ANTI-CD47/ANTI-TIGIT BISPECIFIC ANTIBODY, PREPARATION METHOD THEREFOR AND APPLICATION THEREOF

Abstract

Anti-CD47/anti-TIGIT bispecific antibody, a preparation method thereof and application thereof. The bispecific antibody comprises: (a) a first antigen binding part, comprising heavy chain variable region (V.sub.H) and light chain variable region (V.sub.L), V.sub.H and V.sub.L forming an antigen binding site that specifically binds to CD47; and (b) a second antigen binding part, comprising a single domain antibody (sdAb) that specifically binds to TIGIT, wherein the first antigen binding part and the second antigen binding part are fused with each other. The bispecific antibody can block two modes of tumor immune escape at the same time, thus having a good effect in tumor immunotherapy.

Claims

1. An isolated anti-CD47/anti-TIGIT bispecific antigen binding protein or a fragment thereof comprising (a) a first antigen binding portion comprising a heavy chain variable region (V.sub.H) and a light chain variable region (V.sub.L), wherein the V.sub.H and V.sub.L form an antigen binding site that specifically binds to CD47; and (b) a second antigen binding portion comprising a single domain antibody that specifically binds to TIGIT, wherein the first antigen binding portion and the second antigen binding portion are fused to each other.

2. The isolated bispecific antigen binding protein or a fragment thereof according to claim 1, wherein the V.sub.H of the first antigen binding portion comprises heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3, and the amino acid sequences of the HCDR1, HCDR2, and HCDR3 are as shown in SEQ ID NO:31, SEQ ID NO:32 and SEQ ID NO:33, respectively, or the sequence as shown contains up to three amino acid mutations, respectively; the V.sub.L of the first antigen binding portion comprises light chain complementarity determining regions LCDR1, LCDR2 and LCDR3, and the amino acid sequences of the LCDR1, LCDR2, and LCDR3 are as shown in SEQ ID NO:34, SEQ ID NO:35 and SEQ ID NO:36, respectively, or the sequence as shown contains up to three amino acid mutations, respectively.

3. The isolated bispecific antigen binding protein or a fragment thereof according to claim 1, wherein the single domain antibody of the second antigen binding portion comprises complementarity determining regions CDR1, CDR2, and CDR3, and the amino acid sequences of the CDR1, CDR2, and CDR3 are as shown in SEQ ID NO:39, SEQ ID NO:40, and SEQ ID NO:41, respectively, or the sequence as shown contains up to three amino acid mutations, respectively.

4. The isolated bispecific antigen binding protein or a fragment thereof according to claim 1, wherein the first antigen binding portion is a full-length antibody comprising two heavy chains comprising V.sub.H and two light chains comprising V.sub.L.

5. The isolated bispecific antigen binding protein or a fragment thereof according to claim 1, wherein: a C-terminus of the second antigen binding portion is fused to an N-terminus of at least one heavy chain of the first antigen binding portion or an N-terminus of at least one light chain of the first antigen binding portion; or an N-terminus of the second antigen binding portion is fused to a C-terminus of at least one heavy chain of the first antigen binding portion or a C-terminus of at least one light chain of the first antigen binding portion.

6. (canceled)

7. The isolated bispecific antigen binding protein or a fragment thereof according to claim 1, wherein the first antigen binding portion and the second antigen binding portion are fused via a peptide bond or a peptide linker.

8. The isolated bispecific antigen binding protein or a fragment thereof according to claim 7, wherein the peptide linker is selected from a mutated human IgG1 hinge region or a GS linker.

9. The isolated bispecific antigen binding protein or a fragment thereof according to claim 1, wherein a heavy chain of the first antigen binding portion comprises a sequence having at least 95% identity with an amino acid sequence shown in SEQ ID NO:4, and a light chain of the first antigen binding portion comprises a sequence having at least 95% identity with an amino acid sequence shown in SEQ ID NO:6.

10. The isolated bispecific antigen binding protein or a fragment thereof according to claim 1, wherein the second antigen binding portion comprises a sequence having at least 95% identity with an amino acid sequence shown in SEQ ID NO:38.

11. The isolated bispecific antigen binding protein or a fragment thereof according to claim 1, wherein the first antigen binding portion comprises a human, humanized or chimeric antibody or a fragment thereof, and the second antigen binding portion comprises a camelid, chimeric, humanized, or human single domain antibody.

12. The isolated bispecific antigen binding protein or a fragment thereof according to claim 1, wherein the bispecific antigen binding protein or a fragment thereof comprises anti-CD47 antibodies and anti-TIGIT single domain antibodies, and an N-terminus of the anti-TIGIT single domain antibody is fused to a C-terminus of two heavy chains of the anti-CD47 antibody, wherein the heavy chain fusion polypeptide comprises a sequence having at least 95% identity with an amino acid sequence shown in SEQ ID NO:8 or SEQ ID NO:12, and a light chain polypeptide comprises a sequence having at least 95% identity with the amino acid sequence shown in SEQ ID NO:6; a C-terminus of the anti-TIGIT single domain antibody is fused to an N-terminus of two heavy chains of the anti-CD47 antibody, wherein the heavy chain fusion polypeptide comprises a sequence having at least 95% identity with an amino acid sequence shown in SEQ ID NO:10 or SEQ ID NO:14, and a light chain polypeptide comprises a sequence having at least 95% identity with the amino acid sequence shown in SEQ ID NO:6; an N-terminus of the anti-TIGIT single domain antibody is fused to a C-terminus of two light chains of the anti-CD47 antibody, wherein the light chain fusion polypeptide comprises a sequence having at least 95% identity with an amino acid sequence shown in SEQ ID NO:16 or SEQ ID NO:20, and a heavy chain polypeptide comprises a sequence having at least 95% identity with the amino acid sequence shown in SEQ ID NO:4; or a C-terminus of the anti-TIGIT single domain antibody is fused to an N-terminus of two light chains of the anti-CD47 antibody, wherein the light chain fusion polypeptide comprises a sequence having at least 95% identity with an amino acid sequence shown in SEQ ID NO:18 or SEQ ID NO:22, and a heavy chain polypeptide comprises a sequence having at least 95% identity with the amino acid sequence shown in SEQ ID NO:4.

13.-15. (canceled)

16. An isolated polynucleotide encoding the bispecific antigen binding protein or a fragment thereof according to claim 1.

17. A vector comprising the isolated polynucleotide according to claim 16.

18. A host cell comprising the isolated polynucleotide according to claim 16.

19. A method for producing isolated anti-CD47/anti-TIGIT bispecific antigen binding protein or a fragment thereof, comprising culturing the host cell according to claim 18 under suitable conditions, and recovering antibody or fragments thereof from cells or cell culture fluid.

20. A pharmaceutical composition comprising the bispecific antigen binding protein or a fragment thereof according to claim 1 and a pharmaceutically acceptable carrier.

21. A method of treating a disease associated with abnormal expression of CD47 and/or TIGIT, comprising administrating to the subject an effective amount of the anti-CD47/anti-TIGIT bispecific antigen binding protein or a fragment thereof according to claim 1.

22. The method according to claim 21, wherein the disease associated with abnormal expression of CD47 and/or TIGIT is cancer.

23. The method according to claim 22, wherein the cancer is a solid tumor.

24. The isolated bispecific antigen binding protein or a fragment thereof according to claim 8, wherein an amino acid sequence of the peptide linker is as shown in SEQ ID NO:26 or SEQ ID NO:28.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0038] FIG. 1 Affinity between samples and TIGIT-expressing CHO-K1 cells determined by flow cytometry

[0039] FIG. 2 Affinity between samples and CD47-expressing CHO-K1 cells determined by flow cytometry

[0040] FIG. 3 TIGIT blocking activity of samples determined by the TIGIT/CD155 blocking bioassay system

[0041] FIG. 4 Activity of the bispecific antibody TIGIT-G9-HN determined by the cell phagocytosis test of anti-CD47 antibody

DETAILED DESCRIPTION

[0042] The present invention is described in detail below with reference to specific implementations. It should be understood that the implementations are merely intended to describe the present invention rather than to limit the scope of the present invention. In addition, it should be understood that, after reading the teaching of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application. Unless otherwise specified, the methods and materials in the examples described below are commercially available and conventional products.

