Construction and application of bispecific antibody EpCAM×CD3

Abstract

The present invention provides a bispecific antibody. The bispecific antibody provided by the present invention comprises a single-chain unit and a monovalent unit, wherein the single-chain unit has a specific binding capability against surface antigen CD3 of an immune cell; the monovalent unit has a specific binding capability against the surface antigen EpCAM of a tumor cell; the single-chain unit comprises a single-chain variable fragment ScFv fused with an Fc fragment; and the monovalent unit comprises a light chain and heavy chain pair. The present invention also provides a preparation method of the bispecific antibody and pharmaceutical use of these antibodies.

Claims

1. A bispecific antibody, comprising: (a) a monovalent unit comprising a light chain-heavy chain pair which specifically binds to EpCAM, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 1 and the light chain comprises the amino acid sequence of SEQ ID NO: 3: and (b) a single-chain unit which is a fusion peptide comprising a single-chain variable fragment (scFv) and an FC fragment which comprises a hinge region, a CH2 structural domain and a CH3 structural domain, wherein the single-chain unit has binding specificity to a surface antigen CD3, and wherein the single-chain unit comprises the amino acid sequence of SEQ ID NO: 5.

2. The bispecific antibody of claim 1, wherein the CH2 structural domain of the single-chain unit is positioned between the scFv fragment and the CH3 structural domain and the single-chain unit does not contain a CH1 structural domain.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) Drawings that need to be used in the examples will be briefly introduced below in order to illustrate the technical solution in the examples of the present application more clearly, and it is apparent for those common skilled in the art that the drawings described as below are just some examples recorded in the present invention and other drawings can also be acquired on the basis of those drawings on the premise of not paying creative work, wherein,

(2) FIG. 1 is a structural schematic diagram of the CD3 molecule.

(3) FIG. 2 is a schematic diagram of the EpCAM×CD3 bispecific antibody molecule.

(4) FIG. 3 is an electrophoresis detection PCR product diagram; M: DL10000 nucleic acid molecular marker; 1. Anti-CD3 antibody ScFv-Fc; 2. heavy chain of the anti-EpCAM antibody; and 3. light chain of the anti-EpCAM antibody.

(5) FIG. 4 is a purified bispecific antibody electrophoresis and purity detection result diagram; (4A) denatured SDS-PACE electrophoresis; M: protein molecular weight marker; 1:EpCAM×CD3 bispecific antibody; (4B) non-denatured SDS-PACE electrophoresis; M: protein molecular weight marker; 1:EpCAM×CD3 bispecific antibody; and (4C) HPLC-SEC purity peak shape diagram of EpCAM×CD3.

(6) FIG. 5 is an affinity condition diagram of the EpCAM×CD3 bispecific antibody and HCT116 cells measured based on fluorescence-activated cell sorting, (.square-solid.) EpCAM×CD3 MSBODY; and (.circle-solid.) Anti-EpCAM monoclonal antibody.

(7) FIG. 6 is an affinity condition diagram of the EpCAM×CD3 bispecific antibody and Jurkat cells measured based on fluorescence-activated cell sorting, (.square-solid.) EpCAM×CD3 MSBODY; and (.circle-solid.) Anti-CD3 monoclonal antibody L2K.

(8) FIG. 7 is a result diagram that the EpCAM×CD3 bispecific antibody simultaneously binds to HCT116 cells and Jurkat cells in the process of flow cytometry detection and pulls the two kinds of cells together; (.circle-solid.) EpCAM×CD3 bispecific antibody; (.square-solid.) EpCAM monoclonal antibody; (.box-tangle-solidup.) anti-CD3 monoclonal antibody L2K; and (.Math.) control antibody MCO101.

(9) FIG. 8 is a Tm value result diagram of scanning survey for the EpCAM×CD3 MSBODY bispecific antibody by using a differential scanning calorimeter.

(10) FIG. 9 refers to an activity detection result of the antibody after heat treatment, 9A. binding activity detection with EpCAM; (.circle-solid.) anti-EpCAM monoclonal antibody; (.box-tangle-solidup.) EpCAM×CD3 MSBODY bispecific antibody; 9B. binding activity detection with CD3; (.circle-solid.) anti-CD3 monoclonal antibody L2K; and (.square-solid.) EpCAM×CD3 MSBODY bispecific antibody.

(11) FIG. 10 is a CIK phenotype detection result diagram at the right corner of which double positive NK cells of CD3 and CD56 are located.

(12) FIG. 11 is a killing effect result diagram of effector cells CIK on target cells HCT116 in the presence of different concentrations of antibodies in the flow cytometry detection; (.square-solid.) EpCAM×CD3 MSBODY bispecific antibody; (.box-tangle-solidup.) Mco101:control 4420×CD3 bispecific antibody; (.Math.) Anti-EpCAM: anti-EpCAM monoclonal antibody; and (.circle-solid.) hIgG: human IgG. EpCAM×CD3.

