IMMUNOGLOBULIN Fc REGION VARIANTS COMPRISING STABILITY-ENHANCING MUTATIONS
Fc variants are described comprising one or more amino acid mutations that increase the stability of the Fc variant as compared to a parental Fc that does not include the one or more amino acid mutations, as well as polypeptides comprising an Fc variant and polynucleotides encoding an Fc variant.
1 .- 4 . (canceled)
5 . An Fc variant comprising from one to three stability-enhancing amino acid mutations, the mutations comprising:
(a) one or more mutation selected from: a mutation at position 287 which is a substitution with Phe, His, Met, Trp or Tyr; a mutation at position 308 which is a substitution with Ile, and a mutation at position 309 which is a substitution with Gln or Thr, or
(b) two or more mutations selected from: a mutation at position 250 which is a substitution with Ala, Ile or Val; a mutation at position 287 which is a substitution with Phe, His, Met, Trp or Tyr; a mutation at position 308 which is a substitution with Ile; a mutation at position 309 which is a substitution with Gln or Thr; a mutation at position 428 which is a substitution with Phe, and a pair of mutations at position 242 and position 336 which are both substitutions with Cys, or
(c) three or more mutations comprising: a pair of mutations at position 242 and position 336 which are both substitutions with Cys, and a mutation selected from: a mutation at position 250 which is a substitution with Ala, Ile or Val; a mutation at position 287 which is a substitution with Phe, His, Met, Trp or Tyr; a mutation at position 308 which is a substitution with Ile; a mutation at position 309 which is a substitution with Gln or Thr, and a mutation at position 428 which is a substitution with Phe,
wherein the Fc variant has an increased CH2 domain melting temperature (Tm) as compared to a parental Fc that does not include the one or more stability-enhancing amino acid mutations, and
wherein the numbering of amino acids is according to the EU index.
6 . The Fc variant according to claim 5 comprising a mutation at position 287 which is a substitution with Phe, His, Met, Trp or Tyr.
7 . The Fc variant according to claim 6 , wherein the mutation at position 287 is a substitution with Phe.
8 . The Fc variant according to claim 5 comprising a mutation at position 308 which is a substitution with Ile.
9 . The Fc variant according to claim 5 comprising a mutation at position 309 which is a substitution with Gln or Thr.
10 . The Fc variant according to claim 9 , wherein the mutation at position 309 is a substitution with Gln.
11 . The Fc variant according to claim 5 comprising a mutation at position 250 which is a substitution with Ala, Ile or Val, and a mutation at position 287 which is a substitution with Phe, His, Met, Trp or Tyr.
12 . The Fc variant according to claim 11 , wherein the mutation at position 250 is a substitution with Val.
13 . The Fc variant according to claim 12 , wherein the mutation at position 287 is a substitution with Phe.
14 . The Fc variant according to claim 5 comprising a mutation at position 250 which is a substitution with Ala, Ile or Val, and a mutation at position 309 which is a substitution with Gln or Thr.
15 . The Fc variant according to claim 14 , wherein the mutation at position 250 is a substitution with Val.
16 . The Fc variant according to claim 15 , wherein the mutation at position 309 is a substitution with Gln.
17 . The Fc variant according to claim 5 comprising a mutation at position 250 which is a substitution with Ala, Ile or Val, and a mutation at position 428 which is a substitution with Phe.
18 . The Fc variant according to claim 17 , wherein the mutation at position 250 is a substitution with Val.
19 . The Fc variant according to claim 5 comprising a mutation at position 287 which is a substitution with Phe, His, Met, Trp or Tyr, and a mutation at position 428 which is a substitution with Phe.
20 . The Fc variant according to claim 19 , wherein the mutation at position 287 is a substitution with Phe.
21 . The Fc variant according to claim 5 comprising a pair of mutations at position 242 and position 336 which are both substitutions with Cys.
22 . The Fc variant according to claim 5 comprising a pair of mutations at position 242 and position 336 which are both substitutions with Cys, and a mutation at position 308 which is a substitution with Ile.
23 . The Fc variant according to claim 5 , wherein the stability-enhancing mutations comprised by the Fc variant are selected from: 250V, 287F, 308I, 309Q, 428F, 242C_336C, 287F/428F, 250V/287F, 250V/309Q, 250V/428F and 242C_336C/308I.
24 . The Fc variant according to claim 5 , wherein the stability-enhancing mutations comprised by the Fc variant are selected from: 287F/428F, 250V/287F, 250V/309Q, 250V/428F and 242C_336C/308I.
25 . The Fc variant according to claim 5 , wherein the Fc variant is based on an IgG, IgA, IgD, IgE or IgM Fc.
26 . The Fc variant according to claim 25 , wherein the Fc variant is based on a human IgG, IgA, IgD, IgE or IgM Fc.
27 . The Fc variant according to claim 5 , wherein the Fc variant is based on an IgG Fc.
28 . The Fc variant according to claim 27 , wherein the IgG Fc is an IgG1 Fc.
