ANTIBODY MUTANT AND APPLICATION THEREOF

Abstract

Provided is a mutant of an antibody or fragment thereof, characterized in that the antibody or fragment thereof contains a light-chain constant region, and according to the Kabat numbering system, the amino acid at position 166 is mutated to cysteine.

Claims

1. A variant of antibody or fragment thereof, wherein the antibody or fragment thereof comprises a light chain constant region, and an amino acid at position 166 according to a Kabat numbering system is mutated to cysteine; the antibody preferably is a humanized antibody, a chimeric antibody, more preferably is an IgG antibody.

2. The variant of antibody or fragment thereof of claim 1, wherein the light chain is of a lambda or kappa type.

3. The variant of antibody or fragment thereof of claim 1 or 2, which comprises a heavy chain and a light chain, wherein an amino acid sequence of the heavy chain is shown in SEQ ID NO:1, and an amino acid sequence of the light chain is shown in SEQ ID NO: 3; the amino acid sequence of the heavy chain is shown in SEQ ID NO: 9, and the amino acid sequence of the light chain is shown in SEQ ID NO: 13; or the amino acid sequence of the heavy chain is shown in SEQ ID NO: 17, and the amino acid sequence of the light chain is shown in SEQ ID NO:21.

4. The variant of antibody or fragment thereof of any one of claims 1 to 3, wherein the fragment is a Fab fragment, a Fab′ fragment or a F(ab′).sub.2 fragment, preferably a heavy chain amino acid sequence of the Fab fragment is shown in SEQ ID NO: 25, and a light chain amino acid sequence of the Fab fragment is shown in SEQ ID NO: 29.

5. A bispecific antibody or fusion protein comprising the variant of antibody or fragment thereof of any one of claims 1 to 4.

6. A conjugate comprising the variant of antibody or fragment thereof of any one of claims 1 to 4.

7. The conjugate of claim 6, wherein the conjugate is selected from polyethylene glycol, a cytotoxic agent, an active peptide, a nanobody, a single domain antibody, a Fab fragment, a Fab′ fragment, scFv, a small molecule drug, a chemotherapeutic agent or a radiotherapeutic agent; preferably, the active peptide is Agn2 active peptide, more preferably an Agn2 active peptide as shown in SEQ ID NO: 5; more preferably, the Agn2 active peptide is conjugated to a bevacizumab variant of the present invention via a linker; further preferably, the linker has an amide bond, specifically is formamide-PEG.sub.4-NHS, with a structure as shown in formula I ##STR00004##

8. The conjugate of claim 6, wherein the polyethylene glycol is monomethoxy polyethylene glycol, preferably mPEG2000, mPEG5000, mPEG10000.

9. The conjugate of claim 7 or 8, wherein the polyethylene glycol is further conjugated to a drug molecule, preferably the polyethylene glycol is a linear difunctionalized polyethylene glycol, a linear heterofunctionalized polyethylene glycol or a multi-arm functionalized polyethylene glycol.

10. A pharmaceutical composition comprising the variant of antibody or fragment thereof of any one of claims 1 to 4, the bispecific antibody or fusion protein of claim 5, the conjugate of any one of claims 6 to 9, and optionally, a pharmaceutically acceptable carrier.

11. A method for treatment of cancer (preferably non-small cell lung cancer such as advanced, metastatic or recurrent non-squamous cell non-small cell lung cancer, colorectal cancer such as metastatic colorectal cancer, breast cancer, malignant glioma and renal cell carcinoma, glioblastoma multiforme), comprising administering the variant of antibody or fragment thereof of any one of claims 1 to 4, the bispecific antibody or fusion protein of claim 5, the conjugate of any one of claims 6 to 9, or the pharmaceutical composition of claim 10.

12. Use of the variant of antibody or fragment thereof of any one of claims 1 to 4, the bispecific antibody or fusion protein of claim 5, or the conjugate of any one of claims 6 to 9, or the pharmaceutical composition of claim 10 in the preparation of a medicament or kit for treatment of cancer (preferably non-small cell lung cancer such as advanced, metastatic or recurrent non-squamous cell non-small cell lung cancer, colorectal cancer such as metastatic colorectal cancer, breast cancer, malignant glioma and renal cell carcinoma, glioblastoma multiforme).

13. A kit comprising the variant of antibody or fragment thereof of any one of claims 1 to 4, the bispecific antibody/fusion protein of claim 5, or the conjugate of any one of claims 6 to 9, or the pharmaceutical composition of claim 10, preferably, the kit further comprises an agent to be administered in combination with the antibody or fragment thereof, such as carboplatin or cisplatin.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0023] FIG. 1 shows the SDS-PAGE electropherograms of wild-type (WT, also called “anti-VEGF antibody” in the present invention) antibody, 166 mutant antibody (166C), and 124 mutant antibody (124C) in a reduced and non-reduced state.

[0024] FIG. 2 shows SDS-PAGE electrophoretograms of the anti-VEGF wild-type antibody and the antibody mutant after being coupled with mPEG2000-MAL. It can be seen from this figure that both 166C and 124C can be successfully coupled with the conjugate mPEG2000.

[0025] FIG. 3 shows SDS-PAGE electrophoretograms of the anti-VEGF wild-type antibody and the antibody mutant after being coupled with DC (polypeptide-linker). By using gray scale analysis, it can be seen that 85% of 166C was successfully coupled to DC, while only 75% of 124C was successfully coupled to DC. It can be seen that under the same condition, the 166C antibody mutant showed better conjugation efficiency.

[0026] FIG. 4 shows SDS-PAGE electrophoretograms of the anti-CD20 wild-type antibody and the antibody mutant after being coupled with mPEG2000-MAL.

[0027] FIG. 5 shows SDS-PAGE electrophoretograms of the anti-CD20 wild-type antibody and the antibody mutant after being coupled with DC (polypeptide-linker).

[0028] FIG. 6 shows SDS-PAGE electrophoretograms of the anti-HER2 wild-type antibody and the antibody mutant after being coupled with mPEG2000-MAL.

[0029] FIG. 7 shows SDS-PAGE electrophoretograms of the anti-HER2 wild-type antibody and the antibody mutant after being coupled with DC (polypeptide-linker).

[0030] FIG. 8 shows SDS-PAGE electrophoretograms of the anti-VEGF antibody Fab fragment wild-type antibody and the antibody mutant after being coupled with DC (polypeptide-linker).

[0031] FIG. 9 shows SDS-PAGE electrophoretograms of the anti-VEGF antibody Fab fragment wild-type antibody and the antibody mutant after being coupled with mPEG2000-MAL.

[0032] FIG. 10 shows LC/MS spectrum of the mutant 166C before coupling. FIG. 11 shows LC/MS spectrum of the mutant 166C after being coupled with DC.

[0033] FIG. 12 shows LC/MS spectrum of the mutant 124C before coupling. FIG. 13 shows LC/MS spectrum of the mutant 124C after being coupled with DC.