(13) FIG. 12 a killing effect result diagram of effector cells CIK on target cells NCI-N87 in the presence of different concentrations of antibodies in the flow cytometry detection; (.square-solid.) EpCAM×CD3 MSBODY bispecific antibody; (.box-tangle-solidup.) Mco101:control 4420×CD3 bispecific antibody; (.Math.) Anti-EpCAM: anti-EpCAM monoclonal antibody; and (.circle-solid.) hIgG: human IgG.

(14) FIG. 13 is a killing effect result diagram of effector cells PBMC on target cells HCT116 in the presence of different concentrations of antibodies in the flow cytometry detection; (.square-solid.) EpCAM×CD3 MSBODY bispecific antibody; (.box-tangle-solidup.) Mco101:control 4420×CD3 bispecific antibody; (.Math.) EpCAM: EpCAM monoclonal antibody; and (.circle-solid.) hIgG: human IgG.

(15) FIG. 14 is a killing effect result diagram of effector cells PBMC on target cells NCI-N87 in the presence of different concentrations of antibodies in the flow cytometry detection; (.square-solid.) EpCAM×CD3 MSBODY bispecific antibody; (.box-tangle-solidup.) Mco101:control 4420×CD3 bispecific antibody; (.Math.) EpCAM: EpCAM monoclonal antibody; and (.circle-solid.) hIgG: human IgG.

(16) FIG. 15 refers to a pharmacological experiment result in the bispecific antibody, mice: NOD-SCID; inoculation (i. h): associative inoculation of SW480(5×10.sup.6) and human CIK (5×10.sup.6): administration (i. v): EpCAM mAb: 4 mg/kg; Day (0,2,4), 4420×CD3: 4 mg/kg; Day (0,2,4),EpCAM×CD3(MSBODY)-1: 4 mg/kg; Day (0,2,4),EpCAM×CD3(MSBODY)-2: 2 mg/kg; Day (0,2,4); (.square-solid.) represents PBS which is administrated just through caudal vein; (□) anti-EpCAM monoclonal antibody; (Δ) 4420×CD3 unrelated control bispecific antibody; (.Math.) M701-1:EpCAM×CD3 SMBODY bispecific antibody 4 mg/kg concentration group; and (∘) M701-2: EpCAM×CD3 SMBODY bispecific antibody 2 mg/kg concentration group.

SPECIFIC MODES FOR CARRYING OUT THE INVENTION

(17) The present invention will be further described in detail bellow in conjunction with the specific examples and by reference to the drawings. It should be understood that the examples in the description are just for the purpose of illustrating the present invention, but not limiting the scope of the present invention in any way.

EXAMPLE 1

Construction of Expression Vector of Bispecific Antibody (EpCAM×CD3, M701)

(18) 1. Sequence Design of Bispecific Antibody

(19) The bispecific antibody taking EpCAM and CD3 as targets is named as M701 (as shown in FIG. 2), wherein the anti-EpCAM side is of an IgG form, includes anti-EpCAM heavy chain and light chain and contains Fab and Fc structural domains; the anti-CD3 side is of an ScFv-Fc form and comprises anti-CD3 VH, VL and Fc structural domains. Wherein, Fc of the side of the IgG form is subject to KKW transformation, whereas Fc of the ScFv-Fc side is subject to LDY transformation (the specific Fc transformation process refers to PCT/CN2012/084982), so that each of which is not easy to form a homodimer, but is easy to form a heterodimer, namely the EpCAM×CD3 bispecific antibody. In the meantime, in order to ensure that the bispecific antibody can be expressed in a CHO cell and secreted into a culture medium, a leading peptide sequence of a mouse-derived kappa chain is selected as a secretary signal peptide. The amino acid sequences and the nucleotide sequences of all structural domains and the signal peptide are as shown in SEQ ID No. 1-8.

