Recombinant fusion proteins targeting CD47 and PD-L1, preparation and use thereof
The present application provides a recombinant fusion protein containing an anti-PD-L1 antibody or an antibody fragment thereof, with each paratope of the anti-PD-L1 antibody or antibody fragment thereof linked via a linker to an extracellular Ig-like domain of a signal-regulatory protein (SIRP) at N-terminus of a heavy chain variable region or a light chain variable region constituting the paratope, wherein the recombinant fusion protein can bind to CD47, PD-L1 and FcR simultaneously. The present application also provides a nucleic acid molecule encoding the recombinant fusion protein, an expression vector containing the nucleic acid molecule, a method for producing the recombinant fusion protein and a method for treating a disease associated with over-expression of CD47 and/or PD-L1 using the recombinant fusion protein.
1. A recombinant fusion protein, comprising an anti-PD-L1 antibody or an antigen binding fragment thereof, and a CD47 binding peptide,
wherein the anti-PD-L1 antibody or antibody fragment thereof is linked to the CD47 binding peptide at the N-terminus of a heavy chain variable region or the a chain variable region,
wherein each of the anti-PD-L1 antibody or the antigen binding fragment thereof comprises SEQ ID NO: 19 and SEQ ID NO: 20,
wherein the CD47 binding peptide comprises a signal-regulatory protein (SIRP) extracellular domain comprising SEQ ID NO: 2,
wherein the recombinant fusion protein is capable of binding to CD47 and PD-L1 simultaneously.
2. The recombinant fusion protein of claim 1 , wherein the anti-PD-L1 antibody or antibody fragment thereof is linked to the CD47 binding peptide at the N-terminus of the heavy chain variable region.
3. The recombinant fusion protein of claim 1 , wherein the anti-PD-L1 antibody or antibody fragment thereof is linked to the CD47 binding peptide at N-terminus of the light chain variable region.
4. The recombinant fusion protein of claim 1 , wherein the anti-PD-L1 antibody or antibody fragment thereof is linked to the CD47 binding peptide via a linker.
5. The recombinant fusion protein of claim 4 , wherein the linker is SEQ ID NO: 4.
6. The recombinant fusion protein of claim 2 , comprising SEQ ID NO: 10.
7. The recombinant fusion protein of claim 2 , further comprising SEQ ID NO: 8.
8. The recombinant fusion protein of claim 3 , further comprising a light chain constant region linked to the light chain variable region, wherein the recombinant fusion protein comprises an anti-PD-L1 heavy chain variable region-heavy chain constant region fragment comprising SEQ ID NO: 6, and a CD47binding peptide-linker-anti-PD-L1 light chain variable region-light chain constant region fragment having an amino acid sequence comprising SEQ ID NO: 16.
9. The recombinant fusion protein of claim 1 , comprising a heavy chain constant region comprising SEQ ID NO: 21.
10. An isolated nucleic acid molecule encoding the recombinant fusion protein of claim 1 .
11. An isolated expression vector comprising the nucleic acid molecule of claim 10 .
12. An isolated host cell comprising the expression vector of claim 11 .
13. A pharmaceutical composition, comprising the recombinant fusion protein of claim 11 , and at least one pharmaceutically acceptable excipient.
14. The pharmaceutical composition of claim 13 , further comprising at least one pharmaceutically acceptable adjuvant.
15. A method for treating a disease associated with target cells that are double positive for CD47 and PD-L1 in a subject in need thereof, wherein the disease is non-Hodgkin's lymphoma (NHL) or colon cancer, comprising administering to the subject the pharmaceutical composition of claim 13 .