[0034] FIG. 14 shows inhibition of wild-type antibody (WT), antibody mutants (124C and 166C) and conjugates (124ADC and 166ADC) on the proliferation of endothelial cells HUVEC (proliferation produced by VEGF stimulation). It can be seen from the figure that the activity of the antibody mutant is not changed due to the cysteine mutation at the selected site, indicating that the selected site will not affect the biological activity of the antibody. Similarly, the conjugate generated by conjugating with DC does not affect the activity of the antibody to inhibit cell proliferation.

EXAMPLES

[0035] In order to make the purpose, technical solution, and advantage of the present invention more clear, the present invention will be further described in detail below with reference to specific Examples. It will be understood by those skilled in the art that the following Examples are only for the purpose of illustration, and the protection scope of the present invention shall be based on the appended claims.

[0036] The restriction endonucleases used in the Examples were purchased from Thermo Fisher Scientific (China) Co., Ltd., and the reagents or materials used in the following Examples can be routinely purchased in the art unless otherwise specified. The culture medium of CHO-K1 cells was purchased from sigma.

Example 1 Screening and Expression of Cysteine Mutated Cell Lines

(1) Construction of Expression Vector of Anti-VEGF Antibody and Antibody Mutant

[0037] The DNA encoding the antibody described herein (the corresponding wild-type sequence was obtained from USP Medicines Compendium, specially the heavy chain sequence is shown in SEQ ID NO: 1 and the light chain sequence is shown in SEQ ID NO: 2; based on this, according to kabat numbering, the amino acid at position 166 of the light chain was substituted with cysteine to obtain a variant 166C of the present invention, and the amino acid at position 124 of the light chain was substituted with cysteine to obtain a control antibody 124C) was chemically synthesized (Suzhou GENEWIZ Biotechnology Co., Ltd.), then the antibody heavy chain gene was digested with BstBI and PacI, and the light chain gene was digested with HindIII and EcoRI; T4 ligase was used to ligate the heavy chain gene to an eukaryotic expression vector pCGS3 (Biovector NTCC Inc.) treated by the BstBI and PacI; after successful ligation, the expression vector pCGS3 was digested with HindIII and EcoRI, and then the light chain gene was ligated to the expression vector pCGS3 by T4 ligase, that is, an expression vector containing both heavy and light chains was successfully constructed.

TABLE-US-00001 WT Bevacizumab 166C 124C Encoding Sequence SEQ ID NO: 3 SEQ ID NO: 3 SEQ ID NO: 3 of Heavy Chain Encoding Sequence SEQ ID NO: 4 SEQ ID NO: 5 SEQ ID NO: 6 of Light Chain Amino Acid SEQ ID NO: 1 SEQ ID NO: 1 SEQ ID NO: 1 Sequence of Heavy Chain Amino Acid SEQ ID NO: 2 SEQ ID NO: 7 SEQ ID NO: 8 Sequence of Light Chain

(2) Screening and Purification of Expression Vector of Anti-VEGF Antibody and Antibody Mutant

[0038] The successfully constructed expression vector was transformed into DH5α competent E. coli strain. The transformed DH5α strain was digested with BstBI and PacI, as well as HindIII and EcoRI respectively, and was verified by sequencing, and then positive clones were selected. A plasmid extraction kit (purchased from CoWin Biosciences) was used to lyse the resulting E. coli and to extract the plasmid, thereby obtaining an expression vector containing heavy and light chains.

(3) Expression of Anti-VEGF Antibody and Antibody Mutant in CHO Cells

[0039] The purified expression vector was transfected into CHO-K1 (purchased from ATCC) cells by electroporation; the cells were plated in a 96-well plate, grown for 15 days, and then single clones were picked out, and antibody yield of the cells was determined by ELISA. The first 20% cells were transferred into a 24-well plate. After 7 days of growth, the antibody yield of the cells was determined, and the first 5 to 6 strains were selected and transferred into a shake flask for culture, thereby achieving the expression of anti-VEGF antibody in CHO-K1 cells. After the serum-free culture was stable, the cells were continuously cultured for 7 days, and then collected for purification and follow-up experiments.

(4) Construction of Expression Vector of Anti-CD20 Antibody and Mutant

[0040] The DNA encoding the antibody described herein (the corresponding wild-type sequence was obtained from USP Medicines Compendium, specially the heavy chain sequence is shown in SEQ ID NO: 9 and the light chain sequence is shown in SEQ ID NO: 10; based on this, according to kabat numbering, the amino acid at position 166 of the light chain was substituted with cysteine to obtain a variant 166C of the present invention, and the amino acid at position 124 of the light chain was substituted with cysteine to obtain a control antibody 124C) was chemically synthesized (Suzhou GENEWIZ Biotechnology Co., Ltd.), then the antibody heavy chain gene was digested with BstBI and PacI, and the light chain gene was digested with HindIII and EcoRI; T4 ligase was used to ligate the heavy chain gene to an eukaryotic expression vector pCGS3 (Biovector NTCC Inc.) treated by the BstBI and PacI; after successful ligation, the expression vector pCGS3 was digested with HindIII and EcoRI, and then the light chain gene was ligated to the expression vector pCGS3 by T4 ligase, that is, an expression vector containing both heavy and light chains was successfully constructed.

TABLE-US-00002 WT Rituximab 166C 124C Encoding Sequence SEQ ID SEQ ID SEQ ID of Heavy Chain NO: 11 NO: 11 NO: 11 Encoding Sequence SEQ ID SEQ ID SEQ ID of Light Chain NO: 12 NO: 14 NO: 16 Amino Acid SEQ ID SEQ ID SEQ ID Sequence of Heavy NO: 9 NO: 9 NO: 9 Chain Amino Acid SEQ ID SEQ ID SEQ ID Sequence of Light NO: 10 NO: 13 NO: 15 Chain

(5) Screening and Purification of Expression Vector of Anti-CD20 Antibody and Antibody Mutant

[0041] The successfully constructed expression vector was transformed into DH5α competent E. coli strain. The transformed DH5α strain was digested with BstBI and PacI, as well as HindIII and EcoRI respectively, and was verified by sequencing, and then positive clones were selected. A plasmid extraction kit (purchased from CoWin Biosciences) was used to lyse the resulting E. coli and to extract the plasmid, thereby obtaining an expression vector containing heavy and light chains.

(6) Expression of Anti-CD20 Antibody and Antibody Mutant in CHO Cells

[0042] The purified expression vector was transfected into CHO-K1 (purchased from ATCC) cells by electroporation; the cells were plated in a 96-well plate, grown for 15 days, and then single clones were picked out, and the antibody yield of the cells was determined by ELISA. The first 20% cells were transferred into a 24-well plate. After 7 days of growth, the antibody yield of the cells was determined, and the first 5 to 6 strains were selected and transferred into a shake flask for culture, thereby achieving the expression of anti-CD20 antibody in CHO-K1 cells. After the serum-free culture was stable, the cells were continuously cultured for 7 days, and then collected for purification and follow-up experiments.

