Coiled coil and/or tether containing protein complexes and uses thereof
The invention provides engineered protein complexes constructed using a coiled coil and/or a tether and methods for making, using, and purifying such complexes, such as multispecific antibodies or other multispecific Fc containing complexes.
1. An antibody comprising:
(a) a first polypeptide comprising a VH domain linked to a constant domain and a first coiled coil domain (CC), wherein the first CC comprises a heptad repeat of Formula I:
(X 1 X 2 X 3 X 4 X 5 X 6 X 7 ) n (Formula I)(SEQ ID NO:29)
X 1 is a hydrophobic amino acid residue or Asparagine,
X 2 , X 3 , and X 6 are each any amino acid residue,
X 4 is a hydrophobic amino acid residue, and
X 5 and X 7 are each a charged amino acid residue; and
(b) a second polypeptide comprising a VH domain linked to a constant domain and a second coiled coil domain (CC), wherein the second CC comprises a heptad repeat of Formula II:
(X′ 1 X′ 2 X′ 3 X′ 4 X′ 5 X′ 6 X′ 7 ) n (Formula II) (SEQ ID NO:30)
X′ 1 is a hydrophobic amino acid residue or Asparagine,
X′ 2 , X′ 3 , and X′ 6 are each any amino acid residue,
X′ 4 is a hydrophobic amino acid residue, and
X′ 5 and X′ 7 are each a charged amino acid residue;
wherein n in Formula I and II is greater than or equal to 2; and
wherein, in each heptad repeat, the first CC comprises an X 5 residue that is opposite in charge to the X′ 7 residue in the second CC and the first CC comprises an X 7 residue that is opposite in charge to the X′ 5 residue in the second CC;
wherein the constant domain of a) and the constant domain of b) comprise a CH2 domain and an IgG1 CH3 domain;
wherein the CC is linked to the C-terminal of the constant domain by a linker and wherein the linker is cleavable.
2. The antibody of claim 1 , wherein the first and second polypeptides each comprise a VH and a CH1 domain.
3. The antibody of claim 2 , wherein the first and second polypeptides each further comprise a hinge domain.
4. The antibody of claim 1 , wherein the first and second polypeptides each comprise VH, CH1, hinge, CH2, and CH3 domains positioned relative to each other in an N-terminal to C-terminal direction: VH-CH1-hinge-CH2-CH3.
5. The antibody of claim 1 , wherein said antibody further comprises a third and a fourth polypeptide, wherein said third polypeptide comprises a first VL domain and said fourth polypeptide comprises a second VL domain.
6. The antibody of claim 5 , wherein said VH domain of the first polypeptide is linked to the VL domain of the third polypeptide by a tether and the VH domain of the second polypeptide is linked to the VL domain of the fourth polypeptide by a tether.
7. The antibody of claim 5 , wherein the third polypeptide further comprises a first CL domain wherein said first VL and CL domains are positioned relative to each other within the third polypeptide in an N-terminal to C-terminal direction: VL-CL, and the fourth polypeptide further comprises a second CL domain, and wherein said second VL and CL domains are positioned relative to each other within the fourth polypeptide in an N-terminal to C-terminal direction: VL-CL.
8. The antibody of claim 5 , wherein the sequences of said first VL domain and said second VL domain are the same.
9. The antibody of claim 1 , wherein the N-terminus of the VH of at least one of said first or said second polypeptides is connected to the C-terminus of a CL with a tether.
10. An antibody comprising:
(a) a first polypeptide comprising a VH domain linked to a constant domain comprising a CH2 and an IgG1 CH3 domain and a first coiled coil domain (CC), wherein the first CC comprises a heptad repeat of Formula I:
(X 1 X 2 X 3 X 4 X 5 X 6 X 7 ) n (Formula I) (SEQ ID NO:29)
X 1 is a hydrophobic amino acid residue or Asparagine,
X 2 , X 3 , and X 6 are each any amino acid residue,
X 4 is a hydrophobic amino acid residue, and
X 5 and X 7 are each a charged amino acid residue; and
(b) a second polypeptide comprising a constant domain and a second coiled coil (CC), wherein the constant domain comprises a CH2 and an IgG1 CH3 domain and wherein the second CC comprises a heptad repeat of Formula II:
(X′ 1 X′ 2 X′ 3 X′ 4 X′ 5 X′ 6 X′ 7 ) n (Formula II) (SEQ ID NO:30)
X′ 1 is a hydrophobic amino acid residue or Asparagine,
X′ 2 , X′ 3 , and X′ 6 are each any amino acid residue,
X′ 4 is a hydrophobic amino acid residue, and
X′ 5 and X′ 7 are each a charged amino acid residue;
wherein n in Formula I and II is greater than or equal to 2; and
wherein, in each heptad repeat, the first CC comprises an X 5 residue that is opposite in charge to the X′ 7 residue in the second CC and the first CC comprises an X 7 residue that is opposite in charge to the X′ 5 residue in the second CC;
wherein the CC is linked to the C-terminal of the constant domain by a linker wherein the linker is cleavable.
11. The antibody of claim 10 , wherein the first polypeptide comprises a VH and CH1 domain.
12. The antibody of claim 11 , wherein the first polypeptide further comprises a hinge domain.
13. The antibody of claim 10 , wherein the first polypeptide comprises VH, CH1, hinge, CH2, and CH3 domains positioned relative to each other in an N-terminal to C-terminal direction: VH-CH1-hinge-CH2-CH3.
14. The antibody of claim 10 , wherein the antibody further comprises a third polypeptide, wherein the third polypeptide comprises a VL domain.
