ANTI-PD-1/VEGFA BIFUNCTIONAL ANTIBODY, PHARMACEUTICAL COMPOSITION THEREOF AND USE THEREOF

Abstract

The present application relates to the fields of tumor treatment and molecular immunology, and specifically, to an anti-VEGFA/PD-1 bifunctional antibody, a pharmaceutical composition thereof and use thereof. Specifically, the anti-VEGFA/PD-1 bifunctional antibody comprises a first protein functional region targeting VEGFA and a second protein functional region targeting PD-1. The bifunctional antibody can specifically bind to VEGFA and PD-1, specifically relieve immunosuppression of VEGFA and PD-1 in an organism, and inhibit tumor-induced angiogenesis, thus having good application prospect.

Claims

1. A bispecific antibody, comprising: a first protein functional region targeting VEGFA, and a second protein functional region targeting PD-1; wherein: the first protein functional region is an anti-VEGFA antibody or an antigen-binding fragment thereof, a heavy chain variable region of the anti-VEGFA antibody comprising HCDR1-HCDR3 with amino acid sequences set forth in SEQ ID NOs: 15-17 respectively, and a light chain variable region of the anti-VEGFA antibody comprising LCDR1-LCDR3 with amino acid sequences set forth in SEQ ID NOs: 18-20 respectively; and the second protein functional region is an anti-PD-1 antibody or an antigen-binding fragment thereof, a heavy chain variable region of the anti-PD-1 antibody comprising HCDR1-HCDR3 with amino acid sequences set forth in SEQ ID NOs: 21-23 respectively, and a light chain variable region of the anti-PD-1 antibody comprising LCDR1-LCDR3 with amino acid sequences set forth in SEQ ID NOs: 24-26 respectively.

2. The bispecific antibody according to claim 1, wherein, the anti-VEGFA antibody or the antigen-binding fragment thereof is selected from Fab, Fab′, F(ab′).sub.2, Fd, Fv, dAb, a complementarity determining region fragment, a single chain antibody, a humanized antibody, a chimeric antibody, and a diabody; and/or, the anti-PD-1 antibody or the antigen-binding fragment thereof is selected from Fab, Fab′, F(ab′).sub.2, Fd, Fv, dAb, a complementarity determining region fragment, a single chain antibody, a humanized antibody, a chimeric antibody, and a diabody.

3. The bispecific antibody according to claim 1, wherein, the first protein functional region is an immunoglobulin, a heavy chain variable region of the immunoglobulin comprising HCDR1-HCDR3 with amino acid sequences set forth in SEQ ID NOs: 15-17 respectively, and a light chain variable region of the immunoglobulin comprising LCDR1-LCDR3 with amino acid sequences set forth in SEQ ID NOs: 18-20 respectively; and the second protein functional region is a single chain antibody, a heavy chain variable region of the single chain antibody comprising HCDR1-HCDR3 with amino acid sequences set forth in SEQ ID NOs: 21-23 respectively, and a light chain variable region of the single chain antibody comprising LCDR1-LCDR3 with amino acid sequences set forth in SEQ ID NOs: 24-26 respectively; or, the first protein functional region is a single chain antibody, a heavy chain variable region of the single chain antibody comprising HCDR1-HCDR3 with amino acid sequences set forth in SEQ ID NOs: 21-23 respectively, and a light chain variable region of the single chain antibody comprising LCDR1-LCDR3 with amino acid sequences set forth in SEQ ID NOs: 24-26 respectively; and the second protein functional region is an immunoglobulin, a heavy chain variable region of the immunoglobulin comprising HCDR1-HCDR3 with amino acid sequences set forth in SEQ ID NOs: 15-17 respectively, and a light chain variable region of the immunoglobulin comprising LCDR1-LCDR3 with amino acid sequences set forth in SEQ ID NOs: 18-20 respectively.

4. The bispecific antibody according to claim 3, wherein, the amino acid sequence of the heavy chain variable region of the immunoglobulin is set forth in SEQ ID NO: 5, and the amino acid sequence of the light chain variable region of the immunoglobulin is set forth in SEQ ID NO: 7; and the amino acid sequence of the heavy chain variable region of the single chain antibody is set forth in SEQ ID NO: 9, and the amino acid sequence of the light chain variable region of the single chain antibody is set forth in SEQ ID NO: 11; or, the amino acid sequence of the heavy chain variable region of the single chain antibody is set forth in SEQ ID NO: 9, and the amino acid sequence of the light chain variable region of the single chain antibody is set forth in SEQ ID NO: 11; and the amino acid sequence of the heavy chain variable region of the immunoglobulin is set forth in SEQ ID NO: 5, and the amino acid sequence of the light chain variable region of the immunoglobulin is set forth in SEQ ID NO: 7.

5. The bispecific antibody according to claim 3, wherein the immunoglobulin is an IgG, IgA, IgD, IgE, or IgM.

6. The bispecific antibody according to claim 3, wherein two single chain antibodies are present, and one terminus of each single chain antibody is linked to the C-terminus or the N-terminus of one of the two heavy chains of the immunoglobulin.

7. The bispecific antibody according to claim 3, wherein, the immunoglobulin comprises a non-CDR region derived from a human antibody.

8. The bispecific antibody according to claim 3, wherein, the immunoglobulin comprises constant regions derived from a human antibody.

9. The bispecific antibody according to claim 3, wherein, the heavy chain constant region of the immunoglobulin is human Ig gamma-1 chain C region or human Ig gamma-4 chain C region, and its light chain constant region is human Ig kappa chain C region.

10. The bispecific antibody according to claim 1, wherein the first and second protein functional regions are linked directly or via a linker fragment.

11. The bispecific antibody according to claim 1, wherein the numbers of the first and second protein functional regions are each independently 1, 2 or more.

12. The bispecific antibody according to claim 1, wherein, the bispecific antibody binds to the VEGFA protein with an EC.sub.50 of less than 1 nM, less than 0.5 nM, less than 0.2 nM, less than 0.15 nM, or less than 0.14 nM; and/or, the bispecific antibody binds to the PD-1 protein with an EC.sub.50 of less than 1 nM, less than 0.5 nM, less than 0.2 nM, less than 0.17 nM, less than 0.16 nM, or less than 0.15 nM.

13. An isolated nucleic acid molecule, encoding the bispecific antibody according to claim 1.

14. A vector, comprising the isolated nucleic acid molecule according to claim 13.

15. A host cell, comprising the isolated nucleic acid molecule according to claim 13.

16. A method for preparing the bispecific antibody according to claim 1, comprising: culturing a host cell in a suitable condition, and isolating the bispecific antibody from the cell cultures, wherein the host cell comprises an isolated nucleic acid molecule, and wherein the isolated nucleic acid molecule encodes the bispecific antibody according to claim 1.

17. A conjugate, comprising a bispecific antibody and a conjugated moiety, wherein the bispecific antibody is the bispecific antibody according to claim 1, and the conjugated moiety is a detectable label.

18. A kit, comprising the bispecific antibody according to claim 1.

19. (canceled)

20. A pharmaceutical composition, comprising the bispecific antibody according to claim 1.

21-22. (canceled)

23. An in vivo or in vitro method, comprising administering to a cell an effective amount of the bispecific antibody according to claim 1, wherein the method is selected from: (1) a method for detecting the level of VEGFA in a sample, a method for blocking the binding of VEGFA to VEGFR2, a method for down-regulating the activity or level of VEGFA, a method for relieving the stimulation of VEGFA on vascular endothelial cell proliferation, a method for inhibiting vascular endothelial cell proliferation, or a method for blocking tumor angiogenesis; and/or (2) a method for blocking the binding of PD-1 to PD-L1, a method for down-regulating the activity or level of PD-1, a method for relieving the immunosuppression of PD-1 in an organism, a method for promoting IFN-γ secretion in T lymphocytes, or a method for promoting IL-2 secretion in T lymphocytes.

24. A method for preventing and/or treating a malignant tumor, comprising administering to a subject in need an effective amount of the bispecific antibody according to claim 1.

25.-26. (canceled)

27. The bispecific antibody according to claim 8, wherein the constant regions of the immunoglobulin are selected from constant regions of human IgG1, IgG2, IgG3, and IgG4.

28. The bispecific antibody according to claim 10, wherein the linker fragment is (GGGGS)m, wherein m is 1, 2, 3, 4, 5, or 6.

29. The bispecific antibody according to claim 12, wherein the EC.sub.50 with which the bispecific antibody binds to the VEGFA protein is detected by indirect ELISA; and/or wherein the EC.sub.50 with which the bispecific antibody binds to the PD-1 protein is detected by indirect ELISA.

30. The conjugate according to claim 17, wherein the conjugated moiety is a radioisotope, a fluorescent substance, a luminescent substance, a colored substance, or an enzyme.

31. The kit according to claim 30, wherein the kit further comprises a second antibody capable of specifically binding to the bispecific antibody.

32. The kit according to claim 31, wherein the second antibody further comprises a detectable label.

33. The kit according to claim 32, wherein the detectable label is a radioisotope, a fluorescent substance, a luminescent substance, a colored substance, or an enzyme.

