Stable heterodimeric antibody design with mutations in the Fc domain
The provided scaffolds have heavy chains that are asymmetric in the various domains (e.g. CH2 and CH3) to accomplish selectivity between the various Fc receptors involved in modulating effector function, beyond those achievable with a natural homodimeric (symmetric) Fc molecule, and increased stability and purity of the resulting variant Fc heterodimers. These novel molecules comprise complexes of heterogeneous components designed to alter the natural way antibodies behave and that find use in therapeutics.
1. An isolated heteromultimer comprising a heterodimeric CH3 domain comprising a first CH3 domain polypeptide and a second CH3 domain polypeptide, the first CH3 domain polypeptide comprising amino acid modifications at positions F405 and Y407, and the second CH3 domain polypeptide comprising amino acid modifications at positions T366 and T394, wherein:
(i) the first CH3 domain polypeptide further comprises an amino acid modification at position L351, and
(ii) the second CH3 domain polypeptide further comprises an amino acid modification at position K392,
wherein the amino acid modification at position F405 is F405A, F405T, F405S or F405V; and the amino acid modification at position Y407 is Y407V, Y407A, Y407L or Y407I; the amino acid modification at position T394 is T394W; the amino acid modification at position L351 is L351Y; the amino acid modification at position K392 is K392L, K392M, K392V or K392F, and the amino acid modification at position T366 is T366I, T366L, T366M or T366V,
wherein the heterodimeric CH3 domain has a melting temperature (Tm) of about 70° C. or greater and a purity greater than about 90%, and
wherein the numbering of amino acid residues is according to the EU index as set forth in Kabat.
2. The isolated heteromultimer according to claim 1 , wherein the amino acid modification at position F405 is F405A.
3. The isolated heteromultimer according to claim 1 , wherein the amino acid modification at position T366 is T366I or T366L.
4. The isolated heteromultimer to claim 1 , wherein the amino acid modification at position Y407 is Y407V.
5. The isolated heteromultimer according to claim 1 , wherein the amino acid modification at position F405 is F405A, the amino acid modification at position Y407 is Y407V, the amino acid modification at position T366 is T366I or T366L, and the amino acid modification at position K392 is K392L or K392M.
6. The isolated heteromultimer according to claim 1 , wherein the amino acid modification at position F405 is F405A, the amino acid modification at position Y407 is Y407V, the amino acid modification at position T366 is T366L, and the amino acid modification at position K392 is K392M.
7. The isolated heteromultimer according to claim 1 , wherein the amino acid modification at position F405 is F405A, the amino acid modification at position Y407 is Y407V, the amino acid modification at position T366 is T366L, and the amino acid modification at position K392 is K392L.
8. The isolated heteromultimer according to claim 1 , wherein the amino acid modification at position F405 is F405A, the amino acid modification at position Y407 is Y407V, the amino acid modification at position T366 is T366I, and the amino acid modification at position K392 is K392M.
9. The isolated heteromultimer according to claim 1 , wherein the amino acid modification at position F405 is F405A, the amino acid modification at position Y407 is Y407V, the amino acid modification at position T366 is T366I, and the amino acid modification at position K392 is K392L.
10. The isolated heteromultimer according to claim 1 , wherein the heterodimeric CH3 domain is comprised by an Fc region based on an IgG Fc region.
11. The isolated heteromultimer according to claim 10 , wherein the IgG Fc region is an IgG1 Fc region.
12. The isolated heteromultimer according to claim 1 , wherein the heteromultimer is a bispecific or multispecific antibody.
13. The isolated heteromultimer according to claim 12 , wherein the bispecific or multispecific antibody binds at least one cancer antigen.
14. The isolated heteromultimer according to claim 13 , wherein the bispecific or multispecific antibody is conjugated to a therapeutic agent.
15. The isolated heteromultimer according to claim 1 , wherein the first CH3 domain polypeptide further comprises an amino acid modification at position S400 selected from S400D and S400E.
16. The isolated heteromultimer according to claim 1 , wherein the second CH3 domain polypeptide further comprises the amino acid modification N390R.
17. The isolated heteromultimer according to claim 1 , wherein the first CH3 domain polypeptide further comprises an amino acid modification at position S400 selected from S400D and S400E, and the second CH3 domain polypeptide further comprises the amino acid modification N390R.
18. The isolated heteromultimer according to claim 17 , wherein the amino acid modification at position F405 is F405A, the amino acid modification at position Y407 is Y405V, the amino acid modification at position S400 is S400E, the amino acid modification at position T366 is T366L, and the amino acid modification at position K392 is K392M.
19. An isolated heteromultimer comprising a heterodimeric CH3 domain comprising:
a first CH3 domain polypeptide comprising amino acid modifications L351Y, F405A and Y407V, and
a second CH3 domain polypeptide comprising amino acid modifications T366L, K392M and T394W.
20. An isolated heteromultimer comprising a heterodimeric CH3 domain comprising:
a first CH3 domain polypeptide comprising amino acid modifications L351Y, F405A and Y407V, and
a second CH3 domain polypeptide comprising amino acid modifications T366L, K392L and T394W.
21. An isolated heteromultimer comprising a heterodimeric CH3 domain comprising:
a first CH3 domain polypeptide comprising amino acid modifications L351Y, S400E, F405A and Y407V, and
a second CH3 domain polypeptide comprising amino acid modifications T366L, N390R, K392M and T394W.
22. An isolated heteromultimer comprising a heterodimeric CH3 domain comprising:
a first CH3 domain polypeptide comprising amino acid modifications L351Y, S400E, F405A and Y407V, and
a second CH3 domain polypeptide comprising amino acid modifications T366L, N390R, K392L and T394W.
23. The isolated heteromultimer according to claim 19 , wherein the heterodimeric CH3 domain is comprised by an Fc region based on an IgG1 Fc region.
24. The isolated heteromultimer according to claim 23 , wherein the heteromultimer is a bispecific antibody.
25. The isolated heteromultimer according to claim 20 , wherein the heterodimeric CH3 domain is comprised by an Fc region based on an IgG1 Fc region.
26. The isolated heteromultimer according to claim 25 , wherein the heteromultimer is a bispecific antibody.
27. The isolated heteromultimer according to claim 21 , wherein the heterodimeric CH3 domain is comprised by an Fc region based on an IgG1 Fc region.
28. The isolated heteromultimer according to claim 27 , wherein the heteromultimer is a bispecific antibody.
29. The isolated heteromultimer according to claim 22 , wherein the heterodimeric CH3 domain is comprised by an Fc region based on an IgG1 Fc region.
30. The isolated heteromultimer according to claim 29 , wherein the heteromultimer is a bispecific antibody.