Example 1 Construction and Expression of Anti-CD47/Anti-TIGIT Bispecific Antibodies

[0043] A series of anti-CD47/anti-TIGIT bispecific antibodies were designed using an anti-CD47 monoclonal antibody (mAb) and a TIGIT single domain antibody (sdAb) respectively. The sequences of the two antibodies are shown in Table 1 and Table 2. The TIGIT single domain antibody was fused to the N-terminus or C-terminus of the heavy or light chain of the anti-CD47 monoclonal antibody through two linker sequences (E-linker: EPKSSDKTHTSPPSP or G9-linker: GGGGSGGGS). Each bispecific antibody structure was composed of two identical fusion polypeptide chains and two identical natural polypeptide chains, and the DNA sequence expressing each polypeptide chain was inserted into the pTT5 vector between the EcoRI and HindIII restriction sites. Each plasmid also included a secretion signal sequence for the protein secreted into the growth medium. The TIGIT single domain antibody was fused to the N-terminus of the IgG4-Fc portion with site mutations (S228P and L235E) as a control for biological activity assay in vitro. The plasmids expressing the bispecific antibody protein are shown in Table 3.

TABLE-US-00001 TABLE 1 DNA and amino acid sequences of anti-CD47 monoclonal antibodies SEQ ID DNA sequence NO: DNA GAGGTGCAGCTGGTGCAGTCCGGAGCTGAGGTGAAGAAG 3 sequence of a CCAGGATCCAGCGTGAAGGTGAGCTGCAAGGCTAGCGGC heavy chain of TACTCTTTCACCCACCATTGGATCCACTGGGTGAGGCAG anti-CD47 GCTCCTGGACAGGGACTGGAGTGGATGGGCATGATCGAC antibody GCTTCCGATAGCGAGACAAGACTGTCTCAGAAGTTTAAG GACCGCGTGACCATCACAGCCGATAAGTCTACCTCCACA GCTTACATGGAGCTGTCTTCCCTGAGATCCGAGGACACC GCCGTGTACTATTGTGCTAGGCTGGGCCGGTACTATTTC GATTATTGGGGCCAGGGCACCACAGTGACAGTGAGCTCT GCCAGCACAAAGGGCCCTTCCGTGTTCCCACTGGCTCCC TGCTCCAGAAGCACATCTGAGTCCACCGCCGCTCTGGGC TGTCTGGTGAAGGACTACTTCCCTGAGCCAGTGACCGTG TCCTGGAACAGCGGCGCCCTGACATCTGGCGTGCACACC TTTCCAGCTGTGCTGCAGTCCAGCGGCCTGTACTCCCTG TCTTCCGTGGTGACAGTGCCCAGCTCTTCCCTGGGCACC AAGACATATACCTGCAACGTGGACCATAAGCCTTCCAAT ACCAAGGTGGATAAGAGGGTGGAGAGCAAGTACGGACCA CCTTGCCCACCATGTCCAGCTCCTGAGTTTGAGGGAGGA CCATCCGTGTTCCTGTTTCCTCCAAAGCCTAAGGACACC CTGATGATCAGCCGGACACCTGAGGTGACCTGCGTGGTG GTGGACGTGTCTCAGGAGGATCCAGAGGTGCAGTTCAAC TGGTACGTGGATGGCGTGGAGGTGCACAATGCTAAGACC AAGCCAAGAGAGGAGCAGTTTAATTCCACATACCGCGTG GTGAGCGTGCTGACCGTGCTGCATCAGGATTGGCTGAAC GGCAAGGAGTATAAGTGCAAGGTGTCCAATAAGGGCCTG CCCAGCTCTATCGAGAAGACAATCAGCAAGGCTAAGGGA CAGCCTAGGGAGCCACAGGTGTACACCCTGCCCCCTTCT CAGGAGGAGATGACAAAGAACCAGGTGTCCCTGACCTGT CTGGTGAAGGGCTTCTATCCAAGCGACATCGCTGTGGAG TGGGAGTCTAATGGCCAGCCCGAGAACAATTACAAGACC ACACCACCCGTGCTGGACTCTGATGGCTCCTTCTTTCTG TATTCTAGGCTGACAGTGGATAAGTCCCGGTGGCAGGAG GGCAACGTGTTTAGCTGCTCTGTGATGCACGAGGCCCTG CACAATCATTATACCCAGAAGTCCCTGAGCCTGTCTCTG GGCAAG Amino acid EVQLVQSGAEVKKPGSSVKVSCKASGYSFTHHWIHWVRQ 4 sequence of a APGQGLEWMGMIDASDSETRLSQKFKDRVTITADKSTST heavy chain of AYMELSSLRSEDTAVYYCARLGRYYFDYWGQGTTVTVSS anti-CD47 ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTV antibody (H0) SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT KTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGG PSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFN WYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLN GKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPS QEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEAL HNHYTQKSLSLSLGK DNA GAGATCGTGCTGACCCAGTCTCCAGCCACACTGTCTCTG 5 sequence of a TCCCCAGGAGAGAGGGCCACCCTGAGCTGCCGGGCTTCT light chain of GAGAACGTGGGCACATACATCTCCTGGTATCAGCAGAAG anti-CD47 CCAGGACAGGCTCCTAGGCTGCTGATCTACGGCGCTAGC antibody AATAGATATACCGGCATCCCTGCTCGCTTCAGCGGATCT GGATCCGGCACAGACTTTACCCTGACAATCTCCAGCCTG GAGCCAGAGGATTTCGCCGTGTACTATTGTGGCGAGTCC TACGGCCACCTGTATACCTTTGGCGGCGGCACAAAGGTG GAGATCAAGCGAACGGTGGCTGCACCATCTGTCTTCATC TTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCC TCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAG GCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCG GGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAG GACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGC AAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAA GTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGC TTCAACAGGGGAGAGTGT Amino acid EIVLTQSPATLSLSPGERATLSCRASENVGTYISWYQQK 6 sequence of a PGQAPRLLIYGASNRYTGIPARFSGSGSGTDFTLTISSL light chain of EPEDFAVYYCGESYGHLYTFGGGTKVEIKRTVAAPSVFI anti-CD47 FPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS antibody (L0) GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGEC SEQ ID SEQ ID SEQ ID Sequence NO: Sequence NO: Sequence NO: CDR GYSFTHHW 31 MIDASDSET 32 LGRYYFDY 33 sequence of a IH RLSQKFKD heavy chain of anti-CD47 antibody CDR RASENVGT 34 GASNRYT 35 GESYGHLYT 36 sequence of a YTS light chain of anti-CD47 antibody

TABLE-US-00002 TABLE 2 DNA and amino acid sequences of TIGIT single domain antibodies SEQ Sequence ID NO: DNA GAGGTGCAGCTGGTGGAGTCTGGAGGAGGACTGGTGCAG 37 sequence of CCAGGAGGCAGCCTGAGGCTGTCTTGCGCCGCTTCCGGC a TIGIT TACAAGTATGGCGTGTACTCCATGGGATGGTTCAGGCAG single GCTCCTGGCAAGGGACTGGAGGGCGTGTCCGCCATCTGT domain TCTGGCGGCAGAACCACATACTCTGACTCCGTGAAGGGC antibody AGGTTTACCATCTCCCGGGATAACAGCAACCAGATCCTG TATCTGCAGATGAACTCCCTGAGAGCCGAGGACACCGCC GTGTACTATTGCGCTGCTAGGCCACTGTGGACAGGCGAC TGTGATCTGTCCAGCTCTTGGTATAAGACCTGGGGCCAG GGCACCCTGGTGACAGTGTCCAGC Amino acid EVQLVESGGGLVQPGGSLRLSCAASGYKYGVYSMGWFRQ 38 sequence of APGKGLEGVSAICSGGRTTYSDSVKGRFTISRDNSNQIL a TIGIT YLQMNSLRAEDTAVYYCAARPLWTGDCDLSSSWYKTWGQ single GTLVTVSS domain antibody SEQ ID SEQ ID SEQ Sequence NO: Sequence NO: Sequence ID NO: Amino acid GYKYGVYS 39 AICSGGRTT 40 RPLWTGDCDL 41 sequence of MG YSDSVKG SSSWYKT CDR of a TIGIT single domain antibody

[0044] After being transfected with the expression plasmid, the CHO-3E7 host cells were cultured in an incubator at 37° C. and 100 rpm for 6 days. The supernatant was extracted by centrifugation, and the protein A column was used to purify the bispecific antibody protein.