(20) TABLE-US-00002 Anti-EpCAM Heavy Chain (Amino Acid Sequence SEQ ID NO. 1) EVQLLEQSGAELVRPGTSVKISCKASGYAFTNYWLGWVKQRPGHGLEWIGDIFPGSGNI HYNEKFKGKATLTADKSSSTAYMQLSSLTFEDSAVYFCARLRNWDEPMDYWGQGTTV TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE LLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVY TLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSD LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Anti-EpCAM Heavy Chain (Nucleotide Sequence SEQ ID NO. 1) GAGGTGCAGCTGCTCGAGCAGTCTGGAGCTGAGCTGGTAAGGCCTGGGACTTCA GTGAAGATATCCTGCAAGGCTTCTGGATACGCCTTCACTAACTACTGGCTAGGTTG GGTAAAGCAGAGGCCTGGACATGGACTTGAGTGGATTGGAGATATTTTCCCTGGA AGTGGTAATATCCACTACAATGAGAAGTTCAAGGGCAAAGCCACACTGACTGCAG ACAAATCTTCGAGCACAGCCTATATGCAGCTCAGTAGCCTGACATTTGAGGACTCT GCTGTCTATTTCTGTGCAAGACTGAGGAACTGGGACGAGCCTATGGACTACTGGG GCCAAGGGACCACGGTCACCGTCTCCTCCGCGTCGACCAAGGGCCCATCGGTCTT CCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGC CTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCC TGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTC CCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATC TGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCC AAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGG GGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTC CCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGA GGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAA GCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGT CCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAA AGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCG AGAACCACAGGTCTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAG GTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGT GGGAGAGCAATGGGCAGCCGGAGAACAACTACGATACCACGCCTCCCGTGCTGG ACTCCGACGGCTCCTTCTTCCTCTACAGCGATCTCACCGTGGACAAGAGCAGGTG GCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCAC TACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA Anti-EpCAM Light Chain (Amino Acid Sequence SEQ ID NO. 3) ELVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWAS TRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCQNDYSYPLTFGAGTKLEIKRTVAA PSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDS TYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Anti-EpCAM Light Chain (Nucleotide Sequence SEQ ID NO. 4) GAGCTCGTGATGACACAGTCTCCATCCTCCCTGACTGTGACAGCAGGAGAGAAGG TCACTATGAGCTGCAAGTCCAGTCAGAGTCTGTTAAACAGTGGAAATCAAAAGAA CTACTTGACCTGGTACCAGCAGAAACCAGGGCAGCCTCCTAAACTGTTGATCTAC TGGGCATCCACTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTG GAACAGATTTCACTCTCACCATCAGCAGTGTGCAGGCTGAAGACCTGGCAGTTTA TTACTGTCAGAATGATTATAGTTATCCGCTCACGTTCGGTGCTGGGACCAAGCTTG AGATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAG CAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAG AGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAG GAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACC CTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTC ACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT TAG Anti-CD3 ScFv-Fc (Amino Acid Sequence SEQ ID NO. 5) QVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQRPGQGLEWIGY INPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVYYCARYY DDHYCLDYWGQGTTLTVSSGGGGSGGGGSGGGGSQIVLTQSPAIMSASPG EKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYDTSKLASGVPAHFRGSGS GTSYSLTISGMEAEDAATYYCQQWSSNPFTFGSGTKLEINRGAAAEPKSC DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK CKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG NVFSCSVMHEALHNHYTQKSLSLSPGK Anti-CD3 ScFv-Fc (Nucleotide Sequence SEQ ID NO. 6) CAGGTGCAGCTGGTGCAGAGCGGCGGCGGCGTCGTGCAGCCGGGCAGGTC CCTGAGACTGTCTTGTAAGGCTTCTGGATACACCTTCACTAGATACACAA TGCACTGGGTCAGACAGGCTCCTGGAAAGGGACTCGAGTGGATTGGATAC ATTAATCCTAGCAGAGGTTATACTAACTACAATCAGAAGTTTAAGGACAG ATTCACAATTTCTACTGACAAATCTAAGAGTACAGCCTTCCTGCAGATGG ACTCACTCAGACCTGAGGATACCGGAGTCTATTTTTGTGCTAGATATTAC GATGACCACTACTGTCTGGACTACTGGGGCCAAGGTACCCCGGTCACCGT GAGCTCAGGAGGCGGCGGTTCAGGCGGAGGTGGAAGTGGTGGAGGAGGTT CTGATATTCAGATGACCCAGAGCCCGTCAAGCTTATCTGCTTCTGTCGGA GACAGAGTCACAATCACATGTTCTGCTTCTAGCTCTGTCTCTTACATGAA CTGGTATCAGCAGACACCTGGAAAGGCTCCTAAGCGGTGGATCTACGACA CATCTAAGCTCGCTTCTGGAGTCCCTTCTAGATTCTCTGGTTCTGGCTCT GGAACAGACTACACATTCACAATCTCTTCTCTCCAACCTGAGGACATCGC TACATACTACTGCCAACAGTGGTCTAGCAATCCTTTCACATTCGGACAGG GTACCAAACTGCAGATCACAAGAGGTGCGGCCGCAGAGCCCAAATCTTGT GACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGG ACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCT CCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGAC CCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGC CAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCA GCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAG TGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTC CAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCAT CCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAA GGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCC GGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCT TCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGG AACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACAC GCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA Leading Peptide Sequence (Amino Acid Sequence SEQ ID NO. 7) of Mouse-Derived Kappa Chain METDTLLLWVLLLWVPGSTG Leading Peptide Sequence (Amino Acid Sequence SEQ ID NO. 8) of Mouse-Derived Kappa Chain atggagacagacacactcctgctatgggtactgctgctctgggttccaggttccactggt

