Bispecific antibodies against plasma kallikrein and Factor XII
Described herein are bispecific antibodies that bind to plasma kallikrein (pKal) and Factor XII and methods of producing and using such bi-specific antibodies for treating diseases or disorders associated with the contact system, e.g., hereditary angioedema or thrombosis.
1. A bispecific antibody, comprising:
a first polypeptide that comprises a light chain of a first antibody, the light chain comprising a light chain variable region (V L ) and a light chain constant region (C L ); and
a second polypeptide that comprises a heavy chain of the first antibody, the heavy chain comprising a heavy chain variable region (V H ) and a heavy chain constant region (C H ), wherein the first polypeptide or the second polypeptide further comprises a second antibody, which is a single chain antibody and is fused to the C-terminus of either the first polypeptide or the second polypeptide;
wherein the first antibody binds active plasma kallikrein (pKal) and comprises a heavy chain complementarity determining region (HCDR) 1 set forth as HYIMM (SEQ ID NO: 159), a HCDR2 set forth as GIYSSGGITVYADSVKG (SEQ ID NO: 160), and a HCDR3 set forth as RRIGVPRRDEFDI (SEQ ID NO: 161); and a light chain complementarity determining region (LCDR) 1 set forth as RASQSISSWLA (SEQ ID NO: 162), a LCDR2 set forth as KASTLES (SEQ ID NO: 163), and a LCDR 3 set forth as QQYNTYWT (SEQ ID NO: 164); and
wherein the second antibody binds active Factor XII (FXIIa) and comprises
(i) a heavy chain comprising a HCDR1 set forth as SEQ ID NO: 41, a HCDR2 set forth as SEQ ID NO: 43, and a HCDR3 set forth as SEQ ID NO: 45, and a light chain comprising a LCDR1 set forth as SEQ ID NO: 34, a LCDR2 set forth as SEQ ID NO: 36, and a LCDR3 set forth as SEQ ID NO: 37;
(ii) a heavy chain comprising a HCDR1 set forth as SEQ ID NO: 41, a HCDR2 set forth as SEQ ID NO: 43, and a HCDR3 set forth as SEQ ID NO: 45, and a light chain comprising a LCDR1 set forth as SEQ ID NO: 35, a LCDR2 set forth as SEQ ID NO: 36, and a LCDR3 set forth as SEQ ID NO: 38;
(iii) a heavy chain comprising a HCDR1 set forth as SEQ ID NO: 41, a HCDR2 set forth as SEQ ID NO: 43, and a HCDR3 set forth as SEQ ID NO: 45, and a light chain comprising a LCDR1 set forth as SEQ ID NO: 34, a LCDR2 set forth as SEQ ID NO: 36, and a LCDR3 set forth as SEQ ID NO: 39;
(iv) a heavy chain comprising a HCDR1 set forth as SEQ ID NO: 41, a HCDR2 set forth as SEQ ID NO: 43, and a HCDR3 set forth as SEQ ID NO: 45, and a light chain comprising a LCDR1 set forth as SEQ ID NO: 34, a LCDR2 set forth as SEQ ID NO: 36, and a LCDR3 set forth as SEQ ID NO: 40;
(v) a heavy chain comprising a HCDR1 set forth as SEQ ID NO: 42, a HCDR2 set forth as SEQ ID NO: 44, and a HCDR3 set forth as SEQ ID NO: 45, and a light chain comprising a LCDR1 set forth as SEQ ID NO: 34, a LCDR2 set forth as SEQ ID NO: 36, and a LCDR3 set forth as SEQ ID NO: 37;
(vi) a heavy chain comprising a HCDR1 set forth as SEQ ID NO: 132, a HCDR2 set forth as SEQ ID NO: 133, and a HCDR3 set forth as SEQ ID NO: 134, and a light chain comprising a LCDR1 set forth as SEQ ID NO: 131, a LCDR2 set forth as SEQ ID NO: 36, and a LCDR3 set forth as SEQ ID NO: 37;
(vii) a heavy chain comprising a HCDR1 set forth as SEQ ID NO: 135, a HCDR2 set forth as SEQ ID NO: 136, and a HCDR3 set forth as SEQ ID NO: 134, and a light chain comprising a LCDR1 set forth as SEQ ID NO: 131, a LCDR2 set forth as SEQ ID NO: 36, and a LCDR3 set forth as SEQ ID NO: 37;
(viii) a heavy chain comprising a HCDR1 set forth as SEQ ID NO: 137, a HCDR2 set forth as SEQ ID NO: 138, and a HCDR3 set forth as SEQ ID NO: 134, and a light chain comprising a LCDR1 set forth as SEQ ID NO: 131, a LCDR2 set forth as SEQ ID NO: 36, and a LCDR3 set forth as SEQ ID NO: 37; or
(ix) a heavy chain comprising a HCDR1 set forth as SEQ ID NO: 139, a HCDR2 set forth as SEQ ID NO: 140, and a HCDR3 set forth as SEQ ID NO: 134, and a light chain comprising a LCDR1 set forth as SEQ ID NO: 131, a LCDR2 set forth as SEQ ID NO: 36, and a LCDR3 set forth as SEQ ID NO: 37.