[0045] As mentioned above, the CD47 monoclonal antibody was composed of a heavy chain H0 and a light chain L0. The TIGIT single domain antibody was connected to the N-terminus or C-terminus of the heavy or light chain of the CD47 monoclonal antibody through two linker sequences (E-linker: EPKSSDKTHTSPPSP or G9-linker: GGGGSGGGS) to generate a series of different bispecific antibodies. Firstly, the E-linker linker sequence was used to construct the following fusion proteins: (1) a new polypeptide, called H1, produced by fusing the TIGIT single domain antibody with the C-terminus of the heavy chain H0; (2) a new polypeptide H2 produced by fusing the TIGIT single domain antibody with the N-terminus of the heavy chain H0; (3) a new polypeptide L1 produced by fusing the TIGIT single domain antibody with the C-terminus of the light chain L0; and (4) a new polypeptide L2 produced by fusing the TIGIT single domain antibody with the N-terminus of the light chain L0. Similarly, the G9-linker linker sequence was then used to construct the following fusion proteins: (1) a new polypeptide, called H3, produced by fusing the TIGIT single domain antibody with the C-terminus of the heavy chain H0; (2) a new polypeptide H4 produced by fusing the TIGIT single domain antibody with the N-terminus of the heavy chain H0; (3) a new polypeptide L3 produced by fusing the TIGIT single domain antibody with the C-terminus of the light chain L0; and (4) a new polypeptide L4 produced by fusing the TIGIT single domain antibody with the N-terminus of the light chain L0.

[0046] A series of bispecific antibodies were produced by combining these constructed heavy chain fusion proteins H1, H2, H3, and H4 with the unmodified parental light chain polypeptide chain L0, or combining these constructed light chain fusion proteins L1, L2, L3, and L4 with the unmodified heavy chain polypeptide chain H0. A bispecific antibody TIGIT-E-HC was produced by combining heavy chain fusion protein H1 with parental light chain L0, a bispecific antibody TIGIT-E-HN was produced by combining heavy chain fusion protein H2 with parental light chain L0, a bispecific antibody TIGIT-G9-HC was produced by combining heavy chain fusion protein H3 with parental light chain L0, a bispecific antibody TIGIT-G9-HN was produced by combining heavy chain fusion protein H4 with parental light chain L0, a bispecific antibody TIGIT-E-LC was produced by combining light chain fusion protein L1 with parental heavy chain H0, a bispecific antibody TIGIT-E-LN was produced by combining light chain fusion protein L2 with parental heavy chain H0, a bispecific antibody TIGIT-G9-LC was produced by combining light chain fusion protein L3 with parental heavy chain H0, and a bispecific antibody TIGIT-G9-LN was produced by combining the light chain fusion protein L4 with the parental heavy chain H0. The Fc of human IgG4 was modified by site mutation (S228P and L235E), and then the TIGIT single domain antibody was connected to the N-terminus of the Fc portion of human IgG4 to produce a new fusion protein H5, thereby constructing Fc fusion protein of sdAb-TIGIT-IgG4PE.

TABLE-US-00003 TABLE 3 Plasmids and proteins for construction of bispecific antibodies Amino acid sequence Protein Component Plasmid SEQ ID NO: CD47 H0 pTT5-CD47HC 4 L0 pTT5-CD47LC 6 TIGIT-E-HC H1 pTT5-CD47HC-E-TIGIT 8 L0 pTT5-CD47LC 6 TIGIT-E-HN H2 pTT5-TIGIT-E-CD47HC 10 L0 pTT5-CD47LC 6 TIGIT-E-LC L1 pTT5-CD47LC-E-TIGIT 16 H0 pTT5-CD47HC 4 TIGIT-E-LN L2 pTT5-TIGIT-E-CD47LC 18 H0 pTT5-CD47HC 4 TIGIT-G9-HC H3 pTT5-CD47HC-G9-TIGIT 12 L0 pTT5-CD47LC 6 TIGIT-G9-HN H4 pTT5-TIGIT-G9-CD47HC 14 L0 pTT5-CD47LC 6 TIGIT-G9-LC L3 pTT5-CD47LC-G9-TIGIT 20 H0 pTT5-CD47HC 4 TIGIT-G9-LN L4 pTT5-TIGIT-G9-CD47LC 22 H0 pTT5-CD47HC 4 sdAb-TIGIT-IgG4PE H5 pTT5-sdAb-TIGIT-IgG4PE 24