(7) Construction of Expression Vector of Anti-HER2 Antibody and Antibody Mutant

[0043] The DNA encoding the antibody described herein (the corresponding wild-type sequence was obtained from USP Medicines Compendium, specially the heavy chain sequence is shown in SEQ ID NO: 17 and the light chain sequence is shown in SEQ ID NO: 19; based on this, according to kabat numbering, the amino acid at position 166 of the light chain was substituted with cysteine to obtain a variant 166C of the present invention, and the amino acid at position 124 of the light chain was substituted with cysteine to obtain a control antibody 124C) was chemically synthesized (Suzhou GENEWIZ Biotechnology Co., Ltd.), then the antibody heavy chain gene was digested with BstBI and PacI, and the light chain gene was digested with HindIII and EcoRI; T4 ligase was used to ligate the heavy chain gene to an eukaryotic expression vector pCGS3 (Biovector NTCC Inc.) treated by the BstBI and PacI; after successful ligation, the expression vector pCGS3was digested with HindIII and EcoRI, and then the light chain gene was ligated to the expression vector pCGS3 by T4 ligase, that is, an expression vector containing both heavy and light chains was successfully constructed

TABLE-US-00003 WT Trastuzumab 166C 124C Encoding Sequence SEQ ID SEQ ID SEQ ID of Heavy Chain NO: 18 NO: 18 NO: 18 Encoding Sequence SEQ ID SEQ ID SEQ ID of Light Chain NO: 20 NO: 22 NO: 24 Amino Acid SEQ ID SEQ ID SEQ ID Sequence of Heavy NO: 17 NO: 17 NO: 17 Chain Amino Acid SEQ ID SEQ ID SEQ ID Sequence of Light NO: 19 NO: 21 NO: 23 Chain

(8) Screening and Purification of Expression Vector of Anti-HER2 Antibody and Antibody Mutant

[0044] The successfully constructed expression vector was transformed into DH5α competent E. coli strain. The transformed DH5α strain was digested with BstBI and PacI, as well as HindIII and EcoRI respectively, and was verified by sequencing, and then positive clones were selected. A plasmid extraction kit (purchased from CoWin Biosciences) was used to lyse the resulting E. coli and to extract the plasmid, thereby obtaining an expression vector containing heavy and light chains.

(9) Expression of Anti-HER2 Antibody and Antibody Mutant in CHO Cells

[0045] The purified expression vector was transfected into CHO-K1 (purchased from ATCC) cells by electroporation; the cells were plated in a 96-well plate, grown for 15 days, and then single clones were picked out, and the antibody yield of the cells was determined by ELISA. The first 20% cells were transferred into a 24-well plate. After 7 days of growth, the antibody yield of the cells was determined, and the first 5 to 6 strains were selected and transferred into a shake flask for culture, thereby achieving the expression of anti-HER2 antibody in CHO-K1 cells. After the serum-free culture was stable, the cells were continuously cultured for 7 days, and then collected for purification and follow-up experiments.

(10) Construction of Expression Vector of Anti-VEGF Antibody Fab Fragment and Mutant Thereof

[0046] The DNA encoding the anti-VEGF antibody Fab fragment described herein (the corresponding wild-type sequence was obtained from USP Medicines Compendium, specially the heavy chain sequence of Fab fragment is shown in SEQ ID NO: 25 and the light chain sequence of Fab fragment is shown in SEQ ID NO: 26; based on this, according to kabat numbering, the amino acid at position 166 of the light chain of Fab fragment was substituted with cysteine to obtain a variant 166C of the present invention, and the amino acid at position 124 of the light chain of Fab fragment was substituted with cysteine to obtain a control antibody 124C) was chemically synthesized (Suzhou GENEWIZ Biotechnology Co., Ltd.), then the heavy chain gene of Fab fragment was digested with BstBI and PacI, and the light chain gene of Fab fragment was digested with HindIII and EcoRI; T4 ligase was used to ligate the heavy chain gene of Fab fragment into an eukaryotic expression vector pCGS3 (Biovector NTCC Inc.) treated by the BstBI and PacI; after successful ligation, the expression vector pCGS3 was digested with HindIII and EcoRI, and then the light chain gene of Fab fragment was ligated to the expression vector pCGS3 by T4 ligase, that is, an expression vector containing both heavy and light chains was successfully constructed.

TABLE-US-00004 WTFab (Bevacizumab) 166C 124C Encoding Sequence SEQ ID SEQ ID SEQ ID of Heavy Chain of NO: 27 NO: 27 NO: 27 Fab Fragment Encoding Sequence SEQ ID SEQ ID SEQ ID of Light Chain of NO: 28 NO: 30 NO: 32 Fab Fragment Amino Acid SEQ ID SEQ ID SEQ ID Sequence of Heavy NO: 25 NO: 25 NO: 25 Chain of Fab Fragment Amino Acid SEQ ID SEQ ID SEQ ID Sequence of Light NO: 26 NO: 29 NO: 31 Chain of Fab Fragment

(11) Screening and Purification of Expression Vector of Anti-VEGF Antibody Fab Fragment and Mutant Thereof

[0047] The successfully constructed expression vector was transformed into DH5α competent E. coli strain. The transformed DH5α strain was digested with BstBI and PacI, as well as HindIII and EcoRI respectively, and was verified by sequencing, and then positive clones were selected. A plasmid extraction kit (purchased from CoWin Biosciences) was used to lyse the resulting E. coli and extract the plasmid, thereby obtaining an expression vector containing heavy and light chains.

(12) Expression of Anti-VEGF Antibody Fab Fragment and Mutant Thereof in CHO Cells

[0048] The purified expression vector was transfected into CHO-K1 (purchased from ATCC) cells by electroporation; the cells were plated in a 96-well plate, grown for 15 days, and then single clones were picked out, and the antibody yield of the cells was determined by SDS-PAGE. The first 20% cells were transferred into a 24-well plate. After 7 days of growth, the antibody yield of the cells was determined, and the first 5 to 6 strains were selected and transferred into a shake flask for culture, thereby achieving the expression of antibody in CHO-K1 cells. After the serum-free culture was stable, the cells were continuously cultured for 7 days, and then collected for purification via Ni affinity chromatography column and follow-up experiments.

Example 2 Purification by Pro A Affinity Chromatography Column (HiTrap FF)

[0049] The cells and culture medium were transferred into a 50 mL centrifuge tube, placed in a high-speed refrigerated centrifuge, and centrifuged at 8000 r for 15 min; the supernatant was kept, and the cell debris was discarded; the culture medium was filtered once with a 0.22 μm microfiltration membrane, and the filtrate was collected for later use; the pump, injector, and purification column etc. were washed with 10 column volumes of ultrapure water and PBS; the sample was loaded and eluted with 0.1 M Gly-HCl and the purified antibody protein solution was taken into a EP tube, 1.5 M Tris-HCl was added to adjust the pH to be 7 to 8, and the purified antibody was stored at 2-8° C. The results are shown in FIG. 1.