29 . The Fc variant according to claim 27 , wherein the IgG Fc is a human IgG Fc.
30 . The Fc variant according to claim 5 , wherein the parental Fc comprises one or more amino acid mutations that improve a function of the Fc region.
31 . The Fc variant according to claim 5 , wherein the parental Fc comprises one or more amino acid mutations that improve a function of the Fc region and decrease the CH2 domain Tm of the corresponding wild-type Fc.
32 . The Fc variant according to claim 5 , wherein the CH2 domain Tm of the Fc variant is increased by at least 0.5° C. as compared to the parental Fc.
33 . The Fc variant according to claim 32 , wherein the CH2 domain Tm of the Fc variant is increased by at least 1.0° C., at least 2.0° C., or at least 3.0° C., as compared to the parental Fc.
34 . The Fc variant according to claim 5 , wherein the CH2 domain Tm of the Fc variant is increased by between 0.5° C. and 9.0° C. as compared to the parental Fc.
35 . The Fc variant according to claim 5 , wherein the CH2 domain Tm of the Fc variant is increased by between 2.0° C. and 10.5° C. as compared to the parental Fc.
36 . A polypeptide comprising the Fc variant according to claim 5 and one or more proteinaceous moieties fused or covalently attached to the Fc variant.
37 . The polypeptide according to claim 36 , wherein the one or more proteinaceous moieties comprise an antigen-binding domain, a ligand, a receptor, a receptor fragment, a cytokine or an antigen.
38 . The polypeptide according to claim 37 , wherein at least one of the one or more proteinaceous moieties is an antigen-binding domain.
39 . The polypeptide according to claim 37 , wherein the antigen-binding domain is a Fab or scFv.
40 . The polypeptide according to claim 36 , wherein the polypeptide is an antibody or an antigen-binding antibody fragment.
41 . The polypeptide according to claim 40 , wherein the polypeptide is a therapeutic antibody or antibody fragment.
42 . A polynucleotide or set of polynucleotides encoding the Fc variant according to claim 5 .
43 . A polynucleotide or set of polynucleotides encoding the polypeptide according to claim 36 .
44 . A vector or set of vectors comprising one or more polynucleotides encoding the polypeptide according to claim 36 .
45 . A host cell comprising one or more polynucleotides encoding the polypeptide according to claim 36 .
46 . (canceled)
47 . A method of preparing the polypeptide according to claim 36 comprising transfecting a host cell with one or more polynucleotides encoding the polypeptide, and culturing the host cell under conditions suitable for expression of the polypeptide.
48 . A pharmaceutical composition comprising the polypeptide according to claim 36 .
49 .- 52 . (canceled)
53 . A method of increasing the CH2 domain melting temperature (Tm) of an Fc comprising introducing into a parental Fc one to three stability-enhancing amino acid mutations to provide an Fc variant having an increased CH2 domain Tm as compared to the parental Fc, the mutations comprising:
(a) one or more mutation selected from: a mutation at position 287 which is a substitution with Phe, His, Met, Trp or Tyr; a mutation at position 308 which is a substitution with Ile, and a mutation at position 309 which is a substitution with Gln or Thr, or
(b) two or more mutations selected from: a mutation at position 250 which is a substitution with Ala, Ile or Val; a mutation at position 287 which is a substitution with Phe, His, Met, Trp or Tyr; a mutation at position 308 which is a substitution with Ile; a mutation at position 309 which is a substitution with Gln or Thr; a mutation at position 428 which is a substitution with Phe, and a pair of mutations at position 242 and position 336 which are both substitutions with Cys, or
(c) three or more mutations comprising: a pair of mutations at position 242 and position 336 which are both substitutions with Cys, and a mutation selected from: a mutation at position 250 which is a substitution with Ala, Ile or Val; a mutation at position 287 which is a substitution with Phe, His, Met, Trp or Tyr; a mutation at position 308 which is a substitution with Ile; a mutation at position 309 which is a substitution with Gln or Thr, and a mutation at position 428 which is a substitution with Phe,
wherein the numbering of amino acids is according to the EU index.
54 . The method according to claim 53 , wherein the CH2 domain Tm of the Fc variant is increased by at least 0.5° C. as compared to the parental Fc.
55 . The method according to claim 54 , wherein the CH2 domain Tm of the Fc variant is increased by at least 1.0° C., at least 2.0° C., or at least 3.0° C., as compared to the parental Fc.
56 . The method according to claim 53 , wherein the CH2 domain Tm of the Fc variant is increased by between 0.5° C. and 9.0° C. as compared to the parental Fc.
57 . The method according to claim 53 , wherein the CH2 domain Tm of the Fc variant is increased by between 2.0° C. and 10.5° C. as compared to the parental Fc.
58 . The method according to claim 53 , wherein introducing the stability-enhancing amino acid mutations into the parental Fc provides an Fc variant showing decreased aggregation under mildly acidic conditions as compared to the parental Fc region.
59 . The method according to claim 58 , wherein the stability-enhancing amino acid mutations comprise 250V and 287F.