TABLE-US-00004 DNA sequence of secretion signal peptide (SEQ ID NO: 1) ATGGGCTGGTCCTGCATCATCCTGTTCCTGGTGGCTACCGCCACCGGCGT GCACTCC Amino acid sequence of secretion signal peptide (SEQ ID NO: 2) MGWSCIILFLVATATGVHS DNA sequence of polypeptide chain H1 (SEQ ID NO: 7) GAGGTGCAGCTGGTGCAGTCCGGAGCTGAGGTGAAGAAGCCAGGATCCAG CGTGAAGGTGAGCTGCAAGGCTAGCGGCTACTCTTTCACCCACCATTGGA TCCACTGGGTGAGGCAGGCTCCTGGACAGGGACTGGAGTGGATGGGCATG ATCGACGCTTCCGATAGCGAGACAAGACTGTCTCAGAAGTTTAAGGACCG CGTGACCATCACAGCCGATAAGTCTACCTCCACAGCTTACATGGAGCTGT CTTCCCTGAGATCCGAGGACACCGCCGTGTACTATTGTGCTAGGCTGGGC CGGTACTATTTCGATTATTGGGGCCAGGGCACCACAGTGACAGTGAGCTC TGCCAGCACAAAGGGCCCTTCCGTGTTCCCACTGGCTCCCTGCTCCAGAA GCACATCTGAGTCCACCGCCGCTCTGGGCTGTCTGGTGAAGGACTACTTC CCTGAGCCAGTGACCGTGTCCTGGAACAGCGGCGCCCTGACATCTGGCGT GCACACCTTTCCAGCTGTGCTGCAGTCCAGCGGCCTGTACTCCCTGTCTT CCGTGGTGACAGTGCCCAGCTCTTCCCTGGGCACCAAGACATATACCTGC AACGTGGACCATAAGCCTTCCAATACCAAGGTGGATAAGAGGGTGGAGAG CAAGTACGGACCACCTTGCCCACCATGTCCAGCTCCTGAGTTTGAGGGAG GACCATCCGTGTTCCTGTTTCCTCCAAAGCCTAAGGACACCCTGATGATC AGCCGGACACCTGAGGTGACCTGCGTGGTGGTGGACGTGTCTCAGGAGGA TCCAGAGGTGCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCACAATG CTAAGACCAAGCCAAGAGAGGAGCAGTTTAATTCCACATACCGCGTGGTG AGCGTGCTGACCGTGCTGCATCAGGATTGGCTGAACGGCAAGGAGTATAA GTGCAAGGTGTCCAATAAGGGCCTGCCCAGCTCTATCGAGAAGACAATCA GCAAGGCTAAGGGACAGCCTAGGGAGCCACAGGTGTACACCCTGCCCCCT TCTCAGGAGGAGATGACAAAGAACCAGGTGTCCCTGACCTGTCTGGTGAA GGGCTTCTATCCAAGCGACATCGCTGTGGAGTGGGAGTCTAATGGCCAGC CCGAGAACAATTACAAGACCACACCACCCGTGCTGGACTCTGATGGCTCC TTCTTTCTGTATTCTAGGCTGACAGTGGATAAGTCCCGGTGGCAGGAGGG CAACGTGTTTAGCTGCTCTGTGATGCACGAGGCCCTGCACAATCATTATA CCCAGAAGTCCCTGAGCCTGTCTCTGGGCAAGGAACCTAAGTCTAGCGAC AAAACTCATACCAGCCCCCCTAGTCCAGAGGTGCAGCTGGTGGAGTCTGG AGGAGGACTGGTGCAGCCAGGAGGCAGCCTGAGGCTGTCTTGCGCCGCTT CCGGCTACAAGTATGGCGTGTACTCCATGGGATGGTTCAGGCAGGCTCCT GGCAAGGGACTGGAGGGCGTGTCCGCCATCTGTTCTGGCGGCAGAACCAC ATACTCTGACTCCGTGAAGGGCAGGTTTACCATCTCCCGGGATAACAGCA ACCAGATCCTGTATCTGCAGATGAACTCCCTGAGAGCCGAGGACACCGCC GTGTACTATTGCGCTGCTAGGCCACTGTGGACAGGCGACTGTGATCTGTC CAGCTCTTGGTATAAGACCTGGGGCCAGGGCACCCTGGTGACAGTGTCCA GC Amino acid sequence of polypeptide chain H1 (SEQ ID NO: 8) EVQLVQSGAEVKKPGSSVKVSCKASGYSFTHHWIHWVRQAPGQGLEWMGM IDASDSETRLSQKFKDRVTITADKSTSTAYMELSSLRSEDTAVYYCARLG RYYFDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYF PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTC NVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMI SRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVV SVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPP SQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKEPKSSD KTHTSPPSPEVQLVESGGGLVQPGGSLRLSCAASGYKYGVYSMGWFRQAP GKGLEGVSAICSGGRTTYSDSVKGRFTISRDNSNQILYLQMNSLRAEDTA VYYCAARPLWTGDCDLSSSWYKTWGQGTLVTVSS DNA sequence of polypeptide chain H2 (SEQ ID NO: 9) GAGGTGCAGCTGGTGGAGTCTGGAGGAGGACTGGTGCAGCCAGGAGGCAG CCTGAGGCTGTCTTGCGCCGCTTCCGGCTACAAGTATGGCGTGTACTCCA TGGGATGGTTCAGGCAGGCTCCTGGCAAGGGACTGGAGGGCGTGTCCGCC ATCTGTTCTGGCGGCAGAACCACATACTCTGACTCCGTGAAGGGCAGGTT TACCATCTCCCGGGATAACAGCAACCAGATCCTGTATCTGCAGATGAACT CCCTGAGAGCCGAGGACACCGCCGTGTACTATTGCGCTGCTAGGCCACTG TGGACAGGCGACTGTGATCTGTCCAGCTCTTGGTATAAGACCTGGGGCCA GGGCACCCTGGTGACAGTGTCCAGCGAACCTAAGTCTAGCGACAAAACTC ATACCAGCCCCCCTAGTCCAGAGGTGCAGCTGGTGCAGTCCGGAGCTGAG GTGAAGAAGCCAGGATCCAGCGTGAAGGTGAGCTGCAAGGCTAGCGGCTA CTCTTTCACCCACCATTGGATCCACTGGGTGAGGCAGGCTCCTGGACAGG GACTGGAGTGGATGGGCATGATCGACGCTTCCGATAGCGAGACAAGACTG TCTCAGAAGTTTAAGGACCGCGTGACCATCACAGCCGATAAGTCTACCTC CACAGCTTACATGGAGCTGTCTTCCCTGAGATCCGAGGACACCGCCGTGT ACTATTGTGCTAGGCTGGGCCGGTACTATTTCGATTATTGGGGCCAGGGC ACCACAGTGACAGTGAGCTCTGCCAGCACAAAGGGCCCTTCCGTGTTCCC ACTGGCTCCCTGCTCCAGAAGCACATCTGAGTCCACCGCCGCTCTGGGCT GTCTGGTGAAGGACTACTTCCCTGAGCCAGTGACCGTGTCCTGGAACAGC GGCGCCCTGACATCTGGCGTGCACACCTTTCCAGCTGTGCTGCAGTCCAG CGGCCTGTACTCCCTGTCTTCCGTGGTGACAGTGCCCAGCTCTTCCCTGG GCACCAAGACATATACCTGCAACGTGGACCATAAGCCTTCCAATACCAAG GTGGATAAGAGGGTGGAGAGCAAGTACGGACCACCTTGCCCACCATGTCC AGCTCCTGAGTTTGAGGGAGGACCATCCGTGTTCCTGTTTCCTCCAAAGC CTAAGGACACCCTGATGATCAGCCGGACACCTGAGGTGACCTGCGTGGTG GTGGACGTGTCTCAGGAGGATCCAGAGGTGCAGTTCAACTGGTACGTGGA TGGCGTGGAGGTGCACAATGCTAAGACCAAGCCAAGAGAGGAGCAGTTTA ATTCCACATACCGCGTGGTGAGCGTGCTGACCGTGCTGCATCAGGATTGG CTGAACGGCAAGGAGTATAAGTGCAAGGTGTCCAATAAGGGCCTGCCCAG CTCTATCGAGAAGACAATCAGCAAGGCTAAGGGACAGCCTAGGGAGCCAC AGGTGTACACCCTGCCCCCTTCTCAGGAGGAGATGACAAAGAACCAGGTG TCCCTGACCTGTCTGGTGAAGGGCTTCTATCCAAGCGACATCGCTGTGGA GTGGGAGTCTAATGGCCAGCCCGAGAACAATTACAAGACCACACCACCCG TGCTGGACTCTGATGGCTCCTTCTTTCTGTATTCTAGGCTGACAGTGGAT AAGTCCCGGTGGCAGGAGGGCAACGTGTTTAGCTGCTCTGTGATGCACGA GGCCCTGCACAATCATTATACCCAGAAGTCCCTGAGCCTGTCTCTGGGCA AG Amino acid sequence of polypeptide chain H2 (SEQ ID NO: 10) EVQLVESGGGLVQPGGSLRLSCAASGYKYGVYSMGWFRQAPGKGLEGVSA ICSGGRTTYSDSVKGRFTISRDNSNQILYLQMNSLRAEDTAVYYCAARPL WTGDCDLSSSWYKTWGQGTLVTVSSEPKSSDKTHTSPPSPEVQLVQSGAE VKKPGSSVKVSCKASGYSFTHHWIHWVRQAPGQGLEWMGMIDASDSETRL