Example 3 Detecting the Content of Free Sulfhydryl Groups in the Antibody

[0050] Ellman's method (Thermo Fisher Scientific (China) Co., Ltd.) was used to detect the free sulfhydryl content of the antibody product, and a cell line suitable for further coupling was selected. 0.1 M Na.sub.2HPO.sub.4 solution, DTNB (10 mM) solution, and cysteine standard solution with a final concentration of 0.0059 M to 0.375 M were prepared. At the same time, an antibody sample to be tested was prepared with a concentration of 5 mg/mL. 250 μL 0.1 M Na.sub.2HPO.sub.4 and 5 μL DTNB solution were respectively added into the 1.5 mL EP tube which contains the cysteine standard solution and the sample to be tested, and then the EP tubes were vortexed and shaken for completely mixing, and incubated for 5 min at room temperature in dark. The OD value at 412 nm was detected. A standard curve was established based on the OD value, and the sulfhydryl content of the sample was calculated.

TABLE-US-00005 TABLE 1 Ellman's method was used to detect the free sulfhydryl content of anti-VEGF antibody products (WT, 166C and 124C); wherein, WT substantially does not contain free sulfhydryl groups, while about 100% of sulfhydryl groups in 166C and 124C are free. Sample Free Sulfhydryl % WT 8.05% 166C 106.14% 124C 122.78%

TABLE-US-00006 TABLE 2 Ellman's method was used to detect the free sulfhydryl content of anti-CD20 antibody products (WT, 166C and 124C); wherein, WT substantially does not contain free sulfhydryl groups, while about 100% of sulfhydryl groups in 166C and 124C are free. Sample Free Sulfhydryl % WT 10.21% 166C 100.32% 124C 98.98%

TABLE-US-00007 TABLE 3 Ellman's method was used to detect the free sulfhydryl content of anti-HER2 antibody products (WT, 166C and 124C); wherein, WT substantially does not contain free sulfhydryl groups, while about 100% of sulfhydryl groups in 166C and 124C are free. Sample Free Sulfhydryl % WT 6.11% 166C 110.28% 124C 105.38%

TABLE-US-00008 TABLE 4 Ellman's method was used to detect the free sulfhydryl content of anti-VEGF antibody Fab fragment products (WT, 166C and 124C); wherein, WT substantially does not contain free sulfhydryl groups, while about 50% of sulfhydryl groups in 166C and 124C are free. Sample Free Sulfhydryl % WT 3.22% 166C 60.68% 124C 40.59%

Example 4 Antibody Conjugates and the Detection Thereof

[0051] The conditions for coupling reaction of antibody and conjugate were as follows: the molar ratio of antibody to conjugate (polypeptide or mPEG2000-MAL) was 1:30, at 37° C. for 3 hour to 4 hour, with a reaction buffer of PBS (pH7.2). The SDS-PAGE electrophoretogram for the coupling of anti-VEGF antibody and mPEG2000-MAL (purchased from a chemical reagent company, such as Shanghai ZZBio Co., Ltd., cat. no.: ZZP-MPEG-MAL-2K-01) was shown in FIG. 2.

[0052] The SDS-PAGE electrophoretogram for the coupling of anti-VEGF antibody and DC (polypeptide-linker) (see formula II below for the reaction, wherein 166ADC was obtained by coupling 166C mutant to DC, and 124ADC was obtained by coupling 124C mutant to DC) was shown in FIG. 3. As can be seen by Image J (NIH) grayscale analysis, 85% of 166C was successfully coupled to DC, while only 75% of 124C was successfully coupled to DC. It can be seen that under the same condition, the 166C antibody mutant showed better conjugation efficiency. The construction of DC (polypeptide-linker) was shown as follows:

[0053] The polypeptide was an active polypeptide with Ang2 neutralization effect, with a sequence of Gln-Lys(Ac)-Tyr-Gln-Pro-Leu-Asp-Glu-Lys(Ac)-Asp-Lu-Thr-Leu-Tyr-Asp-Gln-Phe-Met-Leu-Gln-Gln-Gly-CONH.sub.2(SEQ ID NO:9), and was obtained from Hanhua Huang, Jing-Yu Lai, Janet Do, Dingguo Liu, Lingna Li, Joselyn Del Rosario. Specifically Targeting Angiopoietin-2 Inhibits Angiogenesis, Tie2-Expressing Monocyte Infiltration, and Tumor Growth. Clin Cancer Res; 17(5) Mar. 1, 2011, 1001-1011, wherein the underlined Lys was used to be linked to the linker. The linker was formamide-PEG4-NHS, and the structural formula thereof was shown in formula I below:

##STR00002##

[0054] The reaction route of active peptide to linker was as follows

##STR00003##

[0055] The result for the coupling of anti-CD20 antibody (WT, 124C, 166C) to mPEG2000-MAL was shown in FIG. 4;

[0056] The result for the coupling of anti-CD20 antibody (WT, 124C, 166C) to DC (polypeptide-linker) was shown in FIG. 5.

[0057] The result for the coupling of anti-HER2 antibody (WT, 124C, 166C) to mPEG2000-MAL was shown in FIG. 6;

[0058] The result for the coupling of anti-HER2 antibody (WT, 124C, 166C) to DC (polypeptide-linker) was shown in FIG. 7.

[0059] The result for the coupling of anti-VEGF Fab antibody (WT, 124C, 166C) to mPEG2000-MAL was shown in FIG. 8;

[0060] The result for the coupling of anti-VEGF Fab antibody (WT, 124C, 166C) to DC (polypeptide-linker) was shown in FIG. 9.

Mass Spectrometry:

[0061] The sample was pre-treated by an ultrafiltration tub and dissolved in double distilled water. The obtained samples were analyzed by mass spectrometry. The mass spectrometry system was a high-resolution tandem mass spectrometry Triple TOF 4600 LC/MS/MS system of AB Sciex, with electrospray ionization source, and a positive ion mode was used for analysis. Mass spectrometry parameters were as follows: GS1: 55; GS2: 55; CUR: 35; scanning range: 100-2000 Da; Ionspray Voltage (ISVF): 5500 V; ionspray temperature: 350° C.; declustering voltage (DP): 150 V; collision energy (CE): 10 eV. The results were shown in FIG. 10 to FIG. 13. It can be seen from FIG. 10 and FIG. 11 that the main peak of mutant 166C was shifted to the right by 6478.8 Da after coupling with DC, which was the molecular weight of two molecules of DC. It can be known that both the two sulfhydryl groups on 166C were successfully coupled with DC.

[0062] It can be seen from FIG. 12 to FIG. 13 that after coupling with DC, the mutant 124C generated peaks of coupling with zero molecule DC, peaks of coupling with one molecule DC and peaks of coupling with two molecules DC, and thus the coupling uniformity and efficiency were not as high as that of 166C.