34. The pharmaceutical composition according to claim 20, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

35. The method according to claim 24, wherein the malignant tumor is selected from colon cancer, rectal cancer, lung cancer, liver cancer, ovarian cancer, skin cancer, glioma, melanoma, renal tumor, prostate cancer, bladder cancer, gastrointestinal cancer, breast cancer, brain cancer and leukemia.

36. The method according to claim 35, wherein the lung cancer is non-small cell lung cancer.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0173] FIG. 1 shows the SDS-PAGE detection results of bifunctional antibody VP101. The samples of the four lanes from left to right and their respective loading amounts are: antibody in non-reduced protein electrophoresis loading buffer, 1 μg; antibody in reduced protein electrophoresis loading buffer, 1 μg; Marker, 5 μL; BSA, 1 μg.

[0174] FIG. 2 shows the detection results of kinetic characteristic parameters of the binding of antibody VP101 to PD-1.

[0175] FIG. 3 shows the detection results of kinetic characteristic parameters of the binding of antibody BsAbB7 to PD-1.

[0176] FIG. 4 shows the detection results of kinetic characteristic parameters of the binding of antibody BsAbB8 to PD-1.

[0177] FIG. 5 shows the detection results of kinetic characteristic parameters of the binding of antibody 14C12H1L1 to PD-1.

[0178] FIG. 6 shows the detection results of kinetic characteristic parameters of the binding of antibody nivolumab to PD-1.

[0179] FIG. 7 shows the detection results of kinetic characteristic parameters of the binding of antibody VP101 to VEGF.

[0180] FIG. 8 shows the detection results of kinetic characteristic parameters of the binding of antibody BsAbB7 to VEGF.

[0181] FIG. 9 shows the detection results of kinetic characteristic parameters of the binding of antibody BsAbB8 to VEGF.

[0182] FIG. 10 shows the detection results of kinetic characteristic parameters of the binding of antibody bevacizumab to VEGF.

[0183] FIG. 11 shows the binding activity of antibody VP101 to VEGFA detected by indirect ELISA.

[0184] FIG. 12 shows the respective binding activities of antibodies VP101, BsAbB7, BsAbB8 and bevacizumab to VEGFA-his detected by indirect ELISA.

[0185] FIG. 13 shows the binding activity of antibody VP101 to PD-1 detected by indirect ELISA.

[0186] FIG. 14 shows the respective binding activities of antibodies VP101, BsAbB7, BsAbB8, 14C12H1L1 and nivolumab to PD-1 detected by indirect ELISA.

[0187] FIG. 15 shows the activity of antibody VP 101 in competing with VEGFR2 for binding to VEGFA detected by competitive ELISA.

[0188] FIG. 16 shows the activity of antibody VP101 in competing with PD-L1 for binding to PD-1 detected by competitive ELISA.

[0189] FIG. 17 shows the binding EC.sub.50 of antibodies 14C12H1L1 and VP101 to 293T-PD-1 cell surface protein PD-1 detected by FACS.

[0190] FIG. 18 shows the binding EC.sub.50 of antibodies VP101, BsAbB7 and BsAbB8 to 293T-PD-1 cell surface protein PD-1 detected by FACS.

[0191] FIG. 19 shows the activity of antibodies VP101 and 14C12H1L1 in competing with PD-L1 for binding to 293T-PD-1 cell surface protein PD-1 detected by FACS.

[0192] FIG. 20 shows the activity of antibodies 14C12H1L1, VP101, BsAbB7, BsAbB8, 14C12H1L and nivolumab in competing with PD-L1 for binding to 293T-PD-1 cell surface protein PD-1 detected by FACS.

[0193] FIG. 21 shows the neutralization bioactivity of antibodies VP101, BsAbB7 and BsAbB8 in blocking VEGF to activate NFAT signaling pathway.

[0194] FIG. 22 shows the effect of bevacizumab and VP101 on HUVEC cell proliferation.

[0195] FIG. 23 shows the effect of VP101 on secretion of IFN-γ in mixed culture system of DC and PBMC cells.

[0196] FIG. 24 shows the effect of VP101, BsAbB7 and BsAbB8 on secretion of IFN-γ in mixed culture system of DC and PBMC cells.

[0197] FIG. 25 shows the effect of VP101, BsAbB7 and BsAbB8 on secretion of IL-2 in mixed culture system of DC and PBMC cells.

[0198] FIG. 26 shows the effect of antibodies 14C12H1L1 and VP101 on secretion of the cytokine IL-2 induced by mixed culture of PBMC and Raji-PD-L1 cells detected by ELISA.

[0199] FIG. 27 shows effect of antibodies 14C12H1L1 and VP101 on secretion of the cytokine IFN-γ induced by mixed culture of PBMC and Raji-PD-L1 cells detected by ELISA.

[0200] FIG. 28 shows effect of antibodies 14C12H1L1, VP101, BsAbB7 and BsAbB8 on secretion of the cytokine IFN-γ induced by mixed culture of PBMC and Raji-PD-L1 cells detected by ELISA.

[0201] FIG. 29 shows effect of antibodies 14C12H1L1, VP101, BsAbB7 and BsAbB8 on secretion of the cytokine IL-2 induced by mixed culture of PBMC and Raji-PD-L1 cells detected by ELISA.

[0202] FIG. 30 shows the inhibition of tumor growth by VP101 at different concentrations.

[0203] FIG. 31 shows effect of VP101 at different concentrations on body weight of mouse.

DETAILED DESCRIPTION

[0204] The embodiments of the present application will be described in detail below with reference to the examples. Those skilled in the art will understand that the following examples are only used for illustrative purposes, and should not be regarded as limiting the scope of the present invention. The cases without the specific descriptions of techniques or conditions were carried out according to the technologies or conditions described in the literature in the art (e.g., see, Guide to Molecular Cloning Experiments, authored by J. Sambrook et al., and translated by Huang Peitang et al., third edition, Science Press) or according to the product manual. Reagents or instruments used are all commercially available conventional products if the manufacturers thereof are not specified.

[0205] In the following examples, the marketed antibody bevacizumab (trade name Avastin®) for the same target was purchased from Roche as a control antibody, or was prepared according to Preparation Example 4.

[0206] In the following examples, the marketed antibody nivolumab for the same target (trade name Opdivo®) was purchased from BMS as a control antibody.

[0207] In the following examples, the amino acid sequences of the control antibodies BsAbB7 and BsAbB8 were identical to the amino acid sequences of BsAbB7 and BsAbB8 respectively in Chinese Patent Publication CN105175545A.

PREPARATION EXAMPLE 1

Preparation of Fusion Proteins PD-1-mFc, PD-1-hFc and PD-L1-hFc

[0208] The preparation of fusion proteins PD-1-mFc, PD-1-hFc and PD-L1-hFc and the SDS-PAGE electrophoresis detection are carried out by fully referring to Preparation Example 1 of Chinese Patent Publication CN106632674A.

[0209] The amino acid sequences and the encoding nucleotide sequences of the fusion proteins PD-1-mFc, PD-1-hFc and PD-L1-hFc in this preparation example are the same as those of PD-1-mFc, PD-1-hFc and PDL-1-hFc respectively in the Preparation Example 1 of Chinese Patent Publication CN106632674A.

[0210] Fusion proteins PD-1-mFc, PD-1-hFc and PD-L1-hFc were thus obtained.

PREPARATION EXAMPLE 2

Expression and Purification of Fusion Protein VEGFA-His

[0211] 1. Construction of Plasmid VEGFA-His

[0212] PCR amplification was performed using VEGFA human cDNA (purchased from Origene) as a template and the hVEGFA-His fragment was purified and isolated using an ordinary DNA product purification kit. The isolated hVEGFA-His fragment and an expression vector pcDNA3.1 were enzyme-digested with XbaI&HindIII-HF, and a target gene fragment was isolated by gel extraction and ligated with a linear expression vector by T4 ligase. Then all the ligation products were transformed into DH5a chemically competent cells and coated on an Agar plate with Amp. Well separated single colonies were selected for colony PCR identification, PCR positive clones were inoculated to an LB culture medium for culture, and a bacteria solution was taken and sent to Guangzhou Invitrogen Biotechnology for sequencing verification. The alignment of the sequencing results showed that the insertion sequence of the positive recon was completely correct.

[0213] 2. Expression and Purification of Fusion Protein VEGFA-His

[0214] After the recombinant plasmid VEGFA-his was transfected into 293F cells (purchased from Invitrogen) for 7 days according to the manual in lipofectamin transfection kit (purchased from Invitrogen), the culture medium was subjected to high-speed centrifugation, supernatant concentration and buffer exchange into Binding Buffer A, and then loaded onto a HisTrap column, and proteins were linearly eluted with Elution Buffer A. The primary pure sample was subjected to buffer exchange into Binding Buffer B with a HiTrap Desalting column and loaded onto a HiTrap Q column, proteins were linearly eluted with Elution Buffer B, and the target sample was isolated and buffer exchanged into PBS. The purified sample was added to a reduced protein electrophoresis loading buffer for SDS-PAGE electrophoresis detection.

[0215] The fusion protein VEGFA-His was thus obtained.