SQKFKDRVTITADKSTSTAYMELSSLRSEDTAVYYCARLGRYYFDYWGQG TTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNS GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTK VDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVV VDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDW LNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQV SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVD KSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK DNA sequence of polypeptide chain H3 (SEQ ID NO: 11) GAGGTGCAGCTGGTGCAGTCCGGAGCTGAGGTGAAGAAGCCAGGATCCAG CGTGAAGGTGAGCTGCAAGGCTAGCGGCTACTCTTTCACCCACCATTGGA TCCACTGGGTGAGGCAGGCTCCTGGACAGGGACTGGAGTGGATGGGCATG ATCGACGCTTCCGATAGCGAGACAAGACTGTCTCAGAAGTTTAAGGACCG CGTGACCATCACAGCCGATAAGTCTACCTCCACAGCTTACATGGAGCTGT CTTCCCTGAGATCCGAGGACACCGCCGTGTACTATTGTGCTAGGCTGGGC CGGTACTATTTCGATTATTGGGGCCAGGGCACCACAGTGACAGTGAGCTC TGCCAGCACAAAGGGCCCTTCCGTGTTCCCACTGGCTCCCTGCTCCAGAA GCACATCTGAGTCCACCGCCGCTCTGGGCTGTCTGGTGAAGGACTACTTC CCTGAGCCAGTGACCGTGTCCTGGAACAGCGGCGCCCTGACATCTGGCGT GCACACCTTTCCAGCTGTGCTGCAGTCCAGCGGCCTGTACTCCCTGTCTT CCGTGGTGACAGTGCCCAGCTCTTCCCTGGGCACCAAGACATATACCTGC AACGTGGACCATAAGCCTTCCAATACCAAGGTGGATAAGAGGGTGGAGAG CAAGTACGGACCACCTTGCCCACCATGTCCAGCTCCTGAGTTTGAGGGAG GACCATCCGTGTTCCTGTTTCCTCCAAAGCCTAAGGACACCCTGATGATC AGCCGGACACCTGAGGTGACCTGCGTGGTGGTGGACGTGTCTCAGGAGGA TCCAGAGGTGCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCACAATG CTAAGACCAAGCCAAGAGAGGAGCAGTTTAATTCCACATACCGCGTGGTG AGCGTGCTGACCGTGCTGCATCAGGATTGGCTGAACGGCAAGGAGTATAA GTGCAAGGTGTCCAATAAGGGCCTGCCCAGCTCTATCGAGAAGACAATCA GCAAGGCTAAGGGACAGCCTAGGGAGCCACAGGTGTACACCCTGCCCCCT TCTCAGGAGGAGATGACAAAGAACCAGGTGTCCCTGACCTGTCTGGTGAA GGGCTTCTATCCAAGCGACATCGCTGTGGAGTGGGAGTCTAATGGCCAGC CCGAGAACAATTACAAGACCACACCACCCGTGCTGGACTCTGATGGCTCC TTCTTTCTGTATTCTAGGCTGACAGTGGATAAGTCCCGGTGGCAGGAGGG CAACGTGTTTAGCTGCTCTGTGATGCACGAGGCCCTGCACAATCATTATA CCCAGAAGTCCCTGAGCCTGTCTCTGGGCAAGGGTGGAGGCGGTAGTGGA GGCGGTTCAGAGGTGCAGCTGGTGGAGTCTGGAGGAGGACTGGTGCAGCC AGGAGGCAGCCTGAGGCTGTCTTGCGCCGCTTCCGGCTACAAGTATGGCG TGTACTCCATGGGATGGTTCAGGCAGGCTCCTGGCAAGGGACTGGAGGGC GTGTCCGCCATCTGTTCTGGCGGCAGAACCACATACTCTGACTCCGTGAA GGGCAGGTTTACCATCTCCCGGGATAACAGCAACCAGATCCTGTATCTGC AGATGAACTCCCTGAGAGCCGAGGACACCGCCGTGTACTATTGCGCTGCT AGGCCACTGTGGACAGGCGACTGTGATCTGTCCAGCTCTTGGTATAAGAC CTGGGGCCAGGGCACCCTGGTGACAGTGTCCAGC Amino acid sequence of polypeptide chain H3 (SEQ ID NO: 12) EVQLVQSGAEVKKPGSSVKVSCKASGYSFTHHWIHWVRQAPGQGLEWMGM IDASDSETRLSQKFKDRVTITADKSTSTAYMELSSLRSEDTAVYYCARLG RYYFDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYF PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTC NVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMI SRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVV SVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPP SQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKGGGGSG GGSEVQLVESGGGLVQPGGSLRLSCAASGYKYGVYSMGWFRQAPGKGLEG VSAICSGGRTTYSDSVKGRFTISRDNSNQILYLQMNSLRAEDTAVYYCAA RPLWTGDCDLSSSWYKTWGQGTLVTVSS DNA sequence of polypeptide chain H4 (SEQ ID NO: 13) GAGGTGCAGCTGGTGGAGTCTGGAGGAGGACTGGTGCAGCCAGGAGGCAG CCTGAGGCTGTCTTGCGCCGCTTCCGGCTACAAGTATGGCGTGTACTCCA TGGGATGGTTCAGGCAGGCTCCTGGCAAGGGACTGGAGGGCGTGTCCGCC ATCTGTTCTGGCGGCAGAACCACATACTCTGACTCCGTGAAGGGCAGGTT TACCATCTCCCGGGATAACAGCAACCAGATCCTGTATCTGCAGATGAACT CCCTGAGAGCCGAGGACACCGCCGTGTACTATTGCGCTGCTAGGCCACTG TGGACAGGCGACTGTGATCTGTCCAGCTCTTGGTATAAGACCTGGGGCCA GGGCACCCTGGTGACAGTGTCCAGCGGTGGAGGCGGTAGTGGAGGCGGTT CAGAGGTGCAGCTGGTGCAGTCCGGAGCTGAGGTGAAGAAGCCAGGATCC AGCGTGAAGGTGAGCTGCAAGGCTAGCGGCTACTCTTTCACCCACCATTG GATCCACTGGGTGAGGCAGGCTCCTGGACAGGGACTGGAGTGGATGGGCA TGATCGACGCTTCCGATAGCGAGACAAGACTGTCTCAGAAGTTTAAGGAC CGCGTGACCATCACAGCCGATAAGTCTACCTCCACAGCTTACATGGAGCT GTCTTCCCTGAGATCCGAGGACACCGCCGTGTACTATTGTGCTAGGCTGG GCCGGTACTATTTCGATTATTGGGGCCAGGGCACCACAGTGACAGTGAGC TCTGCCAGCACAAAGGGCCCTTCCGTGTTCCCACTGGCTCCCTGCTCCAG AAGCACATCTGAGTCCACCGCCGCTCTGGGCTGTCTGGTGAAGGACTACT TCCCTGAGCCAGTGACCGTGTCCTGGAACAGCGGCGCCCTGACATCTGGC GTGCACACCTTTCCAGCTGTGCTGCAGTCCAGCGGCCTGTACTCCCTGTC TTCCGTGGTGACAGTGCCCAGCTCTTCCCTGGGCACCAAGACATATACCT GCAACGTGGACCATAAGCCTTCCAATACCAAGGTGGATAAGAGGGTGGAG AGCAAGTACGGACCACCTTGCCCACCATGTCCAGCTCCTGAGTTTGAGGG AGGACCATCCGTGTTCCTGTTTCCTCCAAAGCCTAAGGACACCCTGATGA TCAGCCGGACACCTGAGGTGACCTGCGTGGTGGTGGACGTGTCTCAGGAG GATCCAGAGGTGCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCACAA TGCTAAGACCAAGCCAAGAGAGGAGCAGTTTAATTCCACATACCGCGTGG TGAGCGTGCTGACCGTGCTGCATCAGGATTGGCTGAACGGCAAGGAGTAT AAGTGCAAGGTGTCCAATAAGGGCCTGCCCAGCTCTATCGAGAAGACAAT CAGCAAGGCTAAGGGACAGCCTAGGGAGCCACAGGTGTACACCCTGCCCC CTTCTCAGGAGGAGATGACAAAGAACCAGGTGTCCCTGACCTGTCTGGTG AAGGGCTTCTATCCAAGCGACATCGCTGTGGAGTGGGAGTCTAATGGCCA GCCCGAGAACAATTACAAGACCACACCACCCGTGCTGGACTCTGATGGCT CCTTCTTTCTGTATTCTAGGCTGACAGTGGATAAGTCCCGGTGGCAGGAG GGCAACGTGTTTAGCTGCTCTGTGATGCACGAGGCCCTGCACAATCATTA TACCCAGAAGTCCCTGAGCCTGTCTCTGGGCAAG Amino acid sequence of polypeptide chain H4 (SEQ ID NO: 