested by centrifugation. The following experiment was performed as in the previous example, 100ul of PBS resuspended cells were tested on the machine and the binding affinity KD of the diabody to Jurkat cells was calculated by analysis with the software GraphPad prism5.0, with mean fluorescence intensity. The results showed that the CD20 XCD 3 diabody had good binding activity to CD 3-positive Jurkat cells, as shown in FIG. 7, with a KD of 571.3 nM.

3. Double antibody mediated co-binding activity assay

Cultured Raji and Jurkat cells were harvested by centrifugation and washed 2 times with PBS, stained with CFSE and PKH-26, respectively, while diluting bispecific antibody at 10ug/ml, 10-fold gradient dilution was performed to obtain 12 concentration gradients for use, the stained Raji and Jurkat cells were centrifuged to remove supernatant, washed two times with PBS + 1% FBS, and resuspended in PBS + 1% FBS to 4 × 10⁶Each cell/ml, mixed well at 1:1, plated into 96-well plates at 50ul per well (2 × 10)⁵Individual cells) were added with 50ul of diluted bispecific antibody, incubated at room temperature for 1 hour, centrifuged to remove supernatant, washed twice with PBS, finally resuspended in 100ul PBS, tested on the machine, analyzed for the rate of double positive cells, and calculated by analysis using software GraphPad prism5.0, showing that CD20 × CD3 diabody can simultaneously bind to CD20 positive Raji cells and CD3 positive Jurkat cells to form a bifluorescent cell complex, see FIG. 8, showing that the rate varied depending on the antibody concentration to a maximum of about 15%

1. Determination of Tm value of bispecific antibody

The thermal stability of the bispecific antibody was determined by differential scanning calorimetry (MicroCal VP-DSC, GE Co.), the diabody samples were purified and replaced in PBS buffer, and calorimetric scanning data were obtained by scanning from 10 ℃ to 100 ℃ at a heating rate of 60 ℃/h, using PBS buffer as a control. The scanning results show (fig. 9) that the Tm values of the bispecific antibodies are all above 70 ℃, showing good thermostability.

2. Thermal challenge experiments for bispecific antibodies

The single-chain antibody fragment (ScFv) is formed by connecting the heavy chain variable region and the light chain variable region via a connecting peptide (Gly4Ser) 3. However, it has been reported that the inherent instability of ScFv may affect the antibodyThe quality of the drug (Michaelson JS1, etc., Anti-tumor activity of stability-engineered IgG-likespecific antibodies targeting TRAIL-R2 and LTbetaR. MAbs.2009 Mar-Apr; 1(2) 128-41. therefore, we diluted the antibody to 0.4mg/ml, respectively 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, 82 deg.C, PCR instrument processing for 1h, centrifuged each tube 15 ul. to take the supernatant, flow-tested according to the following steps, collected single cell suspension and added to 96 well plate, 3 × 10⁵Wells, addition of various treatment antibodies, and addition of fluorescent secondary antibody, flow-based detection, results shown in FIGS. 10 and 11, show CD20 × CD3 diabody bound to CD20 on Raji cells₅₀A value of 60.69; t binding to CD3 on Jurkat cells₅₀A value of 58.99, all show good thermal stability.

Example 5: dual antibody mediated in vitro cell killing assay

Isolation of PBMC cells and CIK cell culture

Taking fresh anticoagulated blood, centrifuging for 5min at 400g, and discarding the supernatant. Adding 10 times of cell volume of erythrocyte lysate, gently blowing and mixing, and lysing for 4-5 minutes at room temperature or on ice. During the lysis process, the red blood cells are preferably lysed by shaking the cells appropriately. Centrifuge at 400g for 5min at 4 ℃ and discard the red supernatant. If the red blood cells are not completely lysed, steps 2 and 3 are repeated once. Washing for 1-2 times. PBS of 5 times the cell pellet volume was added, the pellet was resuspended, centrifuged at 400g at 4 ℃ for 2-3 minutes, and the supernatant was discarded. The washing can be repeated 1 more time, and the washing is performed 1-2 times. According to the requirement of the experiment, after the cell sediment is re-suspended by PBS with proper 4 ℃, the subsequent experiments such as counting and the like can be carried out.