2. The bispecific antibody of claim 1 , wherein the second antibody is fused to the C-terminus of the second polypeptide.
3. The bispecific antibody of claim 1 , wherein the first antibody is an IgG.
4. The bispecific antibody of claim 3 , wherein the IgG comprises a mutated heavy chain, which, as compared with the wild-type counterpart, has the C-terminal lysine residue deleted or mutated.
5. The bispecific antibody of claim 4 , wherein the heavy chain of the first antibody contains a C-terminal glycine residue, instead of a lysine residue.
6. The bispecific antibody of claim 1 , wherein the second antibody does not contain a KR motif at its C-terminus.
7. The bispecific antibody of claim 1 , wherein the bispecific antibody is tetravalent.
8. The bispecific antibody of claim 2 , wherein the second polypeptide comprises a peptide linker between the heavy chain of the first antibody and the second antibody.
9. The bispecific antibody of claim 1 , wherein the C L is a kappa light chain or a lambda light chain.
10. The bispecific antibody of claim 1 , wherein in the second antibody, the V H is fused to the N-terminus of the V L or fused to the C-terminus of the V L .
11. The bispecific antibody of claim 8 , wherein the second antibody comprises a peptide linker between the V H and V L regions.
12. The bispecific antibody of claim 1 , wherein a disulfide bond is formed between the V H chain and the V L chain in the second antibody.
13. The bispecific antibody of claim 12 , wherein the disulfide bond is formed between a cysteine residue at an amino acid corresponding to position 44 in the V H chain of SEQ ID NO: 123 and a cysteine residue introduced at an amino acid corresponding to position 100 in the V L chain of SEQ ID NO: 127, wherein the sequence of the bispecific antibody comprises any of SEQ ID NOs: 47-50, 123-126, 141-148, and 151-158, wherein the amino acid position is based on Chothia numbering.
14. The bispecific antibody of claim 1 , wherein the V H of the first antibody comprises the amino acid sequence of SEQ ID NO:1; and wherein the V L of the first antibody comprises the amino acid sequence of SEQ ID NO:2.
15. The bispecific antibody of claim 14 , wherein the heavy chain of the first antibody comprises the amino acid sequence of residues 20-470 of SEQ ID NO: 9.
16. The bispecific antibody of claim 15 , wherein the heavy chain of the first antibody comprises the amino acid sequence of SEQ ID NO:9, 149, or 150.
17. The bispecific antibody of claim 1 , wherein the V H of the second antibody comprises any one of the amino acid sequences of SEQ ID NOs:3, 4, and 123-126; and wherein the V L of the second antibody comprises residues 1-111 of any one of the amino acid sequences of SEQ ID NOs:5-8 and 127.
18. The bispecific antibody of claim 1 , wherein the first polypeptide comprises the amino acid sequence of SEQ ID NO:10 and the second polypeptide comprises the amino acid sequence selected from the group consisting of SEQ ID NOs: 11-20, 47-122, 141-148, and 151-158.
19. An isolated nucleic acid or nucleic acid set, comprising a first nucleotide sequence encoding the first polypeptide and a second nucleotide sequence encoding the second polypeptide, wherein the first polypeptide and the second polypeptide are set forth in claim 1 .
20. A pharmaceutical composition comprising (i) a bispecific antibody of claim 1 , and (ii) a pharmaceutically acceptable carrier.
21. A host cell, comprising the isolated nucleic acid or nucleic acid set of claim 19 , wherein the first and second nucleotide sequences are located on at least one nucleic acid molecule, optionally wherein:
(i) a first vector comprises the first nucleotide sequence and the second nucleotide sequence; or
(ii) a first vector comprises the first nucleotide sequence and a second vector comprises the second nucleotide sequence.
22. A method for preparing a bispecific antibody, comprising:
culturing the host cell of claim 21 under conditions allowing for expression of the first polypeptide and the second polypeptide; and
isolating the bispecific antibody that comprises the first polypeptide and the second polypeptide.