14) EVQLVESGGGLVQPGGSLRLSCAASGYKYGVYSMGWFRQAPGKGLEGVSA ICSGGRTTYSDSVKGRFTISRDNSNQILYLQMNSLRAEDTAVYYCAARPL WTGDCDLSSSWYKTWGQGTLVTVSSGGGGSGGGSEVQLVQSGAEVKKPGS SVKVSCKASGYSFTHHWIHWVRQAPGQGLEWMGMIDASDSETRLSQKFKD RVTITADKSTSTAYMELSSLRSEDTAVYYCARLGRYYFDYWGQGTTVTVS SASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSG VHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVE SKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQE DPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQE GNVFSCSVMHEALHNHYTQKSLSLSLGK DNA sequence of polypeptide chain L1 (SEQ ID NO: 15) GAGATCGTGCTGACCCAGTCTCCAGCCACACTGTCTCTGTCCCCAGGAGA GAGGGCCACCCTGAGCTGCCGGGCTTCTGAGAACGTGGGCACATACATCT CCTGGTATCAGCAGAAGCCAGGACAGGCTCCTAGGCTGCTGATCTACGGC GCTAGCAATAGATATACCGGCATCCCTGCTCGCTTCAGCGGATCTGGATC CGGCACAGACTTTACCCTGACAATCTCCAGCCTGGAGCCAGAGGATTTCG CCGTGTACTATTGTGGCGAGTCCTACGGCCACCTGTATACCTTTGGCGGC GGCACAAAGGTGGAGATCAAGCGAACGGTGGCTGCACCATCTGTCTTCAT CTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGT GCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTG GATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGA CAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAG CAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGC CTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTGAACCTAA GTCTAGCGACAAAACTCATACCAGCCCCCCTAGTCCAGAGGTGCAGCTGG TGGAGTCTGGAGGAGGACTGGTGCAGCCAGGAGGCAGCCTGAGGCTGTCT TGCGCCGCTTCCGGCTACAAGTATGGCGTGTACTCCATGGGATGGTTCAG GCAGGCTCCTGGCAAGGGACTGGAGGGCGTGTCCGCCATCTGTTCTGGCG GCAGAACCACATACTCTGACTCCGTGAAGGGCAGGTTTACCATCTCCCGG GATAACAGCAACCAGATCCTGTATCTGCAGATGAACTCCCTGAGAGCCGA GGACACCGCCGTGTACTATTGCGCTGCTAGGCCACTGTGGACAGGCGACT GTGATCTGTCCAGCTCTTGGTATAAGACCTGGGGCCAGGGCACCCTGGTG ACAGTGTCCAGC Amino acid sequence of polypeptide chain L1 (SEQ ID NO: 16) EIVLTQSPATLSLSPGERATLSCRASENVGTYISWYQQKPGQAPRLLIYG ASNRYTGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCGESYGHLYTFGG GTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGECEPKSSDKTHTSPPSPEVQLVESGGGLVQPGGSLRLS CAASGYKYGVYSMGWFRQAPGKGLEGVSAICSGGRTTYSDSVKGRFTISR DNSNQILYLQMNSLRAEDTAVYYCAARPLWTGDCDLSSSWYKTWGQGTLV TVSS DNA sequence of polypeptide chain L2 (SEQ ID NO: 17) GAGGTGCAGCTGGTGGAGTCTGGAGGAGGACTGGTGCAGCCAGGAGGCAG CCTGAGGCTGTCTTGCGCCGCTTCCGGCTACAAGTATGGCGTGTACTCCA TGGGATGGTTCAGGCAGGCTCCTGGCAAGGGACTGGAGGGCGTGTCCGCC ATCTGTTCTGGCGGCAGAACCACATACTCTGACTCCGTGAAGGGCAGGTT TACCATCTCCCGGGATAACAGCAACCAGATCCTGTATCTGCAGATGAACT CCCTGAGAGCCGAGGACACCGCCGTGTACTATTGCGCTGCTAGGCCACTG TGGACAGGCGACTGTGATCTGTCCAGCTCTTGGTATAAGACCTGGGGCCA GGGCACCCTGGTGACAGTGTCCAGCGAACCTAAGTCTAGCGACAAAACTC ATACCAGCCCCCCTAGTCCAGAGATCGTGCTGACCCAGTCTCCAGCCACA CTGTCTCTGTCCCCAGGAGAGAGGGCCACCCTGAGCTGCCGGGCTTCTGA GAACGTGGGCACATACATCTCCTGGTATCAGCAGAAGCCAGGACAGGCTC CTAGGCTGCTGATCTACGGCGCTAGCAATAGATATACCGGCATCCCTGCT CGCTTCAGCGGATCTGGATCCGGCACAGACTTTACCCTGACAATCTCCAG CCTGGAGCCAGAGGATTTCGCCGTGTACTATTGTGGCGAGTCCTACGGCC ACCTGTATACCTTTGGCGGCGGCACAAAGGTGGAGATCAAGCGAACGGTG GCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATC TGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGG CCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAG GAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAG CACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCT GCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAAC AGGGGAGAGTGT Amino acid sequence of polypeptide chain L2 (SEQ ID NO: 18) EVQLVESGGGLVQPGGSLRLSCAASGYKYGVYSMGWFRQAPGKGLEGVSA ICSGGRTTYSDSVKGRFTISRDNSNQILYLQMNSLRAEDTAVYYCAARPL WTGDCDLSSSWYKTWGQGTLVTVSSEPKSSDKTHTSPPSPEIVLTQSPAT LSLSPGERATLSCRASENVGTYISWYQQKPGQAPRLLIYGASNRYTGIPA RFSGSGSGTDFTLTISSLEPEDFAVYYCGESYGHLYTFGGGTKVEIKRTV AAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQ ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC DNA sequence of polypeptide chain L3 (SEQ ID NO: 19) GAGATCGTGCTGACCCAGTCTCCAGCCACACTGTCTCTGTCCCCAGGAGA GAGGGCCACCCTGAGCTGCCGGGCTTCTGAGAACGTGGGCACATACATCT CCTGGTATCAGCAGAAGCCAGGACAGGCTCCTAGGCTGCTGATCTACGGC GCTAGCAATAGATATACCGGCATCCCTGCTCGCTTCAGCGGATCTGGATC CGGCACAGACTTTACCCTGACAATCTCCAGCCTGGAGCCAGAGGATTTCG CCGTGTACTATTGTGGCGAGTCCTACGGCCACCTGTATACCTTTGGCGGC GGCACAAAGGTGGAGATCAAGCGAACGGTGGCTGCACCATCTGTCTTCAT CTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGT GCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTG GATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGA CAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAG CAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGC CTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTGGTGGAGG CGGTAGTGGAGGCGGTTCAGAGGTGCAGCTGGTGGAGTCTGGAGGAGGAC TGGTGCAGCCAGGAGGCAGCCTGAGGCTGTCTTGCGCCGCTTCCGGCTAC AAGTATGGCGTGTACTCCATGGGATGGTTCAGGCAGGCTCCTGGCAAGGG ACTGGAGGGCGTGTCCGCCATCTGTTCTGGCGGCAGAACCACATACTCTG ACTCCGTGAAGGGCAGGTTTACCATCTCCCGGGATAACAGCAACCAGATC CTGTATCTGCAGATGAACTCCCTGAGAGCCGAGGACACCGCCGTGTACTA TTGCGCTGCTAGGCCACTGTGGACAGGCGACTGTGATCTGTCCAGCTCTT GGTATAAGACCTGGGGCCAGGGCACCCTGGTGACAGTGTCCAGC