Example 5 Determination of Affinity of Anti-VEGF Antibody to Antigen

[0063] The affinity of wild-type antibody, antibody mutants and antibody-polypeptide conjugates to VEGF was determined by Biolayer interferometry (BLI) using Pro A probe (ForteBio). Antibody samples were dissolved in PBS with a working concentration of 10 μg/mL; VEGF-165 (Beijing SinoBiological Co., Ltd.) was dissolved in PBS and gradiently diluted to concentrations of 50 nM, 75 nM, 100 nM, 150 nM, 200 nM respectively. The working volume was 200 μL.

[0064] The affinity of wild-type antibody, antibody mutants and antibody-polypeptide conjugates to Ang2 was determined by Biolayer interferometry (BLI) using Pro A probe (ForteBio). Antibody samples were dissolved in PBS with a working concentration of 20 μg/mL; Ang2 (Beijing SinoBiological Co., Ltd.) was dissolved in PBS (0.02% tween-20, 0.1% BSA) and gradiently diluted to concentrations of 25 nM, 50 nM, 75 nM, 100 nM, 200 nM respectively. The working volume was 200 μL.

[0065] The data diagram was fitted by OCTET system and data-processing software, and the intermolecular interaction between the antibody and the VEGF or Ang2 was calculated by the software, and shown in K.sub.D value. The results are shown in Table 5.

[0066] Table 5 shows that there is almost no difference in the binding interaction between the wild-type antibody, antibody mutants and conjugates to the VEGF-165, and the binding interaction of the conjugate to the Ang2 is also within a reasonable range of interaction of neutralization effect.

TABLE-US-00009 TABLE 5 Detection of sample activity K.sub.D of Antibody to Antigen (10.sup.−9) Sample VEGF-165 Ang2 WT 1.65 N.A. 166 1.51 N.A. 124 0.99 N.A. 166ADC 1.96 2.20 124ADC 0.87 4.64

Example 7 Determining the Activity of Anti-VEGF Antibody on Cell Proliferation

[0067] The neutralization effect of antibody on VEGF165 was detected on HUVEC cells (ScienCell Research Laboratories, Inc.). Cells were seeded in a 96-well plate at a density of 4000 cells/50 μL/well. Blank control wells were set up, with no less than 6 control wells. Antibody samples with gradient concentrations (0.001 μg/mL, 0.01 μg/mL, 0.05 μg/mL, 0.1 μg/mL, 0.5 μg/mL, 1μg/mL, 5μg/mL, 10 μg/mL) were prepared by a gradient dilution method. VEGF165 was diluted with a test medium to a concentration of 110 ng/mL. The prepared VEGF165 reagent was mixed with samples at gradient concentration to a final concentration of VEGF165 of 10 ng/mL. The 96-well plate was placed in a CO.sub.2 incubator for 48 hours, and then subjected to a CCK-8 counting. The proliferation rate was calculated based on the OD value of the sample and the control. The results are shown in FIG. 14, indicating the inhibition of wild-type antibody (WT), mutants (124C and 166C) and conjugates (124ADC and 166ADC) on the proliferation of endothelial cells HUVEC (proliferation produced by VEGF stimulation). It can be known from the figure that the activity of antibody mutant was not changed due to the cysteine mutation at the selected site, indicating that the biological activity of the antibody was not affected by the selected sites. Similarly, the conjugate produced by conjugating with DC has no effect on the activity of the antibody to inhibit cell proliferation.

Example 8 Determination of Anti-CD20 Antibody Activity

[0068] The affinity of wild-type antibody, antibody mutants to CD20 was determined by Biolayer interferometry (BLI) using Pro A probe (ForteBio). Antibody samples were dissolved in PBS with a working concentration of 10 μg/mL; CD20 (Beijing SinoBiological Co., Ltd.) was dissolved in PBS and gradiently diluted to concentrations of 25 nM, 50 nM, 75 nM, 100 nM, 150 nM, 200 nM respectively. The working volume was 200 μL.

[0069] The data diagram was fitted by OCTET system and data-processing software, and the intermolecular interaction between the antibody and the CD20 was calculated by the software, and shown in K.sub.D value. The results are shown in Table 6.

TABLE-US-00010 TABLE 6 The affinity of wild-type antibody and antibody mutants to CD20 Sample K.sub.D of Antibody to Antigen (10.sup.−9) WT 5.35 166C 4.86 124C 8.97

Example 9 Determination of Anti-HER2 Antibody Activity

[0070] The affinity of wild-type antibody, antibody mutants and antibody-polypeptide conjugates to HER-FC was determined by Biolayer interferometry (BLI) using Pro A probe (ForteBio). HER-FC (Beijing SinoBiological Co., Ltd.) was dissolved in PBS with a working concentration of 10 μg/mL; the antibody was dissolved in PB S2 and gradiently diluted to concentrations of 25 nM, 50 nM, 75 nM, 150 nM, 300 nM respectively. The working volume was 200 μL.

[0071] The data diagram was fitted by OCTET system and data-processing software, and the intermolecular interaction between the antibody and the HER2 was calculated by the software, and shown in K.sub.D value. The results are shown in Table 7.

TABLE-US-00011 TABLE 7 The affinity of wild-type antibody and antibody mutants to HER2 Sample K.sub.D of Antibody to Antigen (10.sup.−9) WT 3.58 166C 6.87 124C 5.46

REFERENCES

[0072] 1. Doppalapudì V R, Huang J, Liu D, Jin P, Liu B, Li L, Chemical generation of bispecific antibodies. Proc. Natl Acad Sci U S A. (2010) 107:22611-22616.
2. Allen, T. M., Ligand-targeted therapeutics in anticancer therapy; Nat. Rev. Cancer, (2002) 2:750-763.
3. Hamblett, K. J., Effect of Drug Loading on the Antitumor Activity of a Monoclonal Antibody Drug Conjugate, Clin. Cancer Res.,(2004) 10:7063-7070.
4. Junutula J R, Bhakta S, Raab H, et al. Rapid identification of reactive cysteine residues for site-specific labeling of antibody-Fabs. J Immunol Methods, (2008) 332: 41-52.
5. Junutula J R, Flagella K M, Graham R A, et al Engineered thiotrastuzumab-DM1 conjugate with an improved therapeutic index to target human epidermal growth factor receptor 2-positive breast cancer. Clin Cancer Res, (2010) 16: 4769-4778.
6. Junutula J R, Raab H, Clark S, et al. Site-specific conjugation of a cytotoxic drug to an antibody improves the therapeutic index. Nat Biotechnol, (2008) 26: 925-932.
7. Kung Sutherland M S, Walter R B, Jeffrey S C, et al. SGN-CD33A: a novel CD33-targeting antibody-drug conjugate using a pyrrolobenzodiazepine dimer is active in models of drug-resistant AML. Blood, (2013) 122: 1455-1463.