[0216] The amino acid sequence of VEGFA-His is as follows (171 aa):

TABLE-US-00007 (SEQ ID NO: 1) APMAEGGGQNHHEVVKFMDVYQRSYCHPIETLVDIFQEYPDEIEYIFKPS CVPLMRCGGCCNDEGLECVPTEESNITMQIMRIKPHQGQHIGEMSFLQHN KCECRPKKDRARQENPCGPCSERRKHLFVQDPQTCKCSCKNTDSRCKARQ LELNERTCRCDKPRRHHHHHH

[0217] wherein, the underlined part is the amino acid sequence of VEGFA.

[0218] Nucleotide sequence encoding VEGFA-His (513 bp)

TABLE-US-00008 (SEQ ID NO: 2) GCACCCATGGCCGAGGGCGGCGGCCAGAACCACCACGAGGTGGTGAAGTT CATGGACGTGTACCAGAGAAGCTACTGCCACCCCATCGAGACCCTGGTGG ACATCTTCCAGGAGTACCCCGACGAGATCGAGTACATCTTCAAGCCCAGC TGCGTGCCCCTGATGAGATGCGGCGGCTGCTGCAACGACGAGGGCCTGGA GTGCGTGCCCACCGAGGAGAGCAACATCACCATGCAGATCATGAGAATCA AGCCCCACCAGGGCCAGCACATCGGCGAGATGAGCTTCCTGCAGCACAAC AAGTGCGAGTGCAGACCCAAGAAGGACAGAGCCAGACAGGAGAACCCCTG CGGCCCCTGCAGCGAGAGAAGAAAGCACCTGTTCGTGCAGGACCCCCAGA CCTGCAAGTGCAGCTGCAAGAACACCGACAGCAGATGCAAGGCCAGACAG CTGGAGCTGAACGAGAGAACCTGCAGATGCGACAAGCCCAGAAGACATCA TCACCATCACCAC

PREPARATION EXAMPLE 3

Expression and Purification of Fusion Protein VEGFR2-hFc

[0219] 1. Synthesis of Gene VEGFR2-hFc:

[0220] The amino acids corresponding to the extracellular fragment VEGFR2 ECD of gene VEGFR2 (Vascular Endothelial Growth Factor Receptor 2, NCBI GenBank: NP_002244) were fused with TEV and the Fc protein fragment of human IgG (hFc) respectively (SEQ ID NO: 3). Genscript was entrusted to synthesize corresponding encoding nucleotide sequence (SEQ ID NO: 4).

[0221] VEGFR2, Vascular Endothelial Growth Factor Receptor 2, NCBI GenBank NP_002244; hFc: Ig gamma-1 chain C region, ACCESSION: P01857, 106-330;

[0222] Amino acid sequence of fusion protein VEGFR2-hFc: (998 aa)

TABLE-US-00009 (SEQ ID NO: 3) [00001] [00002] [00003] [00004] [00005] [00006] [00007] [00008] [00009] [00010] [00011] [00012] [00013] HTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0223] wherein, the wavy-underlined part is the ECD part of VEGFR2, the framed part is TEV enzyme digestion site, and the solid-underlined part is hFc part.

[0224] Nucleotide sequence encoding fusion protein VEGFR2-hFc: (2997 bp)

TABLE-US-00010 (SEQ ID NO: 4) [00014] [00015] [00016] [00017] [00018] [00019] [00020] [00021] [00022] [00023] [00024] [00025] [00026] [00027] [00028] [00029] [00030] [00031] [00032] [00033] [00034] [00035] [00036] [00037] [00038] [00039] [00040] [00041] [00042] [00043] [00044] [00045] [00046] [00047] [00048] [00049] [00050] [00051] [00052] [00053] [00054] CACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTC TTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGC GTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGA CGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGC ACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAG GAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCAT CTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCC GGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTAT CCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACA AGACCACGCCTCCCGTGTTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCA CCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCAT GAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCCGGGAAATG A

[0225] wherein, the wavy-underlined part is the ECD part of VEGFR2, the framed part is TEV enzyme digestion site, and the solid-underlined part is hFc part.

[0226] 2. Construction of Plasmid pUC57Simple-VEGFR2-hFc:

[0227] The VEGFR2-hFc encoding gene synthesized by Genscript was cloned into an expression vector pUC57simple (provided by Genscript), and a pUC57simple-VEGFR2-hFc plasmid was obtained.

[0228] 3. Construction of Recombinant Plasmid pcDNA3.1-VEGFR2-hFc:

[0229] The plasmid pUC57simple-VEGFR2-hFc was enzyme-digested (Xba I and BamH I), and the fusion gene fragment VEGFR2-hFc isolated by electrophoresis was ligated with expression vector pcDNA3.1 (purchased from Invitrogen) to give pcDNA3.1-VEGFR2-hFc, which was transfected into competent E. coli cell DH5a (purchased from TIANGEN); the transfection and culture were performed according to the manual. The positive pcDNA3.1-VEGFR2-hFc colonies were screened, E. coli was amplified according to a conventional method, and a kit (purchased from Tiangen Biotech (Beijing) Co., Ltd., DP103-03) was then used and a recombinant plasmid pcDNA3.1-VEGFR2-hFc was extracted according to the manual of the kit.

[0230] 4. Transfection of Recombinant Plasmid pcDNA3.1-VEGFR2-hFc into 293F Cells

[0231] The recombinant plasmid pcDNA3.1-VEGFR2-hFc was transfected into 293F cells (purchased from Invitrogen) according to the lipofectamin transfection kit (purchased from Invitrogen).

[0232] 5. SDS-PAGE Electrophoresis Detection of VEGFR2-hFc Protein

[0233] After transfecting the recombinant plasmid pcDNA3.1-VEGFR2-hFc into 293F cells for 7 days, the culture medium was subjected to high-speed centrifugation, microporous membrane vacuum filtration and purification in a Mabselect SuRe column to obtain a VEGFR2-hFc fusion protein sample, and a part of the sample was added into a reduced protein electrophoresis loading buffer for SDS-PAGE electrophoresis detection.

[0234] The fusion protein VEGFR2-hFc was thus obtained.

PREPARATION EXAMPLE 4

Preparation of Anti-VEGFA Antibody Bevacizumab

[0235] Chinese Patent Publication CN1259962A is referred to for the amino acid sequences of the heavy chain variable region and the light chain variable region of the marketed VEGFA monoclonal antibody Avastin (bevacizumab). Genscript was entrusted to synthesize nucleotide sequences encoding the heavy chain variable region and the light chain variable region.

[0236] Amino acid sequence of the heavy chain variable region of bevacizumab: (123 aa)

TABLE-US-00011 (SEQ ID NO: 5) EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKGLEWVGW INTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYP HYYGSSHWYFDVWGQGTLVTVSS

[0237] Nucleotide sequence encoding the heavy chain variable region of bevacizumab: (369 bp)

TABLE-US-00012 (SEQ ID NO: 6) GAGGTGCAGCTGGTCGAGTCCGGGGGGGGGCTGGTGCAGCCAGGCGGGTC TCTGAGGCTGAGTTGCGCCGCTTCAGGGTACACCTTCACAAACTATGGAA TGAATTGGGTGCGCCAGGCACCAGGAAAGGGACTGGAGTGGGTCGGCTGG ATCAACACTTACACCGGGGAACCTACCTATGCAGCCGACTTTAAGCGGCG GTTCACCTTCAGCCTGGATACAAGCAAATCCACTGCCTACCTGCAGATGA ACAGCCTGCGAGCTGAGGACACCGCAGTCTACTATTGTGCTAAATATCCC CACTACTATGGGAGCAGCCATTGGTATTTTGACGTGTGGGGGCAGGGGAC TCTGGTGACAGTGAGCAGC

[0238] Amino acid sequence of the light chain variable region of bevacizumab: (107 aa)

TABLE-US-00013 (SEQ ID NO: 7) DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYF TSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQ GTKVEIK

[0239] Nucleotide sequence encoding the light chain variable region of bevacizumab: (321 bp)

TABLE-US-00014 (SEQ ID NO: 8) GATATTCAGATGACTCAGAGCCCCTCCTCCCTGTCCGCCTCTGTGGGCGA CAGGGTCACCATCACATGCAGTGCTTCACAGGATATTTCCAACTACCTGA ATTGGTATCAGCAGAAGCCAGGAAAAGCACCCAAGGTGCTGATCTACTTC ACTAGCTCCCTGCACTCAGGAGTGCCAAGCCGGTTCAGCGGATCCGGATC TGGAACCGACTTTACTCTGACCATTTCTAGTCTGCAGCCTGAGGATTTCG CTACATACTATTGCCAGCAGTATTCTACCGTGCCATGGACATTTGGCCAG GGGACTAAAGTCGAGATCAAG

[0240] The heavy chain constant regions were all Ig gamma-1 chain C region, ACCESSION: P01857; the light chain constant regions were all Ig kappa chain C region, ACCESSION: P01834.

[0241] The heavy chain cDNA and the light chain cDNA of bevacizumab were cloned into vector pcDNA3.1, and the recombinant expression plasmid of the antibody bevacizumab was obtained. The recombinant plasmid was transfected into 293F cells. The 293F cell culture medium was purified and then detected.

[0242] The anti-VEGFA monoclonal antibody Avastin (bevacizumab) was thus obtained.

PREPARATION EXAMPLE 5

Preparation and Detection of Anti-PD-1 Humanized Antibody 14C12H1L1

[0243] The preparation was carried out according to the Examples 3-4 described in Chinese Patent Publication CN106977602A.