Amino acid sequence of polypeptide chain L3 (SEQ ID NO: 20) EIVLTQSPATLSLSPGERATLSCRASENVGTYISWYQQKPGQAPRLLIYG ASNRYTGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCGESYGHLYTFGG GTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGECGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGY KYGVYSMGWFRQAPGKGLEGVSAICSGGRTTYSDSVKGRFTISRDNSNQI LYLQMNSLRAEDTAVYYCAARPLWTGDCDLSSSWYKTWGQGTLVTVSS DNA sequence of polypeptide chain L4 (SEQ ID NO: 21) GAGGTGCAGCTGGTGGAGTCTGGAGGAGGACTGGTGCAGCCAGGAGGCAG CCTGAGGCTGTCTTGCGCCGCTTCCGGCTACAAGTATGGCGTGTACTCCA TGGGATGGTTCAGGCAGGCTCCTGGCAAGGGACTGGAGGGCGTGTCCGCC ATCTGTTCTGGCGGCAGAACCACATACTCTGACTCCGTGAAGGGCAGGTT TACCATCTCCCGGGATAACAGCAACCAGATCCTGTATCTGCAGATGAACT CCCTGAGAGCCGAGGACACCGCCGTGTACTATTGCGCTGCTAGGCCACTG TGGACAGGCGACTGTGATCTGTCCAGCTCTTGGTATAAGACCTGGGGCCA GGGCACCCTGGTGACAGTGTCCAGCGGTGGAGGCGGTAGTGGAGGCGGTT CAGAGATCGTGCTGACCCAGTCTCCAGCCACACTGTCTCTGTCCCCAGGA GAGAGGGCCACCCTGAGCTGCCGGGCTTCTGAGAACGTGGGCACATACAT CTCCTGGTATCAGCAGAAGCCAGGACAGGCTCCTAGGCTGCTGATCTACG GCGCTAGCAATAGATATACCGGCATCCCTGCTCGCTTCAGCGGATCTGGA TCCGGCACAGACTTTACCCTGACAATCTCCAGCCTGGAGCCAGAGGATTT CGCCGTGTACTATTGTGGCGAGTCCTACGGCCACCTGTATACCTTTGGCG GCGGCACAAAGGTGGAGATCAAGCGAACGGTGGCTGCACCATCTGTCTTC ATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGT GTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGG TGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAG GACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAA AGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGG GCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT Amino acid sequence of polypeptide chain L4 (SEQ ID NO: 22) EVQLVESGGGLVQPGGSLRLSCAASGYKYGVYSMGWFRQAPGKGLEGVSA ICSGGRTTYSDSVKGRFTISRDNSNQILYLQMNSLRAEDTAVYYCAARPL WTGDCDLSSSWYKTWGQGTLVTVSSGGGGSGGGSEIVLTQSPATLSLSPG ERATLSCRASENVGTYISWYQQKPGQAPRLLIYGASNRYTGIPARFSGSG SGTDFTLTISSLEPEDFAVYYCGESYGHLYTFGGGTKVEIKRTVAAPSVF IFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC DNA sequence of polypeptide chain H5 (SEQ ID NO: 23) GAGGTGCAGCTGGTGGAGTCTGGAGGAGGACTGGTGCAGCCAGGAGGCAG CCTGAGGCTGTCTTGCGCCGCTTCCGGCTACAAGTATGGCGTGTACTCCA TGGGATGGTTCAGGCAGGCTCCTGGCAAGGGACTGGAGGGCGTGTCCGCC ATCTGTTCTGGCGGCAGAACCACATACTCTGACTCCGTGAAGGGCAGGTT TACCATCTCCCGGGATAACAGCAACCAGATCCTGTATCTGCAGATGAACT CCCTGAGAGCCGAGGACACCGCCGTGTACTATTGCGCTGCTAGGCCACTG TGGACAGGCGACTGTGATCTGTCCAGCTCTTGGTATAAGACCTGGGGCCA GGGCACCCTGGTGACAGTGTCCAGCGAGAGCAAGTACGGACCACCTTGCC CACCATGTCCAGCTCCTGAGTTTGAGGGAGGACCATCCGTGTTCCTGTTT CCTCCAAAGCCTAAGGACACCCTGATGATCAGCCGGACACCTGAGGTGAC CTGCGTGGTGGTGGACGTGTCTCAGGAGGATCCAGAGGTGCAGTTCAACT GGTACGTGGATGGCGTGGAGGTGCACAATGCTAAGACCAAGCCAAGAGAG GAGCAGTTTAATTCCACATACCGCGTGGTGAGCGTGCTGACCGTGCTGCA TCAGGATTGGCTGAACGGCAAGGAGTATAAGTGCAAGGTGTCCAATAAGG GCCTGCCCAGCTCTATCGAGAAGACAATCAGCAAGGCTAAGGGACAGCCT AGGGAGCCACAGGTGTACACCCTGCCCCCTTCTCAGGAGGAGATGACAAA GAACCAGGTGTCCCTGACCTGTCTGGTGAAGGGCTTCTATCCAAGCGACA TCGCTGTGGAGTGGGAGTCTAATGGCCAGCCCGAGAACAATTACAAGACC ACACCACCCGTGCTGGACTCTGATGGCTCCTTCTTTCTGTATTCTAGGCT GACAGTGGATAAGTCCCGGTGGCAGGAGGGCAACGTGTTTAGCTGCTCTG TGATGCACGAGGCCCTGCACAATCATTATACCCAGAAGTCCCTGAGCCTG TCTCTGGGCAAG Amino acid sequence of polypeptide chain H5 (SEQ ID NO: 24) EVQLVESGGGLVQPGGSLRLSCAASGYKYGVYSMGWFRQAPGKGLEGVSA ICSGGRTTYSDSVKGRFTISRDNSNQILYLQMNSLRAEDTAVYYCAARPL WTGDCDLSSSWYKTWGQGTLVTVSSESKYGPPCPPCPAPEFEGGPSVFLF PPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPRE EQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQP REPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSL SLGK DNA sequence of E-linker linker (SEQ ID NO: 25) GAACCTAAGTCTAGCGACAAAACTCATACCAGCCCCCCTAGTCCA Amino acid sequence of E-linker linker (SEQ ID NO: 26) EPKSSDKTHTSPPSP DNA sequence of G9-linker linker (SEQ ID NO: 27) GGTGGaGGCGGTAGTGGAGGCGGTTCA Amino acid sequence of G9-linker linker (SEQ ID NO: 28) GGGGSGGGS DNA sequence of IgG4 Fc (SEQ ID NO: 29) GAGAGCAAGTACGGACCACCTTGCCCACCATGTCCAGCTCCTGAGTTTGA GGGAGGACCATCCGTGTTCCTGTTTCCTCCAAAGCCTAAGGACACCCTGA TGATCAGCCGGACACCTGAGGTGACCTGCGTGGTGGTGGACGTGTCTCAG GAGGATCCAGAGGTGCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCA CAATGCTAAGACCAAGCCAAGAGAGGAGCAGTTTAATTCCACATACCGCG TGGTGAGCGTGCTGACCGTGCTGCATCAGGATTGGCTGAACGGCAAGGAG TATAAGTGCAAGGTGTCCAATAAGGGCCTGCCCAGCTCTATCGAGAAGAC AATCAGCAAGGCTAAGGGACAGCCTAGGGAGCCACAGGTGTACACCCTGC CCCCTTCTCAGGAGGAGATGACAAAGAACCAGGTGTCCCTGACCTGTCTG GTGAAGGGCTTCTATCCAAGCGACATCGCTGTGGAGTGGGAGTCTAATGG CCAGCCCGAGAACAATTACAAGACCACACCACCCGTGCTGGACTCTGATG GCTCCTTCTTTCTGTATTCTAGGCTGACAGTGGATAAGTCCCGGTGGCAG GAGGGCAACGTGTTTAGCTGCTCTGTGATGCACGAGGCCCTGCACAATCA TTATACCCAGAAGTCCCTGAGCCTGTCTCTGGGCAAG Amino acid sequence of IgG4 Fc (SEQ ID NO: 30) ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQ EDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKE YKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCL VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQ EGNVFSCSVMHEALHNHYTQKSLSLSLGK

