Bispecific antibody targeting human p185 and vascular endothelial growth factor and application thereof

Abstract

A bispecific antibody that simultaneously targets humanized p185 and VEGF, consisting of the four peptide chains: two identical antibody light chains that are the light chains of the antibody that identify the epitope or antigen of p185, and two identical antibody heavy chains that have the amino acid sequence of a recombinant antibody from N- to C-terminus, a light chain sequence of the antibody that recognizes the p185 epitope or the antigen; a constant heavy chain region; a flexible short peptide sequence; and either a single-stranded variable region sequence (ScFv) of anti-VEGF antibody which recognizes the VEGF epitope or antigen, or a receptor domain sequence that binds to VEGF. The bispecific antibody has the ability to bind p185 and VEGF at the same time, inhibits the proliferation of tumor cells, and promotes the expression of IFN-γ by T lymphocytes; it may be applied as anti-tumor antibody drug.

Claims

1. A bispecific antibody that simultaneously binds human p185 and VEGF comprising two identical antibody light chains, and two identical antibody heavy chains, wherein each antibody light chain comprises the amino acid sequence of SEQ ID NO:1 and wherein each antibody heavy chain comprises the amino acid sequence of SEQ ID NO:2.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1 is a schematic drawing showing the structure of the bispecific antibody that simultaneously targets human p185 and vascular endothelial growth factor.

(2) FIG. 2 shows that the bispecific antibody of the present invention, having the light chain sequence of SEQ ID NO: 1 and the recombinant heavy chain sequence of SEQ ID NO: 2 that targets human p185 and VEGF, has a high affinity with human p185 and VEGF proteins, among which, FIG. 2(a) shows the p185 binding ability; FIG. 2(b) shows the VEGF binding ability; FIG. 2(c) shows the p185-VEGF binding ability; and FIG. 2(d) shows the VEGF-p185 binding ability.

(3) FIG. 3 shows that the bispecific antibody of the present invention, having the light chain sequence of SEQ ID NO: 1 and the recombinant heavy chain sequence of SEQ ID NO: 2 that targets human p185 and VEGF, can effectively recognize human p185 and VEGF proteins, among which, FIG. 3(a) shows the recognition at the protein level for VEGF, and FIG. 3(b) shows the recognition at the protein level for p185.

(4) FIG. 4 shows that the bispecific antibody of the present invention, having the light chain sequence of SEQ ID NO: 1 and the recombinant heavy chain sequence of SEQ ID NO: 2 that targets human p185 and VEGF, inhibits the proliferation of vascular endothelial cells, human lung cancer cells, human breast cancer cells and human of gastric cancer cells (detection time: 96 hours after administration).

(5) FIG. 5 shows that the bispecific antibody of the present invention, having the light chain sequence of SEQ ID NO: 1 and the recombinant heavy chain sequence of SEQ ID NO: 2 that targets human p185 and VEGF, promotes IFN-γ secretion by human T lymphocytes.

DETAILED DESCRIPTION OF THE INVENTION

(6) The present invention provides a bispecific antibody that simultaneously targets human p185 and VEGF. The bispecific antibody consists of two identical light chains and two identical heavy chains. The structure of the light chain and the recombinant heavy chain are shown in FIG. 1 as follows: a light chain 1 is to identify p185 antigen epitopes or antigen (amino acid sequence shown in Table 1); recombinant heavy chain sequence 2 (amino acid sequence shown in Table 1): the N-terminus is the light chain sequence 3 that recognizes the p185 epitope antibody, followed by the constant heavy chain 4; the C-terminus is the single-stranded variable region sequence (ScFv) 5 which recognizes an VEGF epitope or antigen, or a receptor domain sequence that binds to VEGF. The N-terminus and C-terminus of the recombinant heavy chain are linked by a flexible peptide 6 that is the linker that links the constant heavy chain 4 and the C-terminus single stranded variable region sequence 5 in FIG. 1.