TABLE-US-00012 Sequences SEQ ID NO: 1 EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKGLEWVGWINTYTGEP TYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGSSHWYFDVWGQGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA VLQSSGLSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEL LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 2 DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRF SGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQL KSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKAD YEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 3 gaagtgcagctggtggaatcaggaggaggactggtgcagccaggaggatctctgagactgtcttgtgctgcttccggatacacctttaccaac tacggaatgaattgggtgagacaggctccaggaaaaggactggagtgggtgggttggattaacacctataccggagagcctacatacgctg ccgatttcaagagaaggttcaccttcagcctggatacctctaagtctaccgcttacctgcagatgaactctctgagagcagaggataccgccgt gtattattgcgctaagtaccctcactactacggctcctcccattggtactttgacgtctggggacagggaacactggtgacagtgtcttccgcttct acaaaaggaccttccgtgttccctctggctccttcttctaagtctacctcaggaggaacagcagctctgggttgtctggtgaaggattacttccca gagccagtgacagtgtcttggaactccggagctctgacatccggagtgcatacatttccagcagtgctccagtcttccggactgtattctctgtct tccgtggtgacagtgccttcttcttctctgggaacacagacctacatctgcaacgtgaaccacaagccttccaacaccaaggtggataagaagg tggagcctaagtcttgcgacaagacccatacttgtcctccttgtccagctccagaactgctgggaggaccatcagtgtttctgtttcctcctaagc ctaaggacaccctgatgatctccagaacaccagaagtgacttgcgtggtggtggatgtgtctcacgaagatccagaggtgaagttcaattggta cgtggacggagtggaagtgcacaacgctaaaaccaagcctagagaggagcagtacaactccacctatagagtggtgtccgtgctgacagtg ctgcatcaggattggctgaacggaaaggagtacaagtgcaaggtgtccaacaaagctctgccagctcctatcgagaagacaatctccaaggc taagggacagcctagagaacctcaggtgtatacactgcctccttctagagaggagatgaccaagaatcaggtgtctctgacttgcctggtgaa gggcttttacccttctgacatcgcagtcgagtgggaatctaacggacagccagagaacaactacaagaccacacctccagtgctggattccga cggatctttcttcctgtactccaagctgaccgtggataagtctcgttggcagcagggtaacgtgttttcttgtagcgtgatgcacgaagctctgcat aatcactacacccagaagtctctgtctctgtctcctggaaagtga SEQ ID NO: 4 gatatccagatgacccagtctccttcctctctgtcagcttcagtgggagatagagtgacaatcacctgttccgcttctcaggatatctccaactacc tgaattggtaccagcagaagccaggaaaggctcctaaggtgctgatctacttcacctcttctctgcattccggagtgccttctagatttagcggct ccggctccggcacagatttcacactgaccatctcttctctgcagccagaagatttcgccacctactattgccagcagtactctaccgtgccttgg acatttggacagggaaccaaggtggagatcaagagaacagtggctgctccttccgtgtttatctttcctccttctgacgaacagctgaaatccgg aacagcttcagtcgtctgcctgctgaacaacttctaccctagagaggccaaagtccagtggaaagtggataacgctctgcagtccggaaattct caggaatccgtgaccgagcaggattctaaggattctacctactccctgtcttctaccctgacactgtctaaggccgattacgagaagcacaagg tgtacgcttgcgaagtgacacatcagggactgtcttctccagtgaccaagtccttcaacagaggcgagtgttga SEQ ID NO: 5 gatatccagatgacccagtctccttcctctctgtcagcttcagtgggagatagagtgacaatcacctgttccgcttctcaggatatctccaactacc tgaattggtaccagcagaagccaggaaaggctcctaaggtgctgatctacttcacctcttctctgcattccggagtgccttctagatttagcggct ccggctccggcacagatttcacactgaccatctcttctctgcagccagaagatttcgccacctactattgccagcagtactctaccgtgccttgg acatttggacagggaaccaaggtggagatcaagagaacagtggctgctccttccgtgtttatctttcctccttctgacgaacagctgaaatccgg aacagcttcagtcgtctgcctgctgaacaacttctaccctagagaggccaaagtccagtggaaagtggataacgctctgcagtccggaaattct caggaatccgtgaccgagtgcgattctaaggattctacctactccctgtcttctaccctgacactgtctaaggccgattacgagaagcacaaggt gtacgcttgcgaagtgacacatcagggactgtcttctccagtgaccaagtccttcaacagaggcgagtgttga SEQ ID NO: 6 gatatccagatgacccagtctccttcctctctgtcagcttcagtgggagatagagtgacaatcacctgttccgcttctcaggatatctccaactacc tgaattggtaccagcagaagccaggaaaggctcctaaggtgctgatctacttcacctcttctctgcattccggagtgccttctagatttagcggct ccggctccggcacagatttcacactgaccatctcttctctgcagccagaagatttcgccacctactattgccagcagtactctaccgtgccttgg acatttggacagggaaccaaggtggagatcaagagaacagtggctgctccttccgtgtttatctttcctccttctgacgaatgtctgaaatccgg aacagcttcagtcgtctgcctgctgaacaacttctaccctagagaggccaaagtccagtggaaagtggataacgctctgcagtccggaaattct caggaatccgtgaccgagcaggattctaaggattctacctactccctgtcttctaccctgacactgtctaaggccgattacgagaagcacaagg tgtacgcttgcgaagtgacacatcagggactgtcttctccagtgaccaagtccttcaacagaggcgagtgttga SEQ ID NO: 7 DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRF SGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQL KSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTECDSKDSTYSLSSTLTLSKAD YEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 8 DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRF SGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDECL KSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKAD YEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 9 QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDT SY NQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTV SAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKAEPKSCDKTHTCPPCPAPELLG GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRD ELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 10 QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFS GSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIKRTVAAPSVFIFPPSDEQL KSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKAD YEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 11 caagtgcaactgcagcagcccggcgccgaactggtgaagcccggcgctagcgtcaagatgagctgtaaggcttccggctacactttcacta gctacaacatgcactgggtcaagcagacacccggaaggggactggaatggatcggcgctatctaccccggcaacggagacacaagctaca accaaaagttcaagggcaaggccactctcactgctgataagagcagctccactgcctacatgcaactgagctctctgactagcgaagattccg ccgtgtactactgcgccagaagcacatactacggcggcgactggtatttcaacgtctggggcgccggcacaacagtgacagtgagcgctgc cagcactaagggaccatccgtgtttcctctcgccccaagctccaagagcacttccggaggcactgccgctctgggctgtctggtcaaggacta cttcccagagccagtgacagtcagctggaatagcggagctctgacaagcggcgtccatacattcccagccgtgctgcagtccagcggactgt