(21) 2. Gene Cloning of Bispecific Antibody

(22) pCHO1.0 was selected as an expression vector for cloning and expressing anti-EpCAM heavy chain and light chain genes, and a pCHO1.0-hygromycin expression vector was obtained by modification through replacing a puromycin gene in a pCHO1.0 vector with a hygromycin resistant gene and was selected to clone and express the anti-CD3 ScFv-Fc fusion gene. The primers in Table 1 were delivered to GENEWIZ, Inc, Suzhou for synthesis after being designed according to a cloning solution. The primers as shown in Table 1 were subject to PCR amplification, a gene plasmid obtained from gene synthesis or subcloned to pCDNA3.1 or pUC57 in the early-stage experiment acted as a template (which was described in PCT/CN2012/084982 patent in detail), and then the anti-EpCAM heavy and light chains were established to the pCHO1.0 expression vector respectively, and the anti-CD3 ScFv-Fc was established onto the pCHO1.0-hyromycin expression vector.

(23) TABLE-US-00003 TABLE 1 Primers Used in Gene Cloning of Bispecific Antibody SEQ Names of ID Fragments Names of Primers NO. Sequences Anti-EpCAM Kozak(EcoR V)F  9 gaggaaggatctcgagctcaagcttgatatcgccgccaccatg LC MK-Leading 10 CAATTgatatcgccgccaccatggagacagacacactcctgctat Sequence (EcoRV)F gggtactgctgctc M701-VL F1 11 tgctatgggtactgctgctctgggttccaggttccactggtgagctcgtg atgacacag hIgK (PacI)R 12 cttatcatgtctggatcgaagcttaattaactaacactctcccctgttgaa g Anti-EpCAM Kozak(Avr II)F 13 cccgaggaggaacggttccgggccgcctagggccgccaccatg HC MK-Leading 14 CAATTcctagggccgccaccatggagacagacacactcctgctat Sequence (AvrII)F gggtactgctgctc M701-VH F1 15 tgctatgggtactgctgctctgggttccaggttccactggtgaggtgca gctgctcgag hIgG1(sbfI)R 16 catagagtataatatagagtatacacctgcaggtcatttacccggagac agggag Anti-CD3 Kozak(Avr II)F 17 cccgaggaggaacggttccgggccgcctagggccgccaccatg ScFv-Fc MK-Leading 18 CAATTcctagggccgccaccatggagacagacacactcctgctat Sequence (AvrII)F gggtactgctgctc L2K-VH(MK)F1 19 gctatgggtactgctgctctgggttccaggttccactggtgatatcaaac tgcagcagt hIgG1(sbfI)R 20 catagagtataatatagagtatacacctgcaggtcatttacccggagac agggag

(24) Initial PCR amplification template DNA: 35 ng template DNA, such as a light chain and a heavy chain of a target antibody; 1 μl of 10 μM forward primer and reverse primer; 2.5 μl of 10×PCR Buffer solution; 1 μl of 10 mM dNTP; 1 μl of 2.5 unit/μl Pyrobest DNA polymerase (Takara, R005A); and distilled water to 25 μl by total volume, all of which were softly mixed in a microfuge tube and rapidly rotated in a microcentrifuge so as to collect the reaction mixture to the bottom of the tube. PCR reaction was performed by using Gene Amp PCR System 9700 (Applied Biosystem) according to the following settings: 5 minutes at 95° C.; and 25 cycles as below: 30 seconds each time at 95° C.; 30 seconds at 56° C.; and 1 minute at 72° C.

(25) Via several cycles of overlap PCR amplification, the Kozak sequence, the leader sequence and restriction enzyme cutting sites EcoRV and Pad were introduced into the light chain (as shown in FIG. 3); and the Kozak sequence, the leader sequence and restriction enzyme cutting sites AvrII and BstZ17I were introduced into the heavy chain by the corresponding primers (as shown in FIG. 3). Firstly, the amplified LC gene fragment was subject to homologous recombination with a pCHO1.0 expression vector suffering restriction enzyme cutting via EcoRV and Pad to obtain the anti-EpCAM light chain-loaded expression vector, and was then subject to homologous recombination with HC after suffering restriction enzyme cutting via AvrII and BstZ17I to obtain the anti-EpCAM pCHO1.0 expression vector of which the plasmid is named as pCHO1.0-anti-EpCAM-HL-KKW.