[0244] The amino acid sequences of the heavy chain variable region and the light chain variable region of humanized antibody 14C12H1L1, and the nucleotide sequence encoding the same are also the same as those described in Examples 3-4 of Chinese Patent Publication CN106977602A, and are also provided herein as follows:

[0245] Amino acid sequence of the heavy chain variable region of humanized antibody 14C12H1L1: (118 aa)

TABLE-US-00015 (SEQ ID NO: 9) EVQLVESGGGLVQPGGSLRLSCAASGFAFSSYDMSWVRQAPGKGLDWVAT ISGGGRYTYYPDSVKGRFTISRDNSKNNLYLQMNSLRAEDTALYYCANRY GEAWFAYWGQGTLVTVSS

[0246] Nucleotide sequence encoding the heavy chain variable region of humanized antibody 14C12H1L1: (354 bp)

TABLE-US-00016 (SEQ ID NO: 10) GAAGTGCAGCTGGTCGAGTCTGGGGGAGGGCTGGTGCAGCCCGGCGGGTC ACTGCGACTGAGCTGCGCAGCTTCCGGATTCGCCTTTAGCTCCTACGACA TGTCCTGGGTGCGACAGGCACCAGGAAAGGGACTGGATTGGGTCGCTACT ATCTCAGGAGGCGGGAGATACACCTACTATCCTGACAGCGTCAAGGGCCG GTTCACAATCTCTAGAGATAACAGTAAGAACAATCTGTATCTGCAGATGA ACAGCCTGAGGGCTGAGGACACCGCACTGTACTATTGTGCCAACCGCTAC GGGGAAGCATGGTTTGCCTATTGGGGGCAGGGAACCCTGGTGACAGTCTC TAGT

[0247] Amino acid sequence of the light chain variable region of humanized antibody 14C12H1L1: (107 aa)

TABLE-US-00017 (SEQ ID NO: 11) DIQMTQSPSSMSASVGDRVTFTCRASQDINTYLSWFQQKPGKSPKTLIYR ANRLVSGVPSRFSGSGSGQDYTLTISSLQPEDMATYYCLQYDEFPLTFGA GTKLELK

[0248] Nucleotide sequence encoding the light chain variable region of humanized antibody 14C12H1L1: (321 bp)

TABLE-US-00018 (SEQ ID NO: 12) GACATTCAGATGACTCAGAGCCCCTCCTCCATGTCCGCCTCTGTGGGCGA CAGGGTCACCTTCACATGCCGCGCTAGTCAGGATATCAACACCTACCTGA GCTGGTTTCAGCAGAAGCCAGGGAAAAGCCCCAAGACACTGATCTACCGG GCTAATAGACTGGTGTCTGGAGTCCCAAGTCGGTTCAGTGGCTCAGGGAG CGGACAGGACTACACTCTGACCATCAGCTCCCTGCAGCCTGAGGACATGG CAACCTACTATTGCCTGCAGTATGATGAGTTCCCACTGACCTTTGGCGCC GGGACAAAACTGGAGCTGAAG

[0249] The anti-PD-1 humanized antibody 14C12H1L1 was thus obtained.

PREPARATION EXAMPLE 6

Preparation and Identification of hIgG

[0250] The sequence of Human Anti-Hen Egg Lysozyme IgG (anti-HEL, i e , human IgG, abbreviated as hIgG) is derived from a variable region sequence of the Fab F10.6.6 sequence in the research published by Acierno et al., which is entitled “Affinity maturation increases the stability and plasticity of the Fv domain of anti-protein antibodies” (Acierno et al., J Mot Biol. 2007; 374(1): 130-46). The preparation method is as follows:

[0251] Nanjing Genscript Biology was entrusted to carry out codon optimization of amino acids and gene synthesis on heavy and light chain (complete sequence or variable region) genes of human IgG antibody, and by referring to the standard technologies introduced in the “Guide to Molecular Cloning Experiments (Third Edition)” and using standard molecular cloning technologies such as PCR, enzyme digestion, DNA gel extraction, ligation transformation, colony PCR or enzyme digestion identification, the heavy and light chain genes were respectively subcloned into the antibody heavy chain expression vector and antibody light chain expression vector of the mammalian expression system, and the heavy and light chain genes of the recombinant expression vector were further sequenced and analyzed. After the sequence was verified to be correct, endotoxin-free expression plasmids were prepared in a large scale, and the heavy and light chain plasmids were transiently co-transfected into HEK293 cells for expression of recombinant antibody. After 7 days of culture, the cell culture medium was collected and affinity purified using an rProtein A column (GE), and the quality of the resulting antibody sample was determined using SDS-PAGE and SEC-HPLC standard analysis techniques.

[0252] The hIgG was thus obtained, and used in Examples 8-9 below.

EXAMPLE 1

Sequence Design, Preparation and Detection of Heavy and Light Chains of Bifunctional Antibody VP101

[0253] 1. Sequence Design

[0254] The structure of the bifunctional antibody VP101 of the present application is in the Morrison form (IgG-scFv), i.e. C-termini of two heavy chains of an IgG antibody are each linked to a scFv fragment of another antibody, and the main composition design of the heavy and light chains is as shown in Table 1 below.

TABLE-US-00019 TABLE 1 Composition design of the heavy and light chains of VP101 Heavy chain Bifunctional IgG Linker scFv Light antibody No. part fragment part chain VP101 Bevacizum Linker1 14C12H1.sub.v- Bevacizumab-L ab-H Linker1-14C12L1.sub.v

[0255] In the Table 1 above:

[0256] (1) Those with “V” labeled at lower right corner refer to the variable region of corresponding heavy chain or the variable region of corresponding light chain. For those without “V” label, the corresponding heavy or light chain is the full length comprising the constant region. The corresponding sequences described in the above preparation examples are referred to for the amino acid sequences of these variable regions or the full length and the nucleotide sequences encoding them.

[0257] (2) The amino acid sequence of Linker1 is GGGGSGGGGSGG GGSGGGGS (SEQ ID NO: 13)

[0258] 2. Expression and purification of antibody VP101

[0259] The heavy chain cDNA sequence and the light chain cDNA sequence of VP101 were each cloned into vector pUC57simple (provided by Genscript) to obtain plasmids pUC57simple-VP101H and pUC57simple-VP101L, respectively.

[0260] Plasmids pUC57simple-VP101H and pUC57simple-VP101L were enzyme-digested (HindIII&EcoRI), and heavy and light chains isolated by electrophoresis were subcloned into vector pcDNA3.1, and recombinant plasmids were extracted to co-transfect 293F cells. After 7 days of cell culture, the culture medium was centrifuged at high speed, and the supernatant was concentrated and loaded onto a HiTrap MabSelect SuRe column. The protein was further eluted in one step with Elution Buffer, and the target sample antibody VP101 was isolated and buffer exchanged into PBS.

[0261] 3. Detection of Antibody VP101

[0262] The purified sample was added to both a reduced protein electrophoresis loading buffer and a non-reduced protein electrophoresis loading buffer, and then boiled for SDS-PAGE electrophoresis detection. The electropherogram of VP101 is shown in FIG. 1. The target protein of the reduced protein sample is at 75 kD and 25 kD, and the target protein of the non-reduced protein sample (single antibody) is at 200 kD.

[0263] Unless otherwise specified, the humanized antibody VP101 used in the following experiments was prepared by the method of this example.

EXAMPLE 2

Detection of Kinetic Parameters of Humanized Antibody VP101

[0264] 1. Detection of kinetic parameters of the binding of humanized antibody VP101 to PD-1-mFc

[0265] The sample dilution buffer was PBS (0.02% Tween-20, 0.1% BSA, pH7.4). 5 μg/mL antibody was immobilized to an AHC sensor with the immobilization height being about 0.4 nM. The sensor was equilibrated in a buffer for 60 s, and the antibody immobilized to the sensor bound to PD-1-mFc at a concentration of 0.62-50 nM (three-fold gradient dilution) for 120 s, and then the antigen and antibody dissociated in the buffer for 300 s. The data were analyzed by 1:1 model fitting to obtain affinity constants. The data acquisition software was Fortebio Data Acquisition 7.0, and the data analysis software was Fortebio Data Analysis 7.0. Kinetic parameters of the binding of antibodies VP101, BsAbB7, BsAbB8, 14C12H1L1 and the control antibody nivolumab to PD-1-mFc are shown in Table 2, and the detection results of the kinetic characteristic parameters are shown in FIG. 2, FIG. 3, FIG. 4, FIG. 5 and FIG. 6, respectively.

TABLE-US-00020 TABLE 2 Kinetic parameters of the binding of humanized antibody VP101, BsAbB7, BsAbB8, 14C12H1L1 and the control antibody nivolumab to PD-1-mFc Sample ID K.sub.D (M) Kon (1/Ms) S E (kon) Kdis (1/s) S E (kdis) Rmax (nm) VP101 1.68E−10 3.22E+05 1.44E+04 5.40E−05 3.16E−05 0.14-0.28 BsAbB7 1.62E−10 3.27E+05 2.60E+04 5.30E−05 6.24E−05 0.01-0.11 BsAbB8 4.06E−10 3.39E+05 2.04E+04 1.37E−04 4.61E−05 0.01-0.13 14C12H1L1 1.64E−10 4.55E+05 1.61E+04 7.47E−05 2.98E−05 0.24-0.28 Nivolumab 2.32E−10 5.85E+05 2.03E+04 1.36E−04 3.47E−05 0.02-0.14

[0266] K.sub.D is affinity constant; kon is binding rate of antigen and antibody; kdis is dissociation rate of antigen and antibody; K.sub.D=kdis/kon.

