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 . (canceled)
2 . An isolated heteromultimer comprising a heterodimer Fc region, wherein the heterodimer Fc region comprises a first Fc polypeptide and a second Fc polypeptide and a variant CH3 domain comprising amino acid modifications to promote heterodimer formation, wherein:
(i) the first Fc polypeptide comprises amino acid modifications at positions F405 and Y407, wherein the amino acid modification at position F405 is F405A, F405S, F405T or F405V, and the amino acid modification at position Y407 is Y407A, Y407I, Y407L or Y407V, and
(ii) the second Fc polypeptide comprises amino acid modifications at positions T366 and T394, wherein the amino acid modification at position T366 is T366I, T366L, T366M or T366V, and the amino acid modification at position T394 is T394W,
wherein the heterodimer Fc region further comprises a variant CH2 domain comprising asymmetric amino acid modifications to promote selective binding of a Fcgamma receptor,
wherein the heterodimer Fc region is based on an IgG Fc region,
and wherein the numbering of amino acid residues is according to the EU index as set forth in Kabat.
3 . The isolated heteromultimer according to claim 2 , wherein the variant CH2 domain selectively binds FcgammaIIIa receptor as compared to a wild-type CH2 domain.
4 . The isolated heteromultimer according to claim 2 , wherein the variant CH3 domain has a melting temperature (Tm) of about 70° C. or greater.
5 .- 7 . (canceled)
8 . The isolated heteromultimer according to claim 2 , wherein the heterodimer Fc region has a purity greater than about 90%.
9 . The isolated heteromultimer according to claim 2 , wherein the heterodimer Fc region has a purity of about 95% or greater.
10 .- 11 . (canceled)
12 . The isolated heteromultimer according to claim 2 , wherein the variant CH3 domain has a Tm of about 74° C. or greater.
13 .- 34 . (canceled)
35 . The isolated heteromultimer according to claim 2 , wherein the amino acid modification at position F405 is F405A.
36 . The isolated heteromultimer according to claim 2 , wherein the second Fc polypeptide further comprises an amino acid modification at position K392, and wherein the amino acid modification at position K392 is K392V, K392M, K392L or K392F.
37 .- 40 . (canceled)
41 . The isolated heteromultimer according to claim 2 , wherein the first Fc polypeptide further comprises the amino acid modification L351Y.
42 . The isolated heteromultimer according to claim 2 , wherein the amino acid modification at position T366 is T366L or T366I.
43 . The isolated heteromultimer according to claim 2 , wherein the amino acid modification at position Y407 is Y407V.
44 . (canceled)
45 . The isolated heteromultimer according to claim 2 , wherein the first Fc polypeptide comprises the amino acid modifications F405A and Y407V, and the second Fc polypeptide comprises the amino acid modifications T366L and T394W.
46 . The isolated heteromultimer according to claim 2 , wherein the first Fc polypeptide comprises the amino acid modifications F405A and Y407V, and the second Fc polypeptide comprises the amino acid modifications T366I and T394W.
47 .- 53 . (canceled)
54 . The isolated heteromultimer according to claim 2 , wherein the first Fc polypeptide further comprises an amino acid modification at position S400, wherein the amino acid modification at position S400 is S400E or S400D.
55 . (canceled)
56 . The isolated heteromultimer according to claim 2 , wherein the second Fc polypeptide further comprises the amino acid modification N390R.
57 . The isolated heteromultimer according to claim 2 , wherein the second Fc polypeptide further comprises an amino acid modification at position K392 selected from K392M and K392L.
58 .- 77 . (canceled)
78 . The isolated heteromultimer according to claim 57 , wherein the first Fc polypeptide comprises the amino acid modifications F405A and Y407V and the second Fc polypeptide comprises the amino acid modifications T366I, K392M and T394W.
79 . The isolated heteromultimer according to claim 57 , wherein the first Fc polypeptide comprises the amino acid modifications F405A and Y407V and the second Fc polypeptide comprises the amino acid modifications T366L, K392M and T394W.
80 . The isolated heteromultimer according to claim 57 , wherein the first Fc polypeptide comprises the amino acid modifications F405A and Y407V and the second Fc polypeptide comprises the amino acid modifications T366L, K392L and T394W.
81 . The isolated heteromultimer according to claim 57 , wherein the first Fc polypeptide comprises the amino acid modifications F405A and Y407V and the second Fc polypeptide comprises the amino acid modifications T366I, K392L and T394W.
82 . The isolated heteromultimer according to claim 2 , wherein the heteromultimer is a bispecific antibody.
83 . The isolated heteromultimer according to claim 2 , wherein the heteromultimer is a multispecific antibody.
84 . A composition comprising the isolated heteromultimer according to claim 2 , and a pharmaceutically acceptable carrier.
85 . A host cell comprising nucleic acid encoding the isolated heteromultimer according to claim 2 .
86 . The isolated heteromultimer according claim 2 , wherein the heteromultimer is a therapeutic antibody.
87 . (canceled)
88 . A method of treating cancer in a patient having a cancer characterized by a cancer antigen, said method comprising administering to said patient a therapeutically effective amount of a heteromultimer of claim 86 .
89 . A method of treating immune disorders in a patient having an immune disorder characterized by an immune antigen, said method comprising administering to said patient a therapeutically effective amount of a heteromultimer of claim 86 .
90 . (canceled)
91 . Nucleic acid encoding the isolated heteromultimer according to claim 2 .
92 . The isolated heteromultimer according to claim 2 , wherein:
(a) the first Fc polypeptide comprises the amino acid modifications L351Y, F405A and Y407V and the second Fc polypeptide comprises the amino acid modifications T366L, K392L and T394W;
(b) the first Fc polypeptide comprises the amino acid modifications L351Y, F405A and Y407V and the second Fc polypeptide comprises the amino acid modifications T366L, K392M and T394W, or
(c) the first Fc polypeptide comprises the amino acid modifications L351Y, S400E, F405A and Y407V and the second Fc polypeptide comprises the amino acid modifications T366L, N390R, K392M and T394W.
93 . The isolated heteromultimer according to claim 2 , wherein the variant CH2 domain comprises one or more asymmetric amino acid modifications selected from: S239D, D265S, S267D, E269K, S298A, K326E, A330L and I332E.
94 . The isolated heteromultimer according to claim 93 , wherein the one or more asymmetric amino acid modifications are selected from: S239D, K326E, A330L and I332E.
95 . The isolated heteromultimer according to claim 93 , wherein the one or more asymmetric amino acid modifications are selected from: S239D, D265S, E269K and I332E.
96 . The isolated heteromultimer according to claim 93 , wherein the one or more asymmetric amino acid modifications are selected from: S239D, D265S and S298A.
97 . The isolated heteromultimer according to claim 93 , wherein the one or more asymmetric amino acid modifications are selected from: S239D, S298A, K326E, A330L and I332E.
98 . The isolated heteromultimer according to claim 93 , wherein the one or more asymmetric amino acid modifications are selected from: S239D, D265S, S298A and I332E.
99 . The isolated heteromultimer according to claim 2 , wherein the heterodimer Fc region is based on an IgG1 Fc region.
100 . The isolated heteromultimer according to claim 2 , wherein the heterodimer Fc region is based on a human IgG1 Fc region.