(26) The anti-CD3ScFv-Fc-loaded expression vector of which the plasmid is named as pCHO1.0-hygromycin-L2K-ScFV-Fc-LDY was obtained through implementing overlap PCR amplification of an anti-CD3ScFv-Fc structural domain, introducing the Kozak sequence, the leader sequence and restriction enzyme cutting sites AvrII and BstZ17I into ScFv-Fc and carrying out homologous recombination on the amplified gene fragment (as shown in FIG. 3) and the pCHO1.0 expression vector suffering restriction enzyme cutting.

EXAMPLE 2

Expression and Purification of Bispecific Antibody

(27) 1. Expression of Bispecific Antibody

(28) Plasmid maxiprep was performed by using an endotoxin-free maxiprep kit (Qiagen, 12391) and specific operations were performed according to the instructions provided by the manufacturer. CHO-S cell culture was performed in a CD CHO culture medium (Gibco, 10743-029) at 37° C. in a 5% CO.sub.2 cell incubator according to the instructions provided by the manufacturer, and after the cells were prepared, plasmids pCHO1.0-anti-EpCAM-HL-KKW and pCHO1.0-Herceptin-L2K-ScFv-Fc-LDY were co-transfected to the CHO-S cells by using a Maxcyte STX electroporation apparatus so as to express the bispecific antibody M701 directed to anti-EpCAM×CD3 according to the instructions (Maxcyte) provided by the manufacturer.

(29) After the second day of co-transfection, the culture temperature drops to 32° C., 3.5% Feed A was replenished every day, and after culture for 14 days, the supernatant was harvested by 800*g centrifugal.

(30) 2. Purification of Bispecific Antibody

(31) The expression supernatant was filtered with a 0.22 uM filter membrane, an antibody with an Fc structural domain was captured from the expression supernatant by using a Mabselect SuRe affinity column (purchased from GE Company, Column Art. No. 18-1153-45 and Filler Art. No. 17-5438-01), passed through the affinity column which was balanced with an equilibration buffer solution (9.5 mM NaH.sub.2PO.sub.4+40.5 mM Na.sub.2HPO.sub.4,pH7.0) and was eluted with an elution buffer solution (50 mM citric acid+100 mM arginine, pH3.2). The target bispecific antibody and byproducts were separated by means of SP cation exchange chromatography, wherein the cation exchange column was purchased from GE Company (Column Art. No. 18-1153-44,17-1087-01); and after the column was balanced with an equilibration buffer solution A (43.8 mM NaH.sub.2PO.sub.4+6.2 mM Na.sub.2HPO.sub.4, pH6.0), a sample was diluted with double pure water, was electrically conducted to a range from 3.0 ms to 3.5 ms and was subject to linear elution of 20 column volumes with an elution buffer solution B (43.8 mM NaH.sub.2PO.sub.4+6.2 mM Na.sub.2HPO.sub.4+1M NaCl, pH6.0) after being combined with an SP column; and finally, Buffer PBS was concentrated and displaced. The purified bispecific antibody had the purity over 95% via SDS-PAGE and SEC detection (as shown in FIG. 3).

EXAMPLE 3

Binding Activity Measurement (FACS) of Bispecific Antibody and Cells

(32) The bispecific antibody of the present invention binds to target antigens on the corresponding cells. As concerned in the present invention, with HCT116 (purchased from American Type Culture Collection ATCC, CCL-247) as an EpCAM positive cell and Jurkat (Jurkat, TIB-152) as a CD3 positive cell, the cell binding activity therebetween was measured by means of the bispecific antibody prepared in the present invention.

(33) 1. Detection of Binding Activity of Bispecific Antibody and the HCT116 Cells Via Fluorescence-Activated Cell Sorting

(34) Enough HCT116 cells are cultured, digested with 0.25% trypsin and then collected by centrifugation. In the meantime, the bispecific antibody was diluted according to the concentration beginning from 10 ug/ml and ten-fold gradient dilution to obtain twelve concentration gradients for later use. The collected cells are washed twice with PBS+1% FBS and resuspended to 4×10.sup.6 cell/ml with PBS+1% FBS and plated in a 96-well plate each well of which was loaded with 50 ul (2×10.sup.5 cells), 50 ul of diluted bispecific antibody was added and cells were incubated for 1 hour at room temperature; and supernatant was removed by centrifugation, cells were washed twice with PBS, then resuspended with a diluted PE-marked anti-human IgG FC antibody (Biolegend, 409304), incubated for 30 minutes at room temperature in a dark place, washed twice with PBS, then resuspended with 100 ul PBS and detected on an instrument, and then, the binding affinity KD value of the bispecific antibody and the HCT116 was analyzed and calculated according to the mean fluorescence intensity by using software GraphPadPrism 5.0. The result displaysed that the EpCAM×CD3 bispecific antibody had favorable binding activity with the EpCAM-positive HCT116 cells, and as shown in FIG. 5, the KD value was 9.602 nM, the KD detection result of Anti-EpCAM was 1.661 nM.