Example 2 Affinity Analysis by FACS

[0047] For the series of constructed bispecific antibody samples, flow cytometry was used to determine the affinity of these samples for antigen. The sample had an initial concentration of 300 nm and was diluted in a 3-fold gradient. And then, the affinity of the samples having different concentrations to the TIGIT antigen or CD47 antigen expressed on CHO-K1 cells were tested, respectively. Next, the geometric mean was used to generate an antibody-antigen binding curve, the original data of the four parameters were plotted using GRAPHPAD Prism V6.02 software, and a best-fit program was compiled to analyze the EC.sub.50.

[0048] For the affinity analysis of TIGIT antigen, after the bispecific antibody produced by fusing the TIGIT single domain antibody to the N-terminus or C-terminus of the heavy or light chain of a CD47 monoclonal antibody (mAb) was incubated on CHO-K1 cells expressing TIGIT antigen, FACS detection showed that compared with the TIGIT single domain antibody control fused to IgG4 Fc (sdAb-TIGIT-IgG4PE), the affinity of the bispecific antibody produced by fusing the TIGIT single domain antibody to the N-terminus of the heavy or light chain of the CD47 monoclonal antibody (mAb) with TIGIT antigen was significantly higher than that of the single domain antibody control (FIG. 1). However, the affinity of the bispecific antibody produced by fusing the TIGIT single domain antibody to the C-terminus of the heavy or light chain of a CD47 monoclonal antibody (mAb) with the TIGIT antigen was lower than that of the single-domain antibody control. Therefore, when the TIGIT single domain antibody was connected to the N-terminus of the CD47 monoclonal antibody, the binding of the TIGIT single domain antibody to the TIGIT antigen would be enhanced, and when the TIGIT single domain antibody was connected to the C-terminus of the CD47 monoclonal antibody, the affinity of the TIGIT single domain antibody with the TIGIT antigen would be reduced.

[0049] For the affinity analysis of CD47 antigen, the bispecific antibody produced by fusing TIGIT single domain antibody to the terminus of the heavy or light chain of a CD47 monoclonal antibody (mAb) was incubated on CHO-K1 cells expressing CD47 antigen, FACS detection showed that compared with the CD47 monoclonal antibody control, the EC.sub.50 values for the binding of all the bispecific antibody samples to the CD47 antigen were higher than the EC.sub.50 values for the binding of the CD47 monoclonal antibody to the CD47 antigen (FIG. 2). This difference in affinity implied that the TIGIT single domain antibody was connected to the CD47 monoclonal antibody, which would interfere with the binding of the CD47 monoclonal antibody to the CD47 antigen to a certain extent, thereby resulting in a decrease in the affinity of the constructed bispecific antibody with the CD47 antigen.

Example 3 Biological Activity Assay In Vitro

[0050] For the biological activity assay in vitro of the CD47/TIGIT bispecific antibody, since there was no analytical system that could detect both CD47 and TIGIT blockers at the same time, the Promega detection kit was used for the TIGIT blocker bioassay, and then the cell phagocytosis test of anti-CD47 antibody was used to determine the activity of the bispecific antibody.

[0051] Promega's TIGIT/CD155 blocking bioassay system could be used to determine the biological activity of antibodies or other biological agents that could block the TIGIT/CD155 interaction. The test consisted of two genetically engineered cell lines: TIGIT effector cells, that was, jurkat T cells expressing human TIGIT and a luciferase reporter gene driven by a natural promoter that responded to TCR activation and CD226 co-stimulation; CD155 aAPC/CHO-K1 cells were CHO-K1 cells that expressed human CD155 and a cell surface protein that could activate the TCR complex in an antigen-independent manner. When the two cell types were co-cultured, TIGIT inhibited CD226 activation and promoter-mediated luminescence. The addition of anti-TIGIT antibody could block the interaction between TIGIT and CD155 or inhibit the ability of TIGIT to prevent CD226 from forming a homodimer, thereby restoring the promoter-mediated luminescence.

[0052] When testing the activity of anti-TIGIT antibodies, the effector cell line Jurkat T cells were first plated in a 96-well plate, and then anti-TIGIT monoclonal antibody samples and the stimulatory cell line CD155 aAPC/CHO-K1 cells were added. The system was incubated at 37° C. for 6 hours. After that, Bio-Glo™ fluorescence detection reagent was added and incubated at room temperature for 5-10 minutes. Finally, a chemical fluorescence signal plate reader was used to read the fluorescence signal in the 96-well plate. In this test, 8 concentrations were used and three replicate wells were set for each concentration. A four-parameter curve was plotted with the relative fluorescence value as the y-axis and the concentration of the antibody sample as the x-axis. GraphPad Prism software was used to analyze the curve and the EC.sub.50 value of the anti-TIGIT monoclonal antibody sample was obtained.

[0053] For the cell phagocytosis test of anti-CD47 antibody, PBMC was firstly extracted from human peripheral blood via a concentration gradient method. After that, a whole monocyte separation kit (Miltenyi Biotech) was used to separate monocytes from PBMC. These monocytes were stimulated into macrophages with GM-CSF over a period of 14 days. At day 14, HL60 cells were stained with PKH26 dye and seeded in a 96-well culture plate, MDM was digested from the culture dish with Accutase, and then MDM was added to the culture plate with HL60 stained by PKH26. In addition, a gradiently diluted anti-CD47 antibody samples were added and incubated at 37° C. for 1 hour to allow the cell phagocytosis reaction to proceed. One hour later, the MDM was digested from the cell culture dish and a fluorescently labeled anti-CD11b antibody was used to stain the MDM. After that, BD FACSCalibur flow cytometry was then used to analyze the cells in the cell plate. The percentage of phagocytosis was calculated by dividing the number of PKH26 and CD11b double positive cells by the number of PKH26 single positive cells. The dose-effect curve graph was plotted with the percentage of phagocytosis as the y-axis and the concentration of anti-CD47 antibody as the x-axis, and GraphPad Prism software was used to analyze and obtain the EC.sub.50 value and other curve parameters.

[0054] The biological activity assay results based on the TIGIT/CD155 blocker showed that the bispecific antibody TIGIT-G9-HN had a higher biological activity than the TIGIT single domain antibody control (sdAb-TIGIT-IgG4PE) (FIG. 3). The TIGIT single domain antibody in this bispecific antibody was fused to the N-terminus of the heavy chain of the CD47 monoclonal antibody (mAb), and the affinity analysis results by FACS showed that its affinity was significantly higher than that of the TIGIT single domain antibody control. The consistency of affinity and biological activity in vitro further confirmed that this bispecific antibody enhanced the activity of the TIGIT single domain antibody.

[0055] The cell phagocytosis test results of anti-CD47 antibody showed that the EC.sub.50 value of the bispecific antibody TIGIT-G9-HN was slightly lower than that of the CD47 control antibody (FIG. 4). The affinity analysis results by FACS showed that its affinity was lower than that of the CD47 monoclonal antibody control, which indicated that the reduction of affinity had little effect on the biological activity in vitro of this bispecific antibody.