[0267] The results show that the antibodies VP101 and BsAbB7 are equivalent in terms of affinity for PD-1-mFc; the affinity constant of VP101 for PD-1-mFc is significantly smaller than that of BsAbB8, suggesting that VP101 has better binding activity; the dissociation rate constant for VP101 and PD-1-mFc was significantly smaller than BsAbB8 and 14C12H1L1, suggesting that VP101 binds to antigen more stably with a dissociation rate slower than that of 14C12H1L1 and BsAbB8.

[0268] 2. Detection of Kinetic Parameters of the Binding of Humanized Antibody VP101 to VEGF-His

[0269] The sample dilution buffer was PBS (0.02% Tween-20, 0.1% BSA, pH7.4). 1 μg/mL VEGF-His was immobilized to the HIS1K sensor for 20 s, then the sensor was equilibrated in a buffer for 60 s, and the VEGF immobilized on the sensor bound to the antibody at a concentration of 12.34-1000 nM (three-fold gradient dilution) for 120 s, and then the antigen and antibody dissociated in the buffer for 300 s. The data were analyzed by 1:1 model fitting to obtain affinity constants. The data acquisition software was Fortebio Data Acquisition 7.0, and the data analysis software was Fortebio Data Analysis 7.0.

[0270] Kinetic parameters of the binding of antibodies VP101, BsAbB7, BsAbB8 and the control antibody bevacizumab to VEGF-His are shown in Table 3, and the detection results of kinetic characteristic parameters are shown in FIG. 7, FIG. 8, FIG. 9 and FIG. 10 respectively.

TABLE-US-00021 TABLE 3 Kinetic parameters of the binding of antibodies VP101, BsAbB7, BsAbB8 and the control antibody bevacizumab to VEGF-His Sample ID K.sub.D (M) Kon (1/Ms) S E (kon) Kdis (1/s) S E (kdis) Rmax (nm) VP101 5.21E−10 1.55E+05 9.67E+03 8.05E−05 4.66E−05 0.39-0.60 BsAbB7 5.14E−10 1.57E+05 9.67E+03 8.05E−05 4.83E−05 0.36-0.53 BsAbB8 6.33E−10 1.71E+05 1.07E+04 1.08E−04 4.64E−05 0.39-0.56 Bevacizumab 7.24E−10 1.23E+05 7.09E+03 8.90E−05 4.53E−05 0.29-0.41

[0271] The results show that the antibodies VP101 and BsAbB7 are equivalent in terms of affinity for the antigen, and the affinity constant of VP101 is significantly smaller than that of BsAbB8 and the control antibody bevacizumab, suggesting that VP101 has better binding activity; the dissociation rate constant of VP101 for VEGF-His is significantly smaller than that of BsAbB8, suggesting that VP101 binds to antigen more stably with a slower dissociation rate than that of BsAbB8.

EXAMPLE 3

Detection of Binding Activity of Antibody VP101 to Antigen by ELISA

[0272] 1. Detection of Binding Activity of Antibody VP101 to Antigen VEGFA-his by Indirect ELISA

[0273] The method is specified as follows:

[0274] The microplate was coated with VEGFA-His and incubated at 37° C. for 2 hours. After being washed, the microplate was blocked with 1% BSA for 2 hours. After being washed, the microplate was added with the gradiently diluted antibody and incubated at 37° C. for 30 minutes. After being washed, the microplate was added with the enzyme-labeled goat anti-human IgG secondary antibody working solution and incubated for 30 minutes at 37° C. After being washed, the microplate was added with TMB chromogenic solution for color developing for 5 minutes in the absence of light, and then stop solution was added to terminate the chromogenic reaction. Then the microplate was put into a microplate reader immediately, and the OD value of each well in the microplate was read at 450 nm. SoftMax Pro 6.2.1 was used to analyze and process the data.

[0275] The detection result of the binding of antibody VP101 to antigen VEGFA-His is shown in FIG. 11. The absorbance intensities at each dose are shown in Table 4. The binding EC.sub.50 of antibody was calculated by curve fitting using antibody concentration as the abscissa and absorbance value as the ordinate, and the results are shown in Table 4 below.

TABLE-US-00022 TABLE 4 Binding of bifunctional antibody to VEGFA-his (Indirect ELISA) Antibody Coating: VEGFA-His concentration (1 μg/mL) (μg/mL) VP101 Bevacizumab 1.0000 3.045 2.943 2.798 2.974 0.3333 3.037 2.861 2.816 2.993 0.1111 2.901 2.689 2.653 2.700 0.0370 2.597 2.460 2.445 2.555 0.0123 2.013 1.914 1.998 2.074 0.0041 1.115 1.086 1.446 1.363 0.0014 0.524 0.496 0.640 0.729 0.0000 0.099 0.091 0.094 0.083 Secondary antibody Goat anti-human IgG (H + L), HRP (1:5000) EC.sub.50 (nM) 0.036 0.035

[0276] The results show that antibody VP101 is able to bind to VEGFA protein efficiently and its binding efficiency is dose-dependent, and the two antibodies are equivalent in terms of binding activity to human VEGFA.

[0277] 2. Detection of Respective Binding Activities of Antibodies VP101, BsAbB7 and BsAbB8 to Antigen VEGFA-His by Indirect ELISA

[0278] The method is specified as follows:

[0279] The microplate was coated with VEGFA-His and incubated overnight at 4° C. After being washed, the microplate was blocked with 1% BSA (dissolved in PBS) for 2 hours. After being washed, the microplate was added with the gradiently diluted antibody and incubated at 37° C. for 30 minutes. After being washed, the microplate was added with the horseradish peroxidase-labeled goat anti-human IgG Fc (Jackson, 109-035-098) working solution and incubated for 30 minutes at 37° C. After being washed, the microplate was added with TMB (Neogen, 308177) for color developing for 5 minutes in the absence of light, and then stop solution was added to terminate the chromogenic reaction. Then the microplate was put into a microplate reader immediately, and the OD value of each well in the microplate was read at 450 nm. SoftMax Pro 6.2.1 was used to analyze and process the data.

[0280] The result of the binding of antibody VP101 to antigen VEGFA-His is shown in FIG. 12. The absorbance intensities at each dose are shown in Table 5. The binding EC.sub.50 of antibody was calculated by curve fitting using antibody concentration as the abscissa and absorbance value as the ordinate, and the results are shown in Table 5 below.

TABLE-US-00023 TABLE 5 Respective binding activities of VP101, BsAbB7, BsAbB8 and bevacizumab to VEGFA-His (Indirect ELISA) Antibody concentration Antibody coating: VEGFA-His 1 μg/mL (μg/mL) VP101 BsAbB7 BsAbB8 Bevacizumab 1.000 3.112 3.090 3.074 3.081 3.070 3.093 3.137 3.138 0.333 3.026 2.961 2.954 2.941 2.946 2.968 3.075 3.086 0.111 2.802 2.684 2.575 2.621 2.631 2.618 2.965 2.999 0.037 1.972 1.876 1.656 1.668 1.756 1.709 2.504 2.503 0.012 0.994 0.915 0.754 0.764 0.809 0.814 1.476 1.454 0.004 0.436 0.391 0.317 0.332 0.347 0.339 0.711 0.700 0.001 0.197 0.177 0.151 0.155 0.159 0.155 0.318 0.311 0 0.083 0.063 0.086 0.076 0.095 0.072 0.066 0.064 Secondary Horseradish peroxidase-labeled goat antibody anti-human IgG Fc, HRP (1:5000) EC.sub.50(nM) 0.130 0.171 0.159 0.092

[0281] The results show that the antibodies VP101, BsAbB7, BsAbB8 and bevacizumab all can bind to the VEGF protein efficiently and their binding efficiency is dose-dependent, and antibody VP101 has a higher binding activity to human VEGF than BsAbB7 and BsAbB8.

[0282] 3. Detection of Binding Activity of Antibody VP101 to Antigen PD-1 by Indirect ELISA

[0283] The method is specified as follows:

[0284] The microplate was coated with human PD-1-mFc and incubated overnight at 4° C. After being blocked with 1% BSA at 37° C. for 2 hours, the microplate was added with antibody, and then incubated at 37° C. for 30 minutes. After the microplate was washed and patted dry, the HRP-labeled goat anti-human IgG (H+L) secondary antibody (Jackson, 109-035-088) was added, and the microplate was incubated at 37° C. for 30 minutes. After the microplate was washed and patted dry, TMB (Neogen, 308177) was added for color developing for 5 minutes, and then stop solution was added to terminate the color development. Then the microplate was put into a microplate reader immediately, and the OD value of each well in the microplate was read at 450 nm. SoftMax Pro 6.2.1 was used to analyze and process the data.

[0285] The detection result of the binding of antibody VP101 to antigen PD-1 is shown in FIG. 13. The absorbance intensities at each dose are shown in Table 6. By quantitative analysis of the bound antibody VP101, the curve simulation was performed to obtain the binding efficiency EC.sub.50 of the antibody, which is shown in Table 6 below.