(35) 2. Detection of Binding Activity of the Bispecific Antibody and Jurkat Cells Via Fluorescence-Activated Cell Sorting

(36) Enough Jurkat suspension cells were cultured and collected by centrifugation. The same as the steps described in the example forementioned, in the following experimental process, the cells resuspended with 100 ul PBS were detected on an instrument, and the binding affinity KD value of the bispecific antibody and the Jurkat cells was analyzed and calculated according to the mean fluorescence intensity by using software GraphPadPrism 5.0. The result displayed that the EpCAM×CD3 bispecific antibody had favorable binding activity with the CD3-positive Jurkat cells, and as shown in FIG. 6, the KD value was 14.27 nM which displayed the favorable affinity.

(37) 3. Co-Binding Experiment of Bispecific Antibody-Mediated Immune Cells and Tumor Cells

(38) Cultured HCT116 and Jurkat cells were collected by centrifugation, washed twice with PBS and stained with CFSE and PKH-26 respectively. In the meantime, the bispecific antibody was diluted according to the concentration beginning from 10 ug/ml and ten-fold gradient dilution to obtain twelve concentration gradients for later use. The stained HCT116 and Jurkat cells were centrifuged to remove supernatant, washed twice with PBS+1% FBS, and then resuspended to 4×10.sup.6 cell/ml with PBS+1% FBS respectively, cells were uniformly mixed according to a ratio of 1:1 and plated in a 96-well plate each well of which was loaded with 50 ul (2×10.sup.5 cells), 50 ul of diluted bispecific antibody was added, and cells were incubated for 1 hour at room temperature; and supernatant was removed by centrifugation, the cells were washed twice with PBS and resuspended with 100 ul PBS finally, and the ratio of double positive cells was analyzed through detection on an instrument and was calculated by using software GraphPadPrism 5.0. The result displayed that in case of no M701, the ratio of bifluorescence via flow cytometer detection was very low (as shown in FIG. 7); under the condition of adding the EpCAM×CD3 bispecific antibody M701, the ratio of bifluorescence via flow cytometer detection reached 27.5%, which indicated that M701 could simultaneously bind to EpCAM-positive HCT cells and CD3-positive Jurkat cells and promote the co-aggregation of the two kinds of cells to form an immune killer complex.

EXAMPLE 4

Determination of Thermal Stability of Bispecific Antibody

(39) 1. Tm Value Determination of Bispecific Antibody

(40) The thermal stability of the bispecific antibody was determined by a differential scanning calorimeter (MicroCal VP-DSC, GE Company), a bispecific antibody sample was displaced in a PBS buffer solution after being purified, and calorimetric scanning data was obtained by scanning at a heating velocity of 60° C./hour from 10° C. to 100° C. with the PBS buffer solution as a control. According to the scanning result displayed in FIG. 8, the Tm value of the bispecific antibody was about 70° C., which showed favorable thermal stability.

(41) 2. Thermal Challenge Experiment of Bispecific Antibody

(42) The single chain antibody fragment (ScFv) was formed by connecting a heavy chain variable region and a light chain variable region through a connecting peptide (Gly.sub.4Ser.sub.3). However, it was reported that the inherent instability of ScFv could possibly affect the quality of an antibody drug (Michaelson JS1, etc., Anti-tumor activity of stability-engineered IgG-like bispecific antibodies targeting TRAIL-R2 and LTbetaR. MAbs. 2009 March-April; 1(2):128-41.). Therefore, the antibody was diluted to 0.4 mg/ml and was respectively treated for 1 h by a PCR instrument at 4° C., 37° C., 42° C., 47° C., 52° C., 57° C., 62° C., 67° C., 72° C., 77° C. and 82° C. with 15 ul each tube. The supernatant was taken by centrifugation, and the flow cytometer detection was performed according to the following steps: collecting a single cell suspension, adding into a 96-well plate with 3×10.sup.5 cell/well, adding various processing antibodies, then adding a fluorescent secondary antibody, and carrying out flow cytometer detection on an instrument, wherein the detection result was as shown in FIG. 9, the thermal stability of the Anti-EpCAM and the EpCAM×CD3 MSBODY bispecific antibody, both of which bound to EpCAM, respectively, was determined as shown in FIG. 9A, and the T.sub.50 values of both were 73.28 and 61.01 respectively; and the thermal stability of the L2K and the EpCAM×CD3 MSBODY bispecific antibody, both of which bound to the CD3 antibody respectively, was determined as shown in FIG. 9B, wherein the T.sub.50 values were 69.33 and 60.30 respectively, both of which displayed better thermal stability.