(7) TABLE-US-00001 TABLE 1 Amino acid sequences of bispecific antibody targeting both human p185 and VEGF Serial Number Name Sequence SEQ ID Light DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAW NO: 1 chain YQQKPGKAPKLLIYSASFINSGVPSRFSGSRSGTD se- FTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEI quence KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFAP REAKVQWKVDNALQSGNSQESVTEQDSKDSTYS LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC SEQ ID Recom- EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIH NO: 2 binant WVRQAPGKGLEWVARIYPTNGYTRYADSVKGRF heavy TISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGD chain GFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSK se- STSGGTAALGCLVKDYYPEPVTVSWNSGALTSGV quence HTFPAVLQSSGLYSLSSVVTYPSSSLGTQTYICNVN 1 HKPSNTKVDKKVEPPKSCDKTHTCPPCPAPELLG GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPVYTLPPSRDELTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGGGGSGGGGSGGGGSEVQLVESGGGLVQP GGSLRLSCAASGYTFTNYGMNWVRQAPGKGLE WVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAY LQMNSLRAEDTAVYYCAKYPHYYGSSHWYFDV WGQGTLVTVSSGGGSGGGSGGGSGGGSDIQMTQ SPSSLSASVGDRVTITCSASQDISNYLNWYQQKPG KAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISS LQPEDFATYYCQQYSTVPWTFGQGTKVEIK SEQ ID Recom- EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIH NO: 3 binant WVRQAPGKGLEWVARIYPTNGYTRYADSVKGRF heavy TISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGD chain GFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSK se- STSGGTAALGCLVKDYFPEPVTVSWNSGALTSG quence HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN 2 HKPSNTKVDKKVEPPKSCDKTHTCPPCPAPELLG GPSVFLFPPKTKDTLMISKTPEVTCVVVDVSHEDP EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGGGGSGGGGSGGGGSDIQMTQSPSSLSAS VGDRVTITCSASQDISNYLNWYQQKPGKAPKVLI YFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFAT YYCQQYSTYPWTFGQGTKVEIKGGGSGGGSGGG SGGGSEVQLVESGGGLVQPGGSLRLSCAASGYTF TNYGMNWVRQAPGKGLEWVGWINTYTGEPTYA ADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYY CAKYPHYYGSSHWYFDVWCOGILVTVSS SEQ ID Recom- EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIH NO: 4 binant WVRQAPGKGLEWVARIYPTNGYTRYADSVKGRF heavy TISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGD chain GFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSK se- STSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV quence HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN 3 HKPSNTKVDKKVEPPKSCDKTHTCPPCPAPELLG GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGGGGSGGGGSGGGGSSDTGRPFVEMYSEI PEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIP DGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHD VVLSPSHGIELSVGEKLVLNCTARTELNVGIDFNW EYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTI DGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEK SEQ ID Recom- EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIH NO: 5 binant WVRQAPGKGLEWVARIYPTNGYTRYADSVKGRF heavy TISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGD chain GFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSK se- STSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV quence HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN 4 HKPSNTKVDKKVEPPKSCDKTHTCPPCPAPELLG GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPGGGGSGGGGSGGGGSSDTGRPFVEMYSEI PEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIP DGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHL YKTNYLTHRQTNTIIDVQISTPRPVKLLRGHTLVL NCTATTPLNTRVQMTWSYPDEKNKRASVRRRIDQ SNSHANIFYSVLTIDKMQNKDKGLYTCRVRSGPSF KSVNTSVHIYDKAFITVKHRK

(8) In the present invention, the DNA fragments encoding the anti-p185 antibody, the anti-VEGF antibody, or the domain sequence of binding VEGF are firstly synthesized, respectively. Then, the antibody light chain sequence and the recombinant heavy chain sequence are respectively cloned into the eukaryotic expression vector, pcDNA, through recombinant DNA (overlapping PCR technique); then, it is transfected into 293F or CHO cells. The supernatant is collected 5-7 days after transfection and then purified by affinity chromatography gel column to obtain the bispecific antibody.

(9) The following examples described the invention in further details which are not intended to limit the scope of protection for the invention.

Example 1

(10) The DNA fragments encoding the anti-p185 antibody, the anti-VEGF antibody and the VEGF binding domain are synthesized. Then, the light chain sequence (SEQ ID NO: 1) and the recombinant heavy chain sequence (SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5) are cloned into the eukaryotic expression vector, pcDNA, respectively. The light chain and the heavy chain DNA are mixed at a mass ratio of 2:1. The DNA mixture (4-10 μg) and 40 μL of 2M CaCl.sub.2 are added to a total volume of 250 μL with ddH.sub.2O. The whole mixture is added into 250 μL 2×HBS (NaCl 16.3 g, KCl 0.74 g. Na.sub.2HPO.sub.4 0.214 g, Glucose 2.4 g and HEPES 10 g, pH=7.05) to form a calcium phosphate-DNA suspension which is added into 293F or CHO cells (logarithmic growth phase). The supernatant is collected 5-7 days after the transfection. After filtration through a 0.45 μm filter, the supernatant is added to the affinity chromatography gel column (Protein A) which is washed with binding buffer (12.15 g Tris dissolved in ddH.sub.2O, pH=7.5, adding 8.78 g NaCl). Then it is eluted by eluent (7.5 g glycine dissolved in ddH.sub.2O, pH=3.5, adding 8.78 g NaCl). The eluted fraction, which is the bispecific antibody as shown in FIG. 1, is neutralized with 1 M Tris-HCl (pH 9.0).