TABLE-US-00024 TABLE 6 Binding of bifunctional antibody to PD-1 (Indirect ELISA) Antibody dilution Antibody coating: PD-1-mFc 0.5 μg/mL gradient VP101 Nivolumab 14C12H1L1 0.333 μg/ml 3.109 3.063 3.137 3.130 3.298 3.278 1:3 3.016 2.926 3.139 3.140 3.245 3.352 1:9 2.461 2.513 2.802 2.758 3.104 3.155  1:27 1.638 1.675 1.949 1.810 2.352 2.549  1:81 0.787 0.791 0.933 0.990 1.382 1.421  1:243 0.301 0.656 0.348 0.375 0.612 0.596  1:729 0.136 0.145 0.159 0.162 0.253 0.247 0 0.068 0.056 0.053 0.053 0.053 0.053 EC.sub.50(nM) 0.06 0.061 0.037

[0286] The results show that antibody VP101 is able to bind to PD-1 protein efficiently and its binding efficiency is dose-dependent.

[0287] 4. Detection of Respective Binding Activities of Antibodies VP101, BsAbB7 and BsAbB8 to Antigen PD-1 by Indirect ELISA

[0288] The method is specified as follows:

[0289] The microplate was coated with human PD-1-mFc and incubated overnight at 4° C. After being blocked with 1% BSA at 37° C. for 2 hours, the microplate was added with antibody, and then incubated at 37° C. for 30 minutes. After the microplate was washed and patted dry, the horseradish peroxidase-labeled goat anti-human IgG Fc (Jackson, 109-035-098) was added, and the microplate was incubated at 37° C. for 30 minutes. After the microplate was washed and patted dry, TMB (Neogen, 308177) was added for color developing for 5 minutes, and then stop solution was added to terminate the color development. Then the microplate was put into a microplate reader immediately, and the OD value of each well in the microplate was read at 450 nm. SoftMax Pro 6.2.1 was used to analyze and process the data.

[0290] The detection result of the binding of antibody VP101 to antigen PD-1 is shown in FIG. 14. The absorbance intensities at each dose are shown in Table 7. By quantitative analysis of the bound antibody VP101, the curve simulation was performed to give the binding efficiency EC.sub.50 of the antibody, which is shown in Table 7 below.

TABLE-US-00025 TABLE 7 Respective binding activities of antibodies VP101, BsAbB7, BsAbB8, 14C12H1L1 and nivolumab to PD-1 (Indirect ELISA) Antibody concentration Antigen coating: PD-1-mFc 0.5 μg/mL (μg/mL) VP101 BsAbB7 BsAbB8 14C12 H1L1 Nivolumab 0.333 2.717 2.709 2.732 2.755 2.716 2.715 2.947 2.966 2.823 2.824 0.111 2.507 2.381 2.318 2.321 2.377 2.409 2.923 2.967 2.747 2.758 0.037 1.709 1.616 1.491 1.457 1.522 1.549 2.656 2.694 2.208 2.293 0.012 0.916 0.822 0.732 0.711 0.797 0.775 2.049 2.060 1.348 1.389 0.004 0.413 0.394 0.333 0.321 0.368 0.351 1.139 1.132 0.629 0.638 0.001 0.195 0.191 0.167 0.174 0.181 0.174 0.552 0.541 0.295 0.295 0.000 0.140 0.123 0.110 0.103 0.117 0.118 0.254 0.248 0.152 0.157 0.000 0.099 0.095 0.089 0.074 0.100 0.081 0.083 0.075 0.078 0.084 Secondary Horseradish peroxidase-labeled goat antibody anti-human IgG Fc, HRP (1:5000) EC.sub.50(nM) 0.146 0.199 0.173 0.045 0.095

[0291] The results show that the antibody VP101 can bind to the PD-1 protein efficiently and its binding efficiency is dose-dependent, and antibody VP101 has a higher binding activity to human PD-1 than BsAbB7 and BsAbB8.

[0292] 5. Detection of Activity of Antibody VP101 in Competing with VEGFR2 for Binding to Antigen VEGFA by Competitive ELISA

[0293] The method is specifically as follows:

[0294] The microplate was coated with VEGF-His and incubated at 37° C. for 2 hours. After being washed, the microplate was blocked with 1% BSA for 1 hour at 37° C. After being washed, the microplate was added with the gradiently diluted antibodies and human VEGFR2 ECD-mFc-bio (final concentration: 0.02 μg/mL) and incubated at room temperature for 2 hours. After being washed, the microplate was added with HRP-labeled streptavidin SA-HRP (1:4000) working solution and incubated at 37° C. for 30 minutes. After being washed, the microplate was added with TMB chromogenic solution for color developing for 5 minutes in the absence of light, and then stop solution was added to terminate the chromogenic reaction. Then the microplate was put into a microplate reader immediately, and the OD value of each well in the microplate was read at 450 nm. SoftMax Pro 6.2.1 was used to analyze and process the data.

[0295] The detection results are shown in FIG. 15. The absorbance intensities at each dose are shown in Table 8. By quantitative analysis of the absorbance intensities of the bound antibodies, the curve simulation was performed to give the binding efficiency EC.sub.50 of the antibodies (Table 8).

TABLE-US-00026 TABLE 8 Detection of antibody in competing with VEGFR2-mFc for binding to the antigen VEGFA-His by competitive ELISA Coating: VEGF-His (2 μg/mL) Antibody concentration (μg/mL) VP101 Bevacizumab 10.000 0.133 0.133 0.103 0.104 3.333 0.161 0.149 0.114 0.109 1.111 0.624 0.563 0.374 0.351 0.370 1.055 1.051 0.905 0.982 0.123 1.059 1.075 0.964 1.049 0.041 1.137 1.068 1.062 1.141 0.014 1.106 1.138 1.010 1.169 0.000 1.155 1.131 1.173 1.153 Receptor VEGFR2 ECD-mFc-bio, 0.02 μg/ml Secondary antibody SA-HRP (1:4000) EC.sub.50 (nM) 5.324 5.086

[0296] The results show that the antibody VP101 can effectively bind to the antigen VEGFA and inhibit the binding of VEGFR2 to VEGFA, and its efficiency in inhibiting the binding of VEGFR2 to VEGFA is dose-dependent.

[0297] 6. Detection of Antibody VP101 in Competing with PD-L1 for Binding to Antigen PD-1 by Competitive ELISA

[0298] The method is specifically as follows:

[0299] The microplate was coated with PD-1-hFc and incubated overnight at 4° C. After the microplate was blocked with 1% BSA for 2 hours, antibodies at different concentrations were each mixed with PD-L1-hFc for 10 minutes (see Table 10 for the dilution concentrations). After incubation at 37° C. for 30 minutes, the microplate was washed and patted dry. Then enzyme-labeled secondary antibody was added, and the microplate was incubated at 37° C. for 30 minutes. After the microplate was washed and patted dry, TMB was added for color developing for 5 minutes, and then stop solution was added to terminate the color development. Then the microplate was put into a microplate reader immediately, and the OD value of each well in the microplate was read at 450 nm (see Table 10). SoftMax Pro 6.2.1 was used to analyze and process the data.

[0300] The detection results are shown in FIG. 16. The absorbance intensities at each dose are shown in Table 9. By quantitative analysis of the bound antibody VP101, the curve simulation was performed to give the binding efficiency EC.sub.50 of the antibody (Table 9).

TABLE-US-00027 TABLE 9 Detection of bifunctional antibody competing with PD-L1 for binding to PD-1 by competitive ELISA Antibody dilution Antigen coating: PD-1-hFc 0.5 μg/mL gradient VP101 Nivolumab 14C12H1L1 5 μg/ml 0.096 0.063 0.058 0.058 0.062 0.063 1:3 0.064 0.077 0.059 0.059 0.061 0.064 1:9 0.166 0.160 0.061 0.062 0.066 0.071  1:27 0.867 0.848 0.284 0.335 0.262 0.193  1:81 1.217 1.149 0.973 1.007 0.968 0.882  1:243 1.196 1.949 1.139 1.144 1.122 1.051  1:729 1.183 1.250 1.127 1.185 1.052 1.059 0 1.153 1.276 0.960 1.071 1.027 1.024 Receptor PD-L1-mFc 0.3 μg/ml Secondary Goat anti-mouse IgG (H + L), antibody HRP conjugated (1:5000) EC.sub.50(nM) 1.216 0.842 0.745

[0301] The results show that antibody VP101 can effectively bind to antigen PD-1 and inhibit the binding of ligand PD-L1 to PD-1, and its efficiency in inhibiting the binding of PD-L1 to PD-1 is dose-dependent.

EXAMPLE 4

Binding of Antibody VP101 to Cell Membrane Surface Antigen

[0302] Firstly, 293T cells expressing PD-1 antigen was constructed, and then the specific binding capacity of the antibody to the cell membrane surface antigen was analyzed and verified by flow cytometry.

[0303] 1. Construction of 293T Cells Expressing PD-1 Antigen

[0304] The vector pLenti6.3-PD-1 of PD-1 (the vector pLenti6.3 was purchased from Invitrogen) was transfected into 293T cells, and clone group 293T-PD-1 cells which stably express PD-1 were obtained by screening.