EXAMPLE 5

Bispecific Antibody-Mediated In Vitro Cell-Killing Detection

(43) 1. PBMC Cell Separation and CIK Cell Culture

(44) Fresh anti-freezing human blood was subject to 400 g centrifugal for 5 min and supernatant was discarded. 10-fold cell volume of red blood cell lysis buffer was added to the human blood, uniformly mixed by slightly blowing and beating, and subject to lysis at room temperature or on ice for 4-5 minutes during which appropriate shaking was needed so as to promote red blood cell lysis. 400 g centrifugal was performed for 5 min at 4 □, and red supernatant was discarded. If the red cell lysis was not complete, the step 2 and step 3 were repeated once. Washing was performed for 1-2 times. 5-fold cell sedimentation volume of PBS was added, cells were resuspended to obtain sediment and subject to 400 g centrifugal for 2-3 minutes at 4□, and then supernatant was discarded. The steps were repeated once if necessary and washing was performed for 1-2 times in total. The cells were resuspended to obtain sediment with appropriate 4□ precooled PBS according to experiment demands, and then subsequent experiments, such as counting can be performed.

(45) CIK cells were cultured according to the following steps: replenishing each portion of cells to 30 ml by using a CIK cell initiation culture solution (a serum-free X-Vivo cell culture solution+750 IU/ml IFN-γ±2% autologous plasma), adding the cells to a 75 cm.sup.2 culture flask, and culturing the cells at 37° C. in a 5.0% CO.sub.2 humidified incubator; after culture for 24 hours, adding 1 ml of CIK cell stimulation factor mixed solution (a serum-free X-Vivo cell culture solution+75 ng/ml anti-human CD3ε, 750 IU/ml IL-2 and 0.6 ng/ml IL-1α), and continuously culturing at 37° C. in the 5.0% CO.sub.2 humidified incubator; as concerned in the following steps, determining the matters, such as replenishing of solutions (serum-free X-Vivo cell culture solution+750 IU/ml IL-2±2% autologous plasma) and bottling according to the growth situation of CIK cells to basically maintain the cells to grow at a density about 2*10.sup.6/ml; and finally, carrying out phenotypic detection, including CD3, CD56, CD4 and CD8, on the collected CIK cells, by using a flow cytometry FC500 and detecting the expression situations of these cell surface antigens in the CIK cells. The detection result was as shown in FIG. 10, the phenotype result displayed that the CIK cell had over 35% CD3 and CD56 double positive, and the cultured cell had favorable NK T cell ratio.

(46) 2. Tumor Cell-Killing Detection of Bispecific Antibody-Effectively Mediated EpCAM Cells

(47) A single-cell suspension was prepared by digesting HCT116 or NCI-N87 cells with trypsin. The HCT116 or NCI-N87 cells were stained with CFSE with the final concentration being 5 uM, and the cells were resuspended to 2×10.sup.5/ml with 10% FBS-1640 cultured by these cells after staining, and cultured over night in a 90-well plate according to 2×10.sup.4 cell/well, namely 100 ul/well. According to the experiment design, the cultured CIK cells were added according to 50 ul/well, control wells were set, and the same volume of culture medium was fed into wells in which no CIK cells need to be added. The corresponding antibody was added with 50 ul/well according to the experiment design while the CIK cells were added, and the same volume of culture medium was fed into wells in which no antibody needs to be added. After 48 hours, the 96-well plate was taken out, cells of each well were digested with trypsin to form the single cell suspension, and correspondingly, all the supernatants and the cell suspension in this process were collected into 1.5 ml EP tubes and subject to 500×g centrifugal for 5 minutes. The supernatant was discarded, and 150 ul 1% FBS-PBS was added to each well, and then cells were resuspended and uniformly mixed. PI (the final concentration of 1 ug/ml) was added 10-15 min before each tube was put on an instrument for fluorescence-activated cell sorting, and the proportion of CFSE and PI double positive cells, namely the death rate of target cells HCT116 or NCI-N87 was detected on the instrument for fluorescence-activated cell sorting (the result was as shown in FIG. 11 and FIG. 12). The cell killing result displayed that the EpCAM×CD3 MSBODY bispecific antibody-mediated CIK cells displayed a favorable killing effect on tumor cells, and both the maximum killing efficiency and EC50 were remarkably higher than those of the Anti-EpCAM monoclonal antibody.