23. A bispecific antibody, which comprises a first antibody binding to active plasma kallikrein (pKal) and a second antibody binding to active Factor FXII (FXIIa), wherein
the first antibody comprises a heavy chain complementarity determining region (HCDR) 1 set forth as HYIMM (SEQ ID NO: 159), a HCDR2 set forth as GIYSSGGITVYADSVKG (SEQ ID NO: 160), and a HCDR3 set forth as RRIGVPRRDEFDI (SEQ ID NO: 161); and a light chain complementarity determining region (LCDR) 1 set forth as RASQSISSWLA (SEQ ID NO: 162), a LCDR2 set forth as KASTLES (SEQ ID NO: 163), and a LCDR 3 set forth as QQYNTYWT (SEQ ID NO: 164); and
wherein the second antibody comprises
(i) a HCDR1 set forth as SEQ ID NO: 41, a HCDR2 set forth as SEQ ID NO: 43, and a HCDR3 set forth as SEQ ID NO: 45, and a LCDR1 set forth as SEQ ID NO: 34, a LCDR2 set forth as SEQ ID NO: 36, and a LCDR3 set forth as SEQ ID NO: 37;
(ii) a HCDR1 set forth as SEQ ID NO: 41, a HCDR2 set forth as SEQ ID NO: 43, and a HCDR3 set forth as SEQ ID NO: 45, and a LCDR1 set forth as SEQ ID NO: 35, a LCDR2 set forth as SEQ ID NO: 36, and a LCDR3 set forth as SEQ ID NO: 38;
(iii) a HCDR1 set forth as SEQ ID NO: 41, a HCDR2 set forth as SEQ ID NO: 43, and a HCDR3 set forth as SEQ ID NO: 45, and a LCDR1 set forth as SEQ ID NO: 34, a LCDR2 set forth as SEQ ID NO: 36, and a LCDR3 set forth as SEQ ID NO: 39;
(iv) a HCDR1 set forth as SEQ ID NO: 41, a HCDR2 set forth as SEQ ID NO: 43, and a HCDR3 set forth as SEQ ID NO: 45, and a LCDR1 set forth as SEQ ID NO: 34, a LCDR2 set forth as SEQ ID NO: 36, and a LCDR3 set forth as SEQ ID NO: 40;
(v) a HCDR1 set forth as SEQ ID NO: 42, a HCDR2 set forth as SEQ ID NO: 44, and a HCDR3 set forth as SEQ ID NO: 45, and a LCDR1 set forth as SEQ ID NO: 34, a LCDR2 set forth as SEQ ID NO: 36, and a LCDR3 set forth as SEQ ID NO: 37;
(vi) a HCDR1 set forth as SEQ ID NO: 132, a HCDR2 set forth as SEQ ID NO: 133, and a HCDR3 set forth as SEQ ID NO: 134, and a LCDR1 set forth as SEQ ID NO: 131, a LCDR2 set forth as SEQ ID NO: 36, and a LCDR3 set forth as SEQ ID NO: 37;
(vii) a HCDR1 set forth as SEQ ID NO: 135, a HCDR2 set forth as SEQ ID NO: 136, and a HCDR3 set forth as SEQ ID NO: 134, and a LCDR1 set forth as SEQ ID NO: 131, a LCDR2 set forth as SEQ ID NO: 36, and a LCDR3 set forth as SEQ ID NO: 37;
(viii) a HCDR1 set forth as SEQ ID NO: 137, a HCDR2 set forth as SEQ ID NO: 138, and a HCDR3 set forth as SEQ ID NO: 134, and a LCDR1 set forth as SEQ ID NO: 131, a LCDR2 set forth as SEQ ID NO: 36, and a LCDR3 set forth as SEQ ID NO: 37; or
(ix) a HCDR1 set forth as SEQ ID NO: 139, a HCDR2 set forth as SEQ ID NO: 140, and a HCDR3 set forth as SEQ ID NO: 134, and a LCDR1 set forth as SEQ ID NO: 131, a LCDR2 set forth as SEQ ID NO: 36, and a LCDR3 set forth as SEQ ID NO: 37.
24. The bispecific antibody of claim 23 , wherein the second antibody comprises a V H chain that comprises any one of the amino acid sequences of SEQ ID NOs:3, 4, and 123-126, and a V L chain that comprises any one of the amino acid sequences of SEQ ID NOs:5-8 and 127.
25. The bispecific antibody of claim 23 , wherein the V H of the first antibody comprises the amino acid sequence of SEQ ID NO:1; and wherein the V L of the first antibody comprises the amino acid sequence of SEQ ID NO:2.
26. A pharmaceutical composition comprising (i) a bispecific antibody of claim 23 , and (ii) a pharmaceutically acceptable carrier.