actctctgagctccgtcgtgactgtgccaagcagctctctgggcacacagacatacatctgcaatgtcaaccacaagcctagcaacacaaagg tggacaagaaggccgaaccaaagagctgcgacaagacacacacatgccctccttgtccagccccagagctgctcggaggaccaagcgtct tcctcttcccacctaagcctaaggacacactgatgatctctaggactccagaggtgacatgcgtcgtcgtcgatgtgtcccacgaggatccaga ggtgaagttcaactggtacgtggacggcgtcgaagtgcacaatgccaagactaagccaagggaggagcagtacaactccacttacagagtc gtgtccgtcctcacagtgctccaccaagattggctgaatggcaaggagtataagtgtaaggtctccaataaagctctgccagctcctatcgaga agactatcagcaaagctaagggccagccaagagagccacaagtgtacactctgcctccatctagggatgagctgactaagaaccaagtgtct ctgacttgcctcgtgaagggcttctatcctagcgacatcgccgtcgagtgggagtccaacggccaaccagaaaacaactacaagactactcct ccagtgctcgacagcgatggctccttctttctgtactccaagctgacagtggacaagtccagatggcagcaaggcaacgtcttcagctgtagc gtgatgcatgaggctctgcacaaccactacactcagaagtctctgtctctgagccccggcaag SEQ ID NO: 12 Cagatcgtcctcagccagagcccagccattctgagcgccagccccggcgagaaggtgactatgacatgcagagcctccagctccgtgagc tacatccactggttccagcagaagcccggctcctcccctaagccttggatctacgctacaagcaatctggcctccggagtcccagtgaggttca gcggcagcggatccggcacttcctactctctgacaatttctagggtggaggctgaggacgctgccacatactactgccagcagtggactagc aaccctcctacattcggcggaggcacaaagctggagatcaagaggactgtggccgctcctagcgtgttcatcttccctccttccgatgagcag ctgaagagcggcactgccagcgtggtctgtctgctgaacaacttctaccctagggaggccaaggtgcaatggaaggtggacaatgctctgca gtccggcaattcccaagagtccgtcacagagcaagattccaaggacagcacatactccctctccagcacactgacactgagcaaggctgact acgagaagcacaaggtgtacgcttgtgaggtgactcatcaaggactgagcagcccagtgactaagagcttcaataggggcgagtgt SEQ ID NO: 13 QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVR FSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIKRTVAAPSVFIFPPS DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTECDSKDSTYSLSSTLTL SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 14 Cagatcgtcctcagccagagcccagccattctgagcgccagccccggcgagaaggtgactatgacatgcagagcctccagctccgtgagc tacatccactggttccagcagaagcccggctcctcccctaagccttggatctacgctacaagcaatctggcctccggagtcccagtgaggttca gcggcagcggatccggcacttcctactctctgacaatttctagggtggaggctgaggacgctgccacatactactgccagcagtggactagc aaccctcctacattcggcggaggcacaaagctggagatcaagaggactgtggccgctcctagcgtgttcatcttccctccttccgatgagcag ctgaagagcggcactgccagcgtggtctgtctgctgaacaacttctaccctagggaggccaaggtgcaatggaaggtggacaatgctctgca gtccggcaattcccaagagtccgtcacagagtgtgattccaaggacagcacatactccctctccagcacactgacactgagcaaggctgacta cgagaagcacaaggtgtacgcttgtgaggtgactcatcaaggactgagcagcccagtgactaagagcttcaataggggcgagtgt SEQ ID NO: 15 QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFS GSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIKRTVAAPSVFIFPPSDECL KSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKAD YEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 16 Cagatcgtcctcagccagagcccagccattctgagcgccagccccggcgagaaggtgactatgacatgcagagcctccagctccgtgagc tacatccactggttccagcagaagcccggctcctcccctaagccttggatctacgctacaagcaatctggcctccggagtcccagtgaggttca gcggcagcggatccggcacttcctactctctgacaatttctagggtggaggctgaggacgctgccacatactactgccagcagtggactagc aaccctcctacattcggcggaggcacaaagctggagatcaagaggactgtggccgctcctagcgtgttcatcttccctccttccgatgagtgtc tgaagagcggcactgccagcgtggtctgtctgctgaacaacttctaccctagggaggccaaggtgcaatggaaggtggacaatgctctgcag tccggcaattcccaagagtccgtcacagagcaagattccaaggacagcacatactccctctccagcacactgacactgagcaaggctgacta cgagaagcacaaggtgtacgcttgtgaggtgactcatcaaggactgagcagcccagtgactaagagcttcaataggggcgagtgt SEQ ID NO: 17 EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRY ADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTV SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGP SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 18 gaagtgcaactggtggagtccggaggcggactggtccaacccggcggctctctgagactgagctgtgctgccagcggcttcaacatcaagg acacttacatccattgggtgaggcaagcccccggcaaaggactggaatgggtggctaggatctacccaactaacggctacactaggtatgcc gacagcgtcaagggaaggttcactatctccgccgacacaagcaagaacactgcctatctgcagatgaactctctgagggctgaggacacag ctgtgtattactgctctaggtggggcggagatggcttctacgctatggactactggggacaaggcacactcgtgacagtgagcagcgctagca ctaaaggcccttccgtgttcccactcgcccctagctccaaaagcactagcggcggaacagctgctctgggctgtctcgtgaaggactatttccc agagccagtgactgtgagctggaatagcggcgctctgacaagcggagtgcacacttttccagccgtgctgcagagcagcggactgtattctc tgtccagcgtcgtcactgtgcctagctcctctctgggcacacagacttacatctgtaacgtgaatcataagccaagcaacactaaggtcgacaa gaaggtcgagccaaagagctgtgacaagacacacacatgcccaccatgtccagccccagagctcctcggaggacctagcgtgttcctcttcc ctcctaagccaaaggacactctcatgatctctaggacaccagaggtcacttgcgtggtggtcgatgtcagccacgaggacccagaggtgaag ttcaattggtacgtggacggcgtggaggtgcataatgccaagacaaagcctagggaggagcagtacaacagcacttacagagtggtctccgt cctcacagtgctgcaccaagattggctcaacggcaaggagtacaagtgcaaggtgtccaacaaggccctcccagcccctatcgagaagact atctccaaggccaagggccagccaagagaacctcaagtgtacacactgcctccatctagggaggagatgacaaagaaccaagtgtctctga catgtctggtgaaaggcttctacccaagcgacatcgccgtggagtgggaaagcaacggccagccagagaacaactacaagactacacctcc agtgctggactccgacggcagcttcttcctctactccaagctcactgtggataagtctaggtggcagcaaggcaatgtgttcagctgttccgtga tgcacgaggctctgcacaaccactacactcagaagtctctgagcctctcccccggcaaa SEQ ID NO: 19 DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSR FSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQ LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 20 Gacatccagatgacacagtccccttcctctctgagcgctagcgtcggcgatagggtgacaatcacttgtagggccagccaagatgtcaacac agccgtggcttggtaccagcagaagcccggcaaggcccctaagctgctcatctatagcgccagctttctgtacagcggcgtccctagcagatt ctccggaagcagatccggcactgacttcacactgactatcagcagcctccagccagaggatttcgccacatactactgccagcagcactaca ctactccacctactttcggccaaggcactaaggtggagattaagaggactgtcgctgccccaagcgtcttcatcttcccacctagcgatgaaca gctcaagagcggcacagctagcgtcgtgtgtctgctgaacaacttctaccctagggaggccaaggtccagtggaaagtggataacgctctgc