(48) 3. Tumor Cell-Killing Detection of Bispecific Antibody-Effectively Mediated PBMC Cells

(49) A single-cell suspension was prepared by digesting HCT116 or NCI-N87 cells with trypsin. The HCT116 or NCI-N87 cells were stained with CFSE with the final concentration being 5 uM (the staining step refers to protocol-1 CFSE staining), and the cells were resuspended to 2×10.sup.5/ml with 10% FBS-1640 cultured by these cells after staining, and cultured over night in a 90-well plate according to 2×10.sup.4 cell/well, namely 100 ul/well. According to the experiment design, the cultured CIK cells were added according to 50 ul/well, control wells were set, and the same volume of culture medium was fed into wells in which no CIK cells need to be added. The corresponding antibody was added with 50 ul/well according to the experiment design while the CIK cells were added, and the same volume of culture medium was fed into wells in which no antibody needs to be added. After 48 hours, the 96-well plate was taken out, cells of each well were digested with trypsin to form the single cell suspension, and correspondingly, all the supernatants and the cell suspension in this process were collected into 1.5 ml EP tubes and subject to 500×g centrifugal for 5 minutes. The supernatant was discarded, and 150 ul 1% FBS-PBS was added to each well, and then cells were resuspended and uniformly mixed. PI (the final concentration of 1 ug/ml) was added 10-15 min before each tube was put on an instrument for fluorescence-activated cell sorting, and the proportion of CFSE and PI double positive cells, namely the death rate of target cells HCT116 or NCI-N87 was detected on the instrument for fluorescence-activated cell sorting (the result was as shown in FIG. 13 and FIG. 14). The cell killing result displayed that the EpCAM×CD3 MSBODY bispecific antibody-mediated CIK cells displayed a favorable killing effect on tumor cells, and both the maximum killing efficiency and EC50 were remarkably higher than those of the Anti-EpCAM monoclonal antibody.

EXAMPLE 6

Pharmacological Detection of Bispecific Antibody for Killing Subcutaneous Xenograft Tumors

(50) A tumor xenograft model was established by mixing 5×10.sup.6 SW480 and 5×10.sup.6 CIK cells and growing at right flanks of female NOD/SCID mice through subcutaneous inoculation (N=8 groups). These mice were randomly grouped within two hours, and then, the mice in an antibody therapy group were administrated with EpCAM×CD3 MSBODY through tail intravenous injection according to the dosages of 2 mg/kg, 1 mg/kg and 0.5 mg/kg. The control groups were as follows: the mice in one group were administrated with 2 mg/kg anti-EpCAM monoclonal antibody and the mice in other group were administrated with MSBODY (4420×CD3) as independent control. The control MSBODY was a constructed by an anti-fluorescein antibody (4-4-20)(Kranz D M, Voss E W Jr., Partial elucidation of an anti-hapten repertoire in BALB/c mice: comparative characterization of several monoclonal antifluorescyl antibodies. Mol Immunol. 1981; 18(10): 889-898). Administration was performed in the second day and the fourth day with unchanged dosage. The animals in the corresponding control group were administrated with PBS through intravenous injection. The volumes of the tumors were measured every three days and calculated from digital caliper measurements as ½×length×width×width (in mm.sup.5).

(51) The antitumor effect estimation of EpCAM×CD3 MSBODY in the body was finished through an adoptive transfer xenograft tumor model. SW480 cells of a gastric cancer cell line were used for establishing a xenograft tumor model on immunodeficient mice NOD/SCID, and the human CIK cells were obtained by simulative culture after the peripheral blood mononuclear cells were separated, and the two kinds of cells were associatively inoculated according to a proportion of 1:1. As shown in FIG. 15, the PBS group, the control anti-EpCAM antibody and the 4420×CD3 antibody for therapy have no remarkable inhibition on the tumor growth, but in the same experiment, no tumor growth was found in an EpCAM×CD3 (2 mg/kg, 4 mg/kg) treatment group, and therefore the tumor growth can be remarkably inhibited via EpCAM×CD3 MSBODY-mediated immune tumor killing. As expected, even CD3 specific molecules were reserved, the MSBODY molecules MC0101 (4420×CD3) lacking EpCAM specificity cannot displayed a remarkable antitumor activity in an in vivo experiment.

(52) It should be understood that the present invention disclosed here is not only limited to describe specific methods, solutions and matters because all of these can change. It also should be realized that terms concerned herein are only for the purpose of describing specific embodiments, but do not have an intend of limiting the scope of the present invention, and the scope of the present invention is only limited by claims attached.

(53) Those skilled in the art should realize or confirm that many equivalents concerned in specific embodiments of the present invention in this text are used within the conventional experiment range. These equivalents are intended to come within the scope of the appended claims.