[0305] 2. Detection of Binding of Antibody to Cell Surface Antigen

[0306] The 293T-PD-1 expressing antigen obtained in the previous step was digested with pancreatin by a conventional pancreatin digestion method, and the number of cells in each collection tube was made to be 2×10.sup.5. Antibody diluting solutions with concentration gradiently diluted with PBSA (1% BSA) were each incubated with 293T-PD-1 cells on ice for 2 hours, and then each tube was added with 100 μL of FITC goat anti-human IgG (1:500) and incubated on ice for 1 hour. Then PBS was used for washing, and 300 μL of PBSA was used to resuspend the cells, and fluorescence signals (MFI) were detected with FITC channel on a flow cytometer.

[0307] The results are shown in FIG. 17, and the MFI values at each concentration are shown in Table 10. By fluorescence quantification analysis and curve fitting of the bound 14C12H1L1 antibody, the binding EC.sub.50 of the VP101 antibody was calculated to be 3.5 nM.

TABLE-US-00028 TABLE 10 Analysis of fluorescence intensity of the binding of VP101 to 293T-PD-1 surface antigen detected by FACS Antibody (nM) 0.14 0.41 1.23 3.70 11.11 33.33 100 EC.sub.50 Bevacizumab 3.2 2.2 2.0 2.3 2.7 3.8 5.7 — Nivolumab 33.3 74.9 171.9 357.9 481.9 498.3 478.4 2.1 14C12H1L1 48.1 99.7 201.5 409.0 600.2 655.4 670.8 2.9 VP101 30.8 61.8 135.7 286.9 487.7 534.0 528.6 3.5

[0308] The results show that the VP101 antibody can effectively bind to the PD-1 antigen on the 293T-PD-1 host cell surface, and its binding efficiency is dose-dependent, and bevacizumab has no binding activity to 293T-PD-1, which indicates that the binding of VP101 to 293T-PD-1 is specific.

[0309] 3. The binding of antibodies VP101, BsAbB7 and BsAbB8 to the cell surface antigen was detected by referring to the experimental procedure described in step 2 of this example.

[0310] The results are shown in FIG. 18, and the MFI values at each concentration are shown in Table 11. By fluorescence quantification analysis and curve fitting of the bound antibody, the binding EC.sub.50 values of nivolumab, 14C12H1L1, VP101, BsAbB7 and BsAbB8 were calculated to be 7.853 nM, 3.607 nM, 7.896 nM, 9.943 nM and 10.610 nM, respectively.

TABLE-US-00029 TABLE 11 Analysis of fluorescence intensities of the binding of VP101, BsAbB7 and BsAbB8 to 293T-PD-1 surface antigen detected by FACS Antibody (nM) 0.014 0.14 0.41 1.23 3.7 11 30 100 EC50(nM) Bevacizumab 1.89 1.90 2.20 1.92 2.04 2.48 2.80 2.43 — Nivolumab 3.91 15.30 34.69 94.04 234.34 533.63 640.15 804.69 7.853 14C12H1L1 7.40 29.55 69.16 175.54 422.53 868.45 831.27 813.58 3.607 VP101 3.47 16.16 38.75 93.08 216.76 509.23 810.37 783.58 7.896 BsAbB7 3.85 14.86 37.45 83.78 202.40 465.10 837.61 846.80 9.943 BsAbB8 4.41 16.77 36.86 89.89 210.40 457.91 804.43 863.35 10.610

[0311] The results show that the VP101 antibody can bind to the membrane surface PD-1 of 293T-PD1 in a dose-dependent manner. Bevacizumab has no binding activity to 293T-PD-1, which indicates that the binding of VP101 to 293T-PD-1 is specific.

EXAMPLE 5

Competitive Binding of Antibody VP101 to Cell Membrane Surface Antigen

[0312] 1. A competitive flow cytometry method was adopted to detect the EC.sub.50 of the VP101 in competing with PD-L1 for binding to the cell membrane surface antigen PD-1, and the method is specified as follows:

[0313] The 293T-PD-1 cells was digested in a conventional way, and divided into several samples with 300,000 cells for each, which were then subjected to centrifugation and washing. Then each tube was added with 100 μL of corresponding gradiently diluted antibody and incubated on ice for 30 minutes; 100 μL of PD-L1-mFc was then added to each tube, and the mixture was mixed well to reach a final concentration of 20 nM, and then incubated on ice for 1 hour. Then 500 μL of 1% PBSA was added, and the mixture was centrifuged at 5600 rpm for 5 minutes to remove the supernatant. 100 μL of FITC coat anti mouse antibody diluted at a ratio of 1:500 was then added into each tube, and the mixture was incubated on ice for 40 minutes in the absence of light after being mixed well. Then the mixture was centrifuged, washed and resuspended, and then transferred to a loading tube for testing.

[0314] The results are shown in FIG. 19, and the MFI values at each concentration are shown in Table 12. By fluorescence quantification analysis and curve fitting, the binding EC.sub.50 values of the antibodies VP101 and 14C12H1L1 were calculated to be 8.33 nM and 4.37 nM, respectively.

TABLE-US-00030 TABLE 12 Analysis of fluorescence intensities of 14C12H1L1 and VP101 in competing for binding to 293T-PD-1 surface antigen detected by FACS Antibody (nM) 0.05 0.14 0.41 1.23 3.70 11.11 33.33 100.00 EC.sub.50 R.sup.2 14C12H1L1 288.17 287.29 277.09 237.22 177.80 12.04 10.32 9.87 4.37 0.988 VP101 272.66 264.39 279.11 272.26 239.18 99.29 17.05 14.91 8.33 0.999

[0315] The results show that the VP101 antibody can effectively block the binding of PDL-1 to PD-1 on the surface of 293T-PD-1 host cells in a dose-dependent manner.

[0316] 2. EC.sub.50 values of VP101, BsAbB7, BsAbB8, 14C12H1L1 and nivolumab in competing with PD-L1 for binding to the cell membrane surface antigen PD-1 were detected by using competitive flow cytometry and referring to the experimental procedure described in step 1 of this example.

[0317] The results are shown in FIG. 20, and the MFI values at each concentration are shown in Table 13. By fluorescence quantification analysis and curve fitting, the competitive binding EC50 values of antibodies VP101, BsAbB7, BsAbB8, 14C12H1L1 and nivolumab were calculated to be 15.04 nM, 22.25 nM, 19.25 nM, 9.21 nM and 9.72 nM, respectively.

TABLE-US-00031 TABLE 13 Analysis of fluorescence intensities of VP101, BsAbB7, BsAbB8, 14C12H1L1 and nivolumab in competing for binding to 293T-PD-1 surface antigen detected by FACS Antibody (nM) 0.14 0.41 1.23 3.7 11.11 33.33 100 300 EC.sub.50 R.sup.2 VP101 1441.94 1380.62 1368.15 1288.34 982.69 112.90 8.49 8.21 15.04 0.9971 BsAbB7 1412.62 1377.27 1339.60 1341.27 1094.35 417.70 9.23 9.18 22.25 0.9985 BsAbB8 1578.36 1521.50 1427.20 1429.85 1137.74 359.69 9.73 9.68 19.25 0.9962 14C12H1L1 1384.08 1551.05 1462.85 1296.64 580.45 12.93 13.37 14.99 9.21 0.9950 Nivolumab 1539.58 1552.37 1483.84 1300.81 713.56 70.92 60.77 56.92 9.72 0.9969

[0318] The results show that the activity of the antibody 14C12H1L1 is equivalent to that of the marketed antibody nivolumab targeting PD-1, and is superior to that of the bifunctional antibody VP101. The activity of the antibody VP101 is superior that of to BsAbB7 and BsAbB8.

EXAMPLE 6

Detection of Neutralization Bioactivity of Antibodies VP101, BsAbB7 and BsAbB8 in Blocking VEGF to Activate NFAT Signaling Pathway

[0319] 1. Construction of 293T-NFAT-(opv)KDR(C7) cells

[0320] KDR (VEGFR2) vector pCDH-KDRFL(OPV)-GFP-Puro (Vector pCDH-GFP-Puro is purchased from Youbio) and NFAT vector pNFAT-luc-hygro (vector pGL4-luc2P-hygro is purchased from Promega) were transfected into 293T cells, and a clone group 293T-NFAT-(opv) KDR(C7) cells stably expressing KDR and NFAT luciferase reporter genes were obtained by screening.

[0321] 2. 293T-NFAT-(opv)KDR(C7) cells were collected and centrifuged for 5 minutes to remove the supernatant; DMEM+10%FBS medium was used to resuspend the cells, and the cell number was counted and the cell viability was detected; then the cell concentration was adjusted to be in a proper range, and 50000 cells/50 μL cell suspension was added into each well of a black 96-well plate;

[0322] Corresponding antibodies (final concentrations being 300, 100, 10, 2, 0.2, 0.02, 0.002 nM) and VEGF (final concentration being 30 ng/mL) were diluted according to the experimental design, and the antibodies targeting VEGF were preincubated with VEGF for 1 hour at room temperature before being added into the cells. Blank and isotype controls (final volume of each well being 100 μL) were designed and incubated in a carbon dioxide incubator at 37° C., 5% CO.sub.2 for 4 hours; 50 μL of Luciferase Assay System was added to each well, and Relative Fluorescence Units (RLUs) were detected by a multi-label microplate tester within 5 minutes.