15. The antibody of claim 14 , wherein said third polypeptide further comprises a CL domain, and the VL and CL domains are positioned relative to each other in an N-terminal to C-terminal direction: VL-CL.
16. The antibody of claim 10 , wherein the N-terminus of the VH of said first polypeptide is connected to the C-terminus of a CL with a tether.
17. The antibody of claim 1 , wherein said hydrophobic amino acid residue in any of X 1 , X′ 1 , X 4 , and X′ 4 is selected from the group consisting of Alanine, Valine, Leucine, Isoleucine, Tryptophan, Phenylalanine, and Methionine.
18. The antibody of claim 1 , wherein said charged amino acid residue in any of X 5 , X′ 5 , X 7 , and X′ 7 is selected from the group consisting of Lysine, Arginine, Histidine, Aspartic Acid, and Glutamic Acid.
19. The antibody of claim 1 , wherein, in at least one heptad repeat of said first CC, X 1 is Asparagine, and wherein the respective X′ 1 is Asparagine in at least one heptad repeat of said second CC.
20. The antibody of claim 1 , wherein
(a) the first CC comprises a heptad repeat wherein
X 1 is Leucine or Asparagine,
X 2 is Alanine or Glutamine,
X 3 is Alanine or Glutamine,
X 4 is Leucine,
X 5 is Glutamic Acid,
X 6 is Lysine or Tryptophan, and
X 7 is Glutamic Acid; and
(b) the second CC comprises a heptad repeat wherein
X′ 1 is Leucine or Asparagine,
X′ 2 is Alanine or Glutamine,
X′ 3 is Alanine or Glutamine,
X′ 4 is Leucine,
X′ 5 is Lysine,
X′ 6 is Lysine or Tryptophan, and
X′ 7 is Lysine.
21. The antibody of claim 1 , wherein n is greater than or equal to 3.
22. The antibody of claim 21 , wherein n is greater than or equal to 4.
23. The antibody of claim 1 , wherein the first CC is linked C-terminal to a CH3 domain of the first polypeptide and the second CC is linked C-terminal to a CH3 domain of the second polypeptide.
24. The antibody of claim 1 , wherein a Lys-C endopeptidase cleavage site is located N-terminal to at least one of said first or said second CC.
25. The antibody of claim 1 , wherein the antibody is multispecific.
26. The antibody of claim 25 , wherein the antibody is capable of binding at least 2 antigens.
27. The antibody of claim 25 , wherein the antibody a capable of binding at least 2 epitopes on the same antigen.
28. The antibody of claim 1 , wherein said antibody is bispecific.
29. The antibody of claim 6 , wherein said tether comprises Glycine (G) and Serine (S) residues.
30. The antibody of claim 6 , wherein said tether is between 15 and 50 amino acids in length.
31. The antibody of claim 30 , wherein said tether is between 20 and 26 amino acids in length.
32. The antibody of claim 6 , wherein said tether comprises GGS repeats.
33. The antibody of claim 6 , wherein said tether is cleavable.
34. The antibody of claim 24 , wherein said antibody comprises a mutation that removes a Lys-C endopeptidase cleavage site.
35. The antibody of claim 34 , wherein said mutation that removes a Lys-C endopeptidase cleavage site is in a hinge domain.
36. The antibody of claim 35 , wherein said antibody has a K222A substitution (EU numbering system).
37. The antibody of claim 1 , wherein said linker is cleavable by one or more of the following endopeptidases: Furin, Thrombin, Genenase, Lys-C, Arg-C, Asp-N, Glu-C, Factor Xa, Tobacco Etch Virus Protease (TEV), Enterokinase, Human Rhinovirus C3 protease (HRV C3), or Kininogenase.
38. The antibody of claim 1 , wherein said linker comprises an Asparagine-Glycine peptide bond.
39. The antibody of claim 38 , wherein said Asparagine-Glycine peptide bond is cleavable by hydroxylamine.
40. The antibody of claim 1 , wherein said antibody comprises a constant region conjugated to a cytotoxic agent.
41. The antibody of claim 1 , wherein said antibody is expressed by a mammalian cell.
42. The antibody of claim 41 , wherein said mammalian cell is a CHO cell.
43. The antibody of claim 1 , wherein said antibody is expressed by a prokaryotic cell.
44. The antibody of claim 43 , wherein said prokaryotic cell is an E. coli cell.
45. A method of producing an antibody, said method comprising the step of culturing a cell comprising a vector encoding the antibody of claim 1 in a culture medium.
46. The method of claim 45 , wherein said method further comprises recovering said antibody from said cell or said culture medium.
47. The method of claim 46 , further comprising the steps of
(a) capturing said antibody on a column comprising Protein A,
(b) eluting said antibody from said column, and
(c) diluting said eluted antibody into a solution containing a chaotropic agent or mild detergent.
48. The method of claim 47 , wherein said chaotropic agent or mild detergent is Arginine, Guanidine-HC1, urea, lithium perchlorate, Histidine, Sodium Dodecyl Sulfate (SDS), Tween, Triton, or NP-40.
49. The antibody of claim 33 , wherein said tether is cleavable by one or more of the following endopeptidases: Furin, Thrombin, Genenase, Lys-C, Arg-C, Asp-N, Glu-C, Factor Xa, Tobacco Etch Virus Protease (TEV), Enterokinase, Human Rhinovirus C3 protease (HRV C3), or Kininogenase.
50. The antibody of claim 33 , wherein said tether comprises an Asparagine-Glycine peptide bond.