Bispecific EGFR/CD16 antigen-binding protein
Described are tetravalent, bispecific EGFR/CD16A antigen-binding proteins for engaging NK-cells towards EGFR-positive cells. EGFR/CD16A antigen-binding proteins with different pharmacokinetic (PK) properties are described. Further described is the use of bispecific EGFR/CD16A antigen-binding proteins for the treatment of an EGFR-positive malignancy, such as EGFR-positive tumors.
1. A multispecific antigen-binding protein comprising antigen-binding sites for EGFR and CD16A, wherein the antigen-binding sites are fused to a polypeptide comprising heavy chain constant domains CH2 and CH3 and wherein the antigen-binding site for EGFR comprises a heavy chain variable domain (VH) and a light chain variable domain (VL) wherein:
(i) VH comprises a heavy chain CDR1 having the amino acid sequence set forth in SEQ ID NO:21; a heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO:22; a heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO:23 and VL comprises a light chain CDR1 having an amino acid sequence set forth in SEQ ID NO:24; a light chain CDR2 having an amino acid sequence set forth in SEQ ID NO:25; and a light chain CDR3 having an amino acid sequence set forth in SEQ ID NO:26; or
(ii) VH has the amino acid sequence set forth in SEQ ID NO:1 and VL has the amino acid sequence set forth in SEQ ID NO:2.
2. The multispecific antigen binding protein of claim 1 , wherein the antigen binding site for CD16A comprises
(i) a variable heavy chain domain (VH) comprising a heavy chain CDR1 having the amino acid sequence set forth in SEQ ID NO:5; a heavy chain CDR2 having the amino acid sequence set forth in SEQ ID NO:6 or 11; a heavy chain CDR3 having the amino acid sequence set forth in SEQ ID NO:7, and a variable light chain domain (VL) comprising a light chain CDR1 having an amino acid sequence set forth in SEQ ID NO:8; a light chain CDR2 having an amino acid sequence set forth in SEQ ID NO:9; and a light chain CDR3 having an amino acid sequence set forth in SEQ ID NO:10; or
(ii) a VH comprising the amino acid sequence set forth in SEQ ID NO:12 or 14 and a VL comprising the amino acid sequence set forth in SEQ ID NO:13.
3. The multispecific antigen-binding protein of claim 1 , wherein the antigen-binding protein comprises at least two antigen-binding sites for EGFR and at least two antigen-binding sites for CD16A.
4. The multispecific antigen-binding protein of claim 3 , wherein the antigen-binding protein consists of two polypeptide chains and each polypeptide chain comprises a first single-chain Fv unit (scFv1) comprising a VL linked by a peptide linker to a VH of a first antigen-binding site, said first single-chain Fv unit (scFv1) being fused by a hinge region N-terminally to a CH2 domain of the CH2-CH3 polypeptide and a second single-chain Fv unit (scFv2) comprising a VL linked by a peptide linker to a VH of a second antigen-binding site, said second single-chain Fv unit (scFv2) being fused by a peptide linker C-terminally to the CH3 domain of the CH2-CH3 polypeptide.
5. The multispecific antigen-binding protein of claim 4 , wherein the scFv1 is an antigen-binding site for CD16A and the scFv2 is an antigen-binding site for EGFR.
6. The multispecific antigen-binding protein of claim 3 , wherein the antibody comprises a F(ab′)2 fragment and a Fc-portion, wherein:
(i) the F(ab′)2 fragment comprises two Fv antigen-binding sites for CD16A and two single-chain Fv (scFv) comprising antigen-binding sites for EGFR are fused to the Fc-portion, wherein each of the scFv is fused C-terminally to the CH3 domain of the Fc-portion; or
(ii) the F(ab′)2 fragment comprises two Fv antigen-binding sites for EGFR and two single-chain Fv (scFv) comprising antigen-binding sites for CD16A are fused to the Fc-portion, wherein each of the scFv is fused C-terminally to the CH3 domain of the Fc-portion.
7. The multispecific antigen-binding protein of claim 6 comprising a heavy chain and a light chain, wherein (i) the heavy chain has the structure VH(CD16A)-CH1-CH2-CH3-VH(EGFR)-VL(EGFR) and the light chain has the structure VL(CD16A)-CL or (ii) the heavy chain has the structure VH(EGFR)-CH1-CH2-CH3-VL(CD16A)-VH(CD16A) and the light chain has the structure VL(EGFR)-CL.
8. The multispecific antigen-binding protein of claim 1 , wherein the antigen-binding protein does not bind to Fc-gamma receptor, but binds to neonatal Fc-receptor.
9. The multispecific antigen-binding protein of claim 1 , wherein the protein comprises a heavy chain having the amino acid sequence set forth in SEQ ID NO:28 and a light chain having the amino acid sequence set forth in SEQ ID NO:29; a heavy chain having the amino acid sequence set forth in SEQ ID NO:43 and a light chain having the amino acid sequence set forth in SEQ ID NO:44; or a heavy chain having the amino acid sequence set forth in SEQ ID NO:45 and a light chain having the amino acid sequence set forth in SEQ ID NO:44.
10. The multispecific antigen-binding protein of claim 1 , further comprising
serum albumin fused to the antigen-binding protein.
11. The multispecific antigen-binding protein of claim 1 for use as a therapeutic compound.
12. The multispecific antigen-binding protein of claim 11 , for use in the treatment of a cancer characterized by EGFR-positive or EGFRvIII-positive cells.
13. The multispecific antigen-binding protein for the use according to claim 12 , wherein the cancer is selected from the group consisting of colorectal cancer, head and neck cancer, lung cancer and glioblastoma.
14. A method for the treatment or amelioration of a proliferative disease or a tumorous disease, the method comprising the step of administering to a subject in need thereof the multispecific antigen-binding protein of claim 1 .
15. The method of claim 14 , wherein the proliferative disease or tumorous disease is characterized by EGFR-positive or EGFRvIII-positive cells.
16. The method of claim 15 , wherein the proliferative disease or tumorous disease is selected from the group consisting of colorectal cancer, head and neck cancer, lung cancer and glioblastoma.