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.

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′) 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′) 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 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 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 50  with which the bispecific antibody binds to the VEGFA protein is detected by indirect ELISA;
 and/or   wherein the EC 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.