[0323] The experimental results are shown in FIG. 21, and the EC.sub.50 values for each antibody are shown in Table 14.

TABLE-US-00032 TABLE 14 Detection of neutralization bioactivity of antibodies VP101, BsAbB7 and BsAbB8 inblocking VEGF to activate NFAT signaling pathway by reporter assay Sample VP101 BsAbB7 BsAbB8 Bevacizumab EC.sub.50 (nM) 1.2400 1.2170 1.7280 0.7730

[0324] The results show that the EC.sub.50 of VP101 is 1.240 nM, the EC.sub.50 of BsAbB7 is 1.217 nM, the EC.sub.50 of BsAbB8 is 1.728 nM, and the EC.sub.50 of bevacizumab is 0.773 nM, and the experimental results show that the activity of VP101 and BsAbB7 in blocking VEGF to activate NFAT signaling pathway is better than that of BsAbB8.

EXAMPLE 7

Experiment of VP101 Antibody Inhibiting VEGFA-Induced HUVEC Cell Proliferation

[0325] HUVEC cells (purchased from Allcell) in a good growth state, after the cell concentration was adjusted to be 1.5×10.sup.4/mL, were inoculated into a 96-well plate at 200 μL/well, and then incubated in an incubator at 37° C., 5% CO.sub.2 for 24 hours. Then it was observed that the cells adhered well, and then culture medium was discarded. 20 nM VEGFA prepared by using 1640 containing 2% FBS was then added into the 96-well plate at 200 μL/well, and antibodies at different concentrations were added, followed by incubation for 72 hours. 72 hours later, the culture medium was discarded and MTT was added. 4 hours later, the MTT was discarded and DMSO was added, and then a microplate reader was used to measure the OD value at 490 nm.

[0326] The results are shown in FIG. 22. The results show that the humanized antibodies VP101 and bevacizumab both can effectively inhibit VEGFA-induced HUVEC cell proliferation in a dose-dependent manner, and the pharmacological activity of VP101 in inhibiting VEGFA-induced HUVEC cell proliferation is higher than that of bevacizumab at the same dose.

EXAMPLE 8

Promotion of Secretion of Cytokines IFN-γ and IL-2 in Mixed Lymphocyte Reaction

[0327] 1. Promotion of secretion of IFN-γ by VP101, 14C12H1L1 and nivolumab in mixed culture system of DC and PBMC cells

[0328] PBMCs were isolated by Ficoll-Paque Plus (GE Healthcare) and added to IL-4 (Peprotech 200-04, 1000 U/mL) and GM-CSF (Peprotech 300-03, 1000 U/mL) for 6 days of induction, and then TNF-α (Peprotech 300-01A, 200 U/mL) was additionally added for 3 days of induction to obtain mature DC cells.

[0329] On the day of co-culture, fresh PBMCs were isolated from peripheral blood of another donor, and the obtained mature DC cells were mixed with the freshly isolated PBMCs of another donor at a ratio of 1:10, and meanwhile antibodies at different concentrations (hIgG as a control) were added. After co-culture for 5-6 days, cell supernatant was collected and assayed for IFN-γ content using an ELISA kit (purchased from Dakewe).

[0330] The effect of VP101 on secretion of IFN-γ in mixed culture system of DC and PBMC cells is shown in FIG. 23. As can be seen in FIG. 23, VP101 can effectively promote secretion of IFN-γ in a dose-dependent manner. In addition, at doses of 30 nM and 300 nM, VP101 has greater activity in promoting secretion of IFN-γ than equivalent 14C12H1L1, and at dose level of 30 nM, it has greater activity in promoting secretion of IFN-γ than equivalent nivolumab.

[0331] 2. Promotion of secretion of IL-2 and IFN-γ by VP101, BsAbB7 and BsAbB8b in mixed culture system of DC and PBMC cells

[0332] Step 1 in this example was referred to for the experimental method, namely PBMCs were isolated by Ficoll-Paque Plus (GE Healthcare) and added to IL-4 (Peprotech 200-04, 1000 U/mL) and GM-CSF (Peprotech 300-03, 1000 U/mL) for 6 days of induction, and then TNF-α (Peprotech 300-01A, 200 U/mL) was additionally added for 3 days of induction to obtain mature DC cell.

[0333] On the day of co-culture, fresh PBMCs were isolated from peripheral blood of another donor, and the obtained mature DC cells were mixed with the freshly isolated PBMCs of another donor at a ratio of 1:10, and meanwhile antibodies at different concentrations (hIgG as a control) were added; after co-culture for 5-6 days, cell supernatant was collected and assayed for IL-2 and IFN-γ content using an ELISA kit (purchased from Dakewe).

[0334] The effect of VP101 on secretion of IFN-γ in mixed culture system of DC and PBMC cells is shown in FIG. 24. As can be seen from FIG. 24, VP101 can effectively promote secretion of IFN-γ in a dose-dependent manner. The pharmacological activity of VP101 in promoting secretion of IFN-γ is significantly better than that of BsAbB7 and BsAbB8.

[0335] The effect of VP101 on secretion of IL-2 in mixed culture system of DC and PBMC cells is shown in FIG. 25. As can be seen from FIG. 25, VP101 can effectively promote secretion of IL-2 in a dose-dependent manner, and the pharmacological activity of VP101 in promoting secretion of IL-2 is better than that of BsAbB7 and BsAbB8.

[0336] 3. Promotion of secretion of IL-2 and IFN-γ by VP101, 14C12H1L1 and nivolumab in mixed culture system of PBMC and Raji-PD-L1 cells

[0337] PD-L1 was stably transfected into Raji cells through lentivirus infection, and Raji-PD-L1 cells stably expressing PD-L1 were obtained after dosing and screening; PBMCs, after two days of stimulation by SEB, were cultured in together with mitomycin C-treated Raji-PD-L1.

[0338] The results are shown in FIGS. 26 and 27. The results show that VP101 can effectively promote secretion of IL-2 and IFN-γ, and at dose level of 300 nM, the activity of VP101 in promoting secretion of IL-2 is significantly better than that of equivalent 14C12H1L1 and nivolumab.

[0339] The isotype control antibody to be studied was Human Anti-Hen Lysozyme (anti-HEL, i.e., human IgG, abbreviated as hIgG), and it was prepared as described in Preparation Example 6 above.

[0340] 4. The promotion of secretion of IL-2 and IFN-γ by VP101, BsAbB7 and BsAbB8 in mixed culture system of PBMC and Raji-PD-L1 cells was studied by referring to the experimental method described in step 3 of this example.

[0341] The results of secretion of IFN-γ are shown in FIG. 28. The results show that VP101 can effectively promote secretion of IFN-γ in a dose-dependent manner. At the same time, VP101 is significantly better than BsAbB7 at the same dose, while the pharmacological activity of VP101 is significantly better than that of BsAbB8 at dose levels of 3 nM and 30 nM.

[0342] The results of secretion of IL-2 are shown in FIG. 29. The results show that VP101 can effectively promote secretion of IL-2 in a dose-dependent manner. At the same time, the pharmacological activity of VP101 is significantly better than that of BsAbB7 at doses of 3 nM and 300 nM, while VP101 is equivalent to BsAbB8 at the same dose.

EXAMPLE 9

Experiment of Inhibition of Tumor Growth In Vivo by VP101

[0343] To detect the in vivo tumor-inhibiting activity of VP101, U87MG cells (human glioma cells, purchased from ATCC) were first inoculated subcutaneously into 5-7 week old female Scid Beige mice (purchased from Vital River), and the modeling and specific mode of administration were shown in Table 15. After the administration, the length and width of each group of tumors were measured, and the tumor volume was calculated.

TABLE-US-00033 TABLE 15 Dosing regimen of treating U87MG tumor xenograft Scid Beige mouse model with VP101 Grouping n Tumor xenograft Condition of administration Isotype control 7 U-87MG, 5 million Isotype control antibody, hIgG, 40 mg/kg cells/mouse 40 mg/kg, injected intravenously subcutaneously on days 0, 7 and 13 Bevacizumab 8 Bevacizumab 30 mg/kg, 30mg/kg injected intravenously on days 0, 7 and 13 VP101 7 VP101 40 mg/kg, 40mg/kg injected intravenously on days 0, 7 and 13 VP101 7 VP101 4 mg/kg, 4mg/kg injected intravenously on days 0, 7 and 13

[0344] The results are shown in FIG. 30. The results show that compared with an isotype control antibody hIgG (the preparation method is the same as that of the Preparation Example 6), bevacizumab and VP101 at different doses can effectively inhibit the growth of mouse tumors, and the high-dose VP101 is better than that of low-dose VP 101 in inhibiting tumors.

[0345] Furthermore, as shown in FIG. 31, VP101 does not affect the body weight of tumor-bearing mouse.

[0346] While the content of the present application has provided complete and clear description of its disclosed embodiments, it is not limited thereto. For those skilled in the art, modifications and replacements to the present invention are possible with the guidance of these descriptions, and such modifications and replacements are included within the scope of the present invention. The full scope of the present application is given by the appended claims and any equivalent thereof.