Culturing CIK cells, supplementing 30ml each with CIK cell priming medium (serum-free X-Vivo cell culture medium +750IU/ml IFN-gamma + -2% autologous plasma), and adding to 75cm²Placing in a culture flask with saturated humidity, 37 deg.C, and 5.0% CO₂Culturing in incubator for 24 hr, adding CIK cell stimulating factor mixture (serum-free X-Vivo cell culture solution +75ng/ml Anti-human CD3, 750IU/ml IL-2, 0.6ng/ml IL-1 α), and further placing at saturated humidity, 37 deg.C, and 5.0% CO₂Culturing in an incubator. The next step is to determine the fluid replacement (blood-free) according to the growth of CIK cellsClear X-Vivo broth +750IU/ml IL-2 + -2% autologous plasma), and vials were used to maintain cell growth at a concentration of approximately 2X 10^ 6. Finally, the collected CIK cells were phenotypically examined by flow cytometer FC500, which included: CD3, CD56, CD4 and CD8, and the expression of the cell surface antigens in CIK cells is detected. The detection results are shown in FIG. 12, and the phenotype results show that the CIK cells have 18.1% of CD3 and CD56 double positives, and the cultured cells have a good NK T cell rate.

2. Double-antibody effective mediated CIK cell killing tumor cell detection

Raji single cell suspensions were collected, stained with CFSE at a final concentration of 5uM (see protocol-1 CFSE staining), and after staining the cells were resuspended to 2X 10^5/ml in 10% FBS-1640 from the cell culture, and incubated overnight in 96-well plates at 2X 10^ 4/well, i.e., 100 ul/well. Experimental design the wells to which the cultured CIK cells were added, 50 ul/well, control wells were set, and wells to which CIK cells were not added were supplemented with the same volume of medium. The corresponding antibody, 50 ul/well, was added at the same time as the addition of CIK cells according to the experimental design, and the wells without the addition of antibody were supplemented with the same volume of medium. After 48h the 96 well plate was removed and all supernatants and cell suspensions during this period were collected in 1.5ml EP tubes and centrifuged 500 g.times.5 min. The supernatant was discarded, and 150ul of 1% FBS-PBS was added to each well to resuspend and mix the cells. PI (final concentration of 1ug/ml) is added into each tube 10-15min before the flow type machine to detect CFSE by the flow type machine, and the proportion of PI double positive cells in CFSE positive cells is the death rate of target cells Raji. The detection result is shown in figure 13, the cell killing result shows that the CD20 multiplied by CD3MSBODY bispecific antibody mediated CIK cell killing tumor cells shows good killing effect, and the maximum killing efficiency and EC50 of the cell killing result are obviously stronger than those of rituximab.

3. Double-antibody efficient mediated PBMC cell killing tumor cell detection

A Raji single cell suspension was prepared. Staining with CFSE at a final concentration of 5uM (see protocol-1 CFSE staining for the staining procedure), after staining, resuspending the cells to 2X 10^5/ml with 10% FBS-1640 of the cell culture, and culturing overnight with 2X 10^ 4/well, i.e., 100 ul/well in 96-well plates. Experimental design PBMC cells were added at 50 ul/well, control wells were set, and wells without PBMC cells were supplemented with the same volume of medium. PBMC cells were added with the corresponding antibody, 50 ul/well as designed for the experiment, and wells without antibody were supplemented with the same volume of medium. After 48h the 96 well plate was removed and all supernatants and cell suspensions during this period were collected in 1.5ml EP tubes and centrifuged 500 g.times.5 min. The supernatant was discarded, and 150ul of 1% FBS-PBS was added to each well to resuspend and mix the cells. PI (final concentration of 1ug/ml) is added into each tube 10-15min before the flow type machine to detect CFSE by the flow type machine, and the proportion of PI double positive cells in CFSE positive cells is the death rate of target cells Raji. The detection result is shown in figure 14, the cell killing result shows that the CD20 multiplied by CD3MSBODY bispecific antibody mediates that PBMC cells kill tumor cells and shows good killing effect, and the maximum killing efficiency and EC50 of the cell killing result are obviously stronger than those of rituximab.