agagcggcaattcccaagagagcgtcacagagcaagatagcaaagacagcacttactctctgagcagcacactgactctgtccaaggccga ctacgagaagcacaaggtctatgcttgtgaggtcactcaccaaggactgtccagcccagtcactaagagcttcaataggggcgagtgc SEQ ID NO: 21 DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSR FSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQ LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTECDSKDSTYSLSSTLT LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 22 Gacatccagatgacacagtccccttcctctctgagcgctagcgtcggcgatagggtgacaatcacttgtagggccagccaagatgtcaacac agccgtggcttggtaccagcagaagcccggcaaggcccctaagctgctcatctatagcgccagctttctgtacagcggcgtccctagcagatt ctccggaagcagatccggcactgacttcacactgactatcagcagcctccagccagaggatttcgccacatactactgccagcagcactaca ctactccacctactttcggccaaggcactaaggtggagattaagaggactgtcgctgccccaagcgtcttcatcttcccacctagcgatgaaca gctcaagagcggcacagctagcgtcgtgtgtctgctgaacaacttctaccctagggaggccaaggtccagtggaaagtggataacgctctgc agagcggcaattcccaagagagcgtcacagagtgcgatagcaaagacagcacttactctctgagcagcacactgactctgtccaaggccga ctacgagaagcacaaggtctatgcttgtgaggtcactcaccaaggactgtccagcccagtcactaagagcttcaataggggcgagtgc SEQ ID NO: 23 DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSR FSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDECL KSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 24 Gacatccagatgacacagtccccttcctctctgagcgctagcgtcggcgatagggtgacaatcacttgtagggccagccaagatgtcaacac agccgtggcttggtaccagcagaagcccggcaaggcccctaagctgctcatctatagcgccagctttctgtacagcggcgtccctagcagatt ctccggaagcagatccggcactgacttcacactgactatcagcagcctccagccagaggatttcgccacatactactgccagcagcactaca ctactccacctactttcggccaaggcactaaggtggagattaagaggactgtcgctgccccaagcgtcttcatcttcccacctagcgatgaatg cctcaagagcggcacagctagcgtcgtgtgtctgctgaacaacttctaccctagggaggccaaggtccagtggaaagtggataacgctctgc agagcggcaattcccaagagagcgtcacagagcaagatagcaaagacagcacttactctctgagcagcacactgactctgtccaaggccga ctacgagaagcacaaggtctatgcttgtgaggtcactcaccaaggactgtccagcccagtcactaagagcttcaataggggcgagtgc SEQ ID NO: 25 EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKGLEWVGWINTYTGEP TYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGSSHWYFDVWGQGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA VLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT SEQ ID NO: 26 DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRF SGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQL KSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKAD YEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 27 Gaggtgcaactggtcgaaagcggaggcggactggtgcagcccggcggatctctgaggctgagctgtgccgccagcggctacactttcact aactacggcatgaattgggtgaggcaagcccccggcaaaggactggagtgggtcggatggatcaacacttacactggcgagccaacttacg ccgccgacttcaagaggaggttcacattcagcctcgacactagcaagagcacagcctatctgcagatgaacagcctcagagctgaggacact gccgtgtactactgcgctaagtatcctcactactacggcagcagccactggtatttcgatgtgtggggccaaggcacactcgtcactgtcagca gcgcttccacaaaaggccctagcgtgttccctctcgcccctagctccaagagcacatccggaggaacagctgctctcggctgtctggtgaag gattacttcccagagccagtgactgtcagctggaatagcggcgctctgacaagcggcgtgcacacattcccagccgtgctgcagagctccgg actgtactccctcagcagcgtggtcacagtccctagcagctctctgggcactcagacatacatctgcaacgtgaaccacaagccaagcaaca ctaaggtcgacaagaaggtggagcctaagagctgcgacaagactcacaca SEQ ID NO: 28 Gatatccagatgacccagtctccttcctctctgtcagcttcagtgggagatagagtgacaatcacctgttccgcttctcaggatatctccaactac ctgaattggtaccagcagaagccaggaaaggctcctaaggtgctgatctacttcacctcttctctgcattccggagtgccttctagatttagcggc tccggctccggcacagatttcacactgaccatctcttctctgcagccagaagatttcgccacctactattgccagcagtactctaccgtgccttgg acatttggacagggaaccaaggtggagatcaagagaacagtggctgctccttccgtgtttatctttcctccttctgacgaacagctgaaatccgg aacagcttcagtcgtctgcctgctgaacaacttctaccctagagaggccaaagtccagtggaaagtggataacgctctgcagtccggaaattct caggaatccgtgaccgagcaggattctaaggattctacctactccctgtcttctaccctgacactgtctaaggccgattacgagaagcacaagg tgtacgcttgcgaagtgacacatcagggactgtcttctccagtgaccaagtccttcaacagaggcgagtgt SEQ ID NO: 29 DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRF SGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQL KSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTECDSKDSTYSLSSTLTLSKAD YEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 30 gatatccagatgacacagtccccttcctctctgagcgctagcgtgggcgatagggtgacaattacttgtagcgccagccaagatatcagcaact atctgaactggtaccagcagaagcccggcaaggccccaaaggtcctcatctacttcactagcagcctccacagcggcgtcccatccagattta gcggcagcggcagcggaactgacttcacactgacaatcagctctctgcagccagaggatttcgccacttactactgccagcagtactccaca gtcccttggacatttggccaaggcactaaggtggagatcaagagaacagtggccgcccctagcgtcttcatcttcccaccttccgatgagcag ctgaagagcggaacagcttccgtggtctgtctgctgaacaacttctacccaagagaggccaaggtccagtggaaagtggacaacgctctgca gagcggcaattcccaagagtccgtcacagagtgcgattccaaggacagcacatactctctgagcagcacactgactctgagcaaggccgac tacgagaagcacaaggtctacgcttgtgaggtcactcaccaaggactgagcagcccagtgactaagtccttcaataggggcgagtgctga SEQ ID NO: 31 DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRF SGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDECL KSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKAD YEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 32 gatatccagatgacccagtctccttcctctctgtcagcttcagtgggagatagagtgacaatcacctgttccgcttctcaggatatctccaactacc tgaattggtaccagcagaagccaggaaaggctcctaaggtgctgatctacttcacctcttctctgcattccggagtgccttctagatttagcggct ccggctccggcacagatttcacactgaccatctcttctctgcagccagaagatttcgccacctactattgccagcagtactctaccgtgccttgg acatttggacagggaaccaaggtggagatcaagagaacagtggctgctccttccgtgtttatctttcctccttctgacgaatgtctgaaatccgg aacagcttcagtcgtctgcctgctgaacaacttctaccctagagaggccaaagtccagtggaaagtggataacgctctgcagtccggaaattct caggaatccgtgaccgagcaggattctaaggattctacctactccctgtcttctaccctgacactgtctaaggccgattacgagaagcacaagg tgtacgcttgcgaagtgacacatcagggactgtcttctccagtgaccaagtccttcaacagaggcgagtgt