Novel recombinant bi-functional fusion protein, preparation method therefor and use thereof

Abstract

Provided is a bi-functional fusion protein as well as its preparation method and use. The bi-functional fusion protein is composed of a monoclonal anti-human CD20 antibody linked to a first extracellular domain of human SIRPα. It is shown in in vitro tests that the bi-functional fusion protein is capable of binding to human CD20 and CD47 simultaneously, thus killing CD20+ tumor cells via antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity, and is also capable of blocking CD47-SIRPα interaction such that macrophages are activated to attack tumor cells, The bi-functional fusion protein has evident anti-tumor activities.

We claim: 
     
         1 . A recombinant bi-functional fusion protein, comprising an antibody or an antibody fragment specifically binding to CD20, and a peptide specifically binding to CD47,
 wherein the peptide is linked to the antibody or the antibody fragment,   wherein the antibody or antibody fragment specifically binding to CD20 comprises a heavy chain variable region of SEQ ID NO.: 5, and the light chain contains a light chain variable region of SEQ ID NO.: 7 and   wherein the peptide specifically binding to CD47 comprises a sequence having at least 95% identity to SEQ ID NO: 9.   
     
     
         2 . A polynucleotide encoding the recombinant bi-functional fusion protein according to  claim 1 . 
     
     
         3 . A vector containing the polynucleotide according to  claim 2 . 
     
     
         4 . A genetically engineered host cell containing the vector according to  claim 3 . 
     
     
         5 . An immunoconjugate containing (a) the recombinant bi-functional fusion protein according to  claim 1 , and (b) a second molecule conjugated to the recombinant bi-functional fusion protein selected from the group consisting of a detectable marker, a drug, a toxin, a cytokine, a radionuclide, and an enzyme. 
     
     
         6 . A pharmaceutical composition containing: (i) the recombinant bi-functional fusion protein according to  claim 1 , and (ii) a pharmaceutically acceptable carrier. 
     
     
         7 . A method for treating a CD20+ tumor, comprising administering to a subject in need thereof a therapeutically effective amount of the pharmaceutical composition according to  claim 6 . 
     
     
         8 . A method for preparing the recombinant bi-functional fusion protein, comprising the steps of:
 (a) culturing the host cell according to  claim 4  under conditions suitable for protein expression; and   (b) isolating the recombinant bi-functional fusion protein from the cell culture.   
     
     
         9 . The recombinant bi-functional fusion protein according to  claim 1 , wherein the antibody is selected from the group consisting of Rituximab, Obinutuzumab, and Ofatumumab. 
     
     
         10 . The recombinant bi-functional fusion protein according to  claim 1 , wherein the peptide is linked to the N-terminus of the heavy chain of the antibody. 
     
     
         11 . The recombinant bi-functional fusion protein according to  claim 1 , wherein the peptide is linked to the C-terminus of the heavy chain of the antibody. 
     
     
         12 . The method according to  claim 7 , wherein the tumor is selected from the group consisting of stomach cancer, liver cancer, leukemia, kidney tumor, lung cancer, small intestinal cancer, bone cancer, prostatic cancer, colorectal cancer, breast cancer, large intestinal cancer, prostatic cancer, cervical cancer, adrenal cancer, and bladder cancer. 
     
     
         13 . A genetically engineered host cell having its genome integrated with the polynucleotide according to  claim 2 . 
     
     
         14 . The recombinant bi-functional fusion protein according to  claim 1 , wherein the peptide specifically binding to CD47 comprises the sequence of SEQ ID NO: 9.