Example 6: drug effect detection of bispecific antibody killing subcutaneous transplantation tumor

The pharmacodynamic evaluation of CD20 × CD3MSBODY is completed on a blood spreading tumor model established on the basis of CD20 expression positive raji cells, and the model establishment method is 1x10⁶The drug effect evaluation is that the mice are randomly grouped on day 3 by inoculating raji cells, wherein human T cell cells coated with CD20 × CD3MSBODY are transfused to the antibody treatment group, and 1.6x10⁷One (obtained by culturing anti-human CD3 antibody and IL-2-induced human PBMC), two controls were reinfused with PBS and non-antibody-coated human T cells (all reinfused mice had IL-22000U/mouse), and the mice were reinfused in the same manner on days 6, 9, 12, and 14 after the first reinfusion treatment. The state and the weight of the mice are monitored in real time in the whole treatment process, the mice are observed every day after about 15 days, and the survival rate of each group is counted.

The results obtained after pharmacodynamic evaluation of CD20 × CD3MSBODY based on the above experimental model are shown in fig. 15, where the T cell control group infused with PBS and without CD20 × CD3MSBODY started to die 25 days after inoculation, the PBS group died completely after 28 days, the T cell group died completely after 35 days, and the mice treated with CD20 × CD3MSBODY were observed continuously for 100% survival rate and normal health status for 40 days. This demonstrates that T cells bind and accumulate around CD20 positive tumor cells after being mediated by CD20 xcd 3MSBODY, and that cytotoxic killing of tumor cells by CD3 stimulation of T cell production is achieved, whereas control groups did not achieve timely and complete killing and clearance of tumor cells without T cells or with T cells without CD20 xcd 3MSBODY mediation. The result is consistent with the theoretical design, and the T cells mediated by the CD20 multiplied by the CD3MSBODY can kill CD20 positive tumor cells specifically and play a killing role more than that of simple T cells.

It is to be understood that the invention disclosed is not limited to the particular methodology, protocols, and materials described, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims.

Those skilled in the art will also recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims (12)

1. A bispecific antibody, characterized in that said antibody comprises: (a) a monovalent unit that is a light-heavy chain pair having specific binding capacity for a tumor cell surface antigen that is CD 20; and (b) a single chain unit which is a fusion peptide comprising a single chain variable fragment ScFv and an Fc fragment having a hinge region, a CH2 domain and a CH3 domain, wherein the fusion peptide has specific binding ability to the immune cell surface antigen CD 3;

the single chain unit comprises the antibody anti-CD3 directed to CD3, and the monovalent unit comprises the antibody anti-CD 20 directed to CD 20;

the amino acid sequence of the anti-CD 20 heavy chain is the amino acid sequence shown in the sequence number 1, the amino acid sequence of the anti-CD 20 light chain is the amino acid sequence shown in the sequence number 3, and the amino acid sequence of the anti-CD 3ScFv-Fc is the amino acid sequence shown in the sequence number 5; and the cysteine at position 224 of the anti-CD 20 heavy chain is disulfide-linked to the cysteine at position 213 of the light chain of anti-CD 20, the cysteine at positions 230 and 233 of the anti-CD 20 heavy chain is disulfide-linked to the cysteine at positions 255 and 258 of the anti-CD 3ScFv-Fc, respectively, the anti-CD 20 heavy chain is salt-bridged at positions 396 and 413 to the anti-CD 3ScFv-Fc at positions 428 and 397, and the anti-CD 20 heavy chain is protuberance-into-hole linked at position 370 to the anti-CD 3ScFv-Fc at position 436.

2. The bispecific antibody according to claim 1, characterized in that: the CH2 domain of the single chain unit is located between the ScFv fragment and the CH3 domain; does not contain a CH1 domain.

3. The bispecific antibody according to claim 1, characterized in that: the single-chain variable fragment consists of a light chain variable region and a heavy chain variable region domain, both of which are targeted to epitope CD 3.

4. The bispecific antibody according to claim 1, characterized in that: in a monovalent unit, both the light chain constant region domain and the light chain variable region domain of the light chain target the tumor epitope CD 20; the heavy chain constant domain CH1 and the heavy chain variable domain of the heavy chain also target the tumor epitope CD 20; the light chain is bound to the heavy chain by a disulfide bond; the heavy chain is bound to the fusion peptide by one or more disulfide bonds.