Example 2 Enzyme-Linked Immunosorbent Assay (ELISA)

(11) The human p185 and VEGF proteins are coated into 96 well plates for 16 hours and being incubated with PBS buffer containing 1% bovine serum albumin (BSA) at 37° C. for 2 hours. After washing with PBST, it is incubated with the commercially available p185 and VEGF antibody as well as the antibody of the present invention at 37° C. for 2 hours. Following by the PBST washing, anti-human IgG Fab antibody is added for 1 hour. After washing with PBST, tetramethyl benzidine is added for 5 minutes to terminate the reaction. The affinity is measured with the absorbance at 450 nm.

(12) The ELISA results are shown in FIG. 2. The results suggest that the bispecific antibody targeting both human p185 and VEGF has high affinity with human p185 (FIG. 2(a)), VEGF (FIG. 2(b)), p185-VEGF (FIG. 2(c)), and VEGF-p185 (FIG. 2(d)).

Example 3 Western Blotting Assay

(13) (1) The Recognition of VEGF by the Bispecific Antibody

(14) The HUVEC cells which are morphological health and with the fusion rate of 80% are collected. After centrifugation, the cell lysate is added. The cell lysate is incubated at 95° C. for 10 minutes and is separated by polyacrylamide gel electrophoresis. The protein is transferred into the nitrocellulose membrane (NC membrane) and blocked with 5% milk at room temperature for 1 hour. The bispecific antibody, commercially available VEGF or 1-actin antibody is added and incubated at room temperature for 1 hour. After washing with PBST, the antibody with horseradish peroxidase is added and incubated for 1 hour at room temperature. After washing with PBST, the chemiluminescent reagent is used for developing in the darkroom.

(15) (2) The Recognition of p185 by the Bispecific Antibody

(16) The MDA-MB-453 cells which are morphological health and with the fusion rate of 80% are collected. After centrifugation, the cell lysate is added. The cell lysate is incubated at 95° C. for 10 minutes and is separated by polyacrylamide gel electrophoresis. The protein is transferred into the nitrocellulose membrane (NC membrane) and blocked with 5% milk at room temperature for 1 hour. The bispecific antibody, commercially available p185 or β-actin antibody is added and incubated at room temperature for 1 hour. After washing with PBST, the antibody with horseradish peroxidase is added and incubated for 1 hour at room temperature. After washing with PBST, the chemiluminescent reagent is used for developing in the darkroom.

(17) The results of the western blotting assay are shown in FIG. 3. The results indicate that the bispecific antibody targeting both human p185 and VEGF efficiently recognizes p185 (FIG. 3(b) and VEGF (FIG. 3(a).

Example 4 Cell Proliferation Assay

(18) Cells, including HUVEC cells, human lung cancer cells, human breast cancer cells, and human gastric cancer cells, are seeded in the 96-well plate and cultured in medium containing 0.5% fetal bovine serum overnight. Each cell lines include five groups, i.e., control group, commercially p185 antibody group, commercially VEGF antibody group, commercially p185 and VEGF antibody group, and the bispecific antibody). Cells are cultured for 72 hours. The proliferation of cells is detected through commercially available cell proliferation assay kit (CCK8 Kit).

(19) The results of cell proliferation test are shown in FIG. 4. The results suggest that the bispecific antibody targeting both human p185 and VEGF effectively inhibits the proliferation of HUVEC cells, human lung cancer cells, human breast cancer cells, and human gastric cancer cells.

Example 5 T Cell Stimulation Test

(20) The suspension of T lymphocyte is added into a 96-well plate. The test includes two groups which are added with the medium and the bispecific antibody. Cells are cultured for 72 hours. The enzyme-linked immunosorbent assay (ELISA) kit is used to detect the concentration of IFN-γ.

(21) The results of T cell stimulation test are shown in FIG. 5. The results indicate that the bispecific antibody targeting both human p185 and VEGF promotes the IFN-γ expression of T lymphocytes.

Comparative Example 1

(22) The connection of conventional p185 antibody and conventional VEGF antibody with the same procedure of the present invention is not able to simultaneously target both p185 and VEGF. Some connections are with low expression, while some connections could not be expressed due to the spatial structure. Some connections which are able to express are with decreased binding ability with p185 and/or VEGF.