5. The bispecific antibody according to claim 1, characterized in that: the heavy chain in said monovalent unit comprises a human or humanized Fc fragment comprising a human IgG Fc fragment; the Fc fragment of the fusion peptide comprises a human or humanized Fc fragment comprising a human IgG Fc fragment.

6. The bispecific antibody according to claim 5, characterized in that: the human IgG Fc fragment of the monovalent unit and the IgG Fc of the single-chain unit are linked by a salt bridge and a knob-in-hole structure.

7. A method for the preparation of said bispecific antibody according to any one of claims 1 to 6, characterized in that said method comprises the steps of:

(1) respectively constructing heavy chains and light chains of the monovalent units on a first expression vector and constructing the single chain units on a second expression vector; the first expression vector is pCHO1.0 and the second expression vector is pCHO1.0-hygromycin;

(2) co-transfecting the first and second expression vectors together into a cell, the cell being a CHO-S cell, culturing and taking the supernatant;

(3) separating the expression supernatant to obtain a purified bispecific antibody; the separating step comprises: protein A affinity chromatographic column captures all antibodies with Fc structural domains from the expression supernatant, realizes the separation of the target bispecific antibody and byproducts by SP cation exchange chromatography, passes through a Q column, and finally concentrates and replaces buffer PBS.

8. The method according to claim 7, characterized in that in step (1) of the method:

the monovalent unit is anti-CD 20 antibody, primers for amplifying a light chain are Kozak (EcoR V) F, MK-leader sequence (EcoRV) F, Rituxan-VL F1 and hIgK (PacI) R, and the Kozak sequence, the leader sequence and enzyme cutting sites EcoR V and PacI are introduced into the light chain through overlapping PCR amplification; primers for amplifying the heavy chain are Kozak (Avr II) F, MK-leader sequence (AvrII) F, Rituxan-VH F1 and hIgG1(sbfI) R, and Kozak sequence, leader sequence and cleavage site AvrII and BstZL7I are introduced into the heavy chain by overlapping PCR amplification; carrying out homologous recombination on the amplified LC gene fragment and a pCHO1.0 expression vector cut by EcoR V and PacI enzyme to obtain an expression vector filled with an anti-CD 20 light chain; then carrying out enzyme digestion on AvrII and BstZL7I, and then carrying out homologous recombination on the AvrII and BstZL7I and HC to obtain a pCHO1.0 expression vector of anti-CD 20, wherein the plasmid is named as pCHO1.0-anti-CD 20-HL-LDY;

the single chain unit is an anti-CD 3ScFv-Fc antibody, primers used for amplifying the anti-CD 3ScFv-Fc antibody are Kozak (Avr II) F, MK-leader sequence (AvrII) F, L2K-VH (MK) F1 and hIgG1(sbfI) R, an anti-CD 3ScFv-Fc domain is amplified through overlapping PCR, the Kozak sequence, the leader sequence, the enzyme cutting site AvrII and BstZL7I are introduced into ScFv-Fc, the amplified gene fragment and the enzyme-cut pCHO1.0-hygromycin expression vector are subjected to homologous recombination to obtain the expression vector filled with the anti-CD 3ScFv-Fc, and the plasmid is named as pCHO1.0-hygromycin-L2K-ScFv-Fc-KKW.

9. Use of the bispecific antibody of any one of claims 1-6 in the manufacture of a medicament for the treatment of lymphoma caused by expression of a CD 20-specific antigen, or for killing a CD 20-expressing lymphoma cell.

10. Use of a bispecific antibody prepared by the method of any one of claims 7-8 in the preparation of a medicament for the treatment of lymphoma caused by expression of CD 20-specific antigen, or for killing lymphoma cells expressing CD 20.

11. Use of the bispecific antibody of any one of claims 1-6 in the manufacture of a medicament for screening for or evaluating the efficacy of a medicament for the treatment of a lymphoma expressing a CD20 specific antigen.

12. Use of a bispecific antibody prepared by the method of any one of claims 7-8 in the preparation of a medicament for screening for or evaluating the efficacy of a medicament for the treatment of a lymphoma expressing a CD20 specific antigen.

Images