FC POLYPEPTIDE VARIANTS HAVING AN INCREASED HALF-LIFE

1 . Variant of a parent polypeptide comprising an Fc fragment, the variant having an improved half-life relative to the parent polypeptide, and comprising:

At least one mutation of the Fc fragment increasing the sialylation of the Fc; and

At least one mutation of the Fc fragment increasing the binding of the Fc to FcRn.

2 . Variant according to claim 1 , comprising at least three mutations of the Fc fragment comprising:

A mutation A) of at least one amino acid chosen from amino acids in position 240, 241, 242, 243, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 290, 291 292, 293, 294, 295, 296, 298, 299, 300, 301, 302, 303, 304 or 305; and

A mutation B selected from the group consisting of 378V, 378T, 434Y and 434S; and

At least one C mutation selected from the group consisting of 226G, 228L, 228R, 230S, 230T, 230L, 241L, 264E, 307P, 315D, 330V, 362R, 378V, 378T, 389T, 389K, 434Y and 434S,

it being understood that:

mutations A, B and C do not take place on the same amino acid,

the amino acid position numbering of the Fc fragment being that of the EU index or equivalent in Kabat.

3 . Variant according to claim 2 , comprising:

i) a mutation A of at least one amino acid selected from amino acids in position 240, 241, 242, 243, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 290, 291 292, 293, 294, 295, 296, 298, 299, 300, 301, 302, 303, 304 or 305; and

ii) at least one combination of mutations selected from the group consisting of 226G/315D/434Y, 230S/315D/434Y, 230T/315D/434Y, 230T/264E/434S, 230T/389T/434S, 241L/264E/378V, 241L/264E/434S, 250A/389K/434Y, 259I/315D/434Y, 264E/378T/396L, 264E/378V/416K, 264E/378V/434S, 264E/396L/434S, 294del/307P/434Y, 307P/378V/434Y, 315D/330V/434Y, 315D/382V/434Y and 378V/383N/434Y, it being understood that mutation A can not take place on the same amino acid as one of the amino acids of mutation ii), and

that the amino acid position numbering of the Fc fragment is that of the EU index or equivalent in Kabat.

4 . Variant according to claim 2 , comprising:

i) a mutation A of at least one amino acid selected from amino acids in position 240, 241, 242, 243, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 290, 291, 292, 293, 294, 295, 296, 298, 299, 300, 301, 302, 303, 304 or 305; and

ii) at least one combination of mutations selected from the group consisting of 307A/315D/330V/382V/389T/434Y, 256N/378V/383N/434Y, 315D/330V/361D/378V/434Y, 259I/315D/434Y, 230S/315D/428L/434Y, 241L/264E/307P/378V/433R, 250A/389K/434Y, 305A/315D/330V/395A/434Y, 264E/386R/396L/434S/439R, 315D/330V/362R/434Y, 294del/307P/434Y, 305A/315D/330V/389K/434Y, 315D/327V/330V/397M/434Y, 230T/241L/264E/265G/378V/421T, 264E/396L/415N/434S, 227L/264E/378V/434S, 264E/378T/396L, 230T/315D/362R/426T/434Y, 226G/315D/330V/434Y, 230L/241L/243L/264E/307P/378V, 250A/315D/325S/330V/434Y, 290E/315D/342R/382V/434Y, 241L/315D/330V/392R/434Y, 241L/264E/307P/378V/434S, 230T/264E/403T/434S, 264E/378V/416K, 230T/315D/362E/434Y, 226G/315D/434Y, 226G/315D/362R/434Y, 226G/264E/347R/370R/378V/434S, 3081/315D/330V/382V/434Y, 230T/264E/378V/434S, 231T/241L/264E/378T/397M/434S, 230L/264E/378V/434S, 230T/315D/330V/386K/434Y, 226G/315D/330V/389T/434Y, 267R/307P/378V/421T/434Y, 230S/315D/387T/434Y, 230S/264E/352S/378V/434S and 230T/303A/322R/389T/404L/434S,

it being understood that mutation A can not take place on the same amino acid as one of the amino acids of mutation ii), and that the amino acid position numbering of the Fc fragment is that of the EU index or equivalent in Kabat.

5 . Variant according to claim 2 , comprising;

i) a mutation A of at least one amino acid selected from amino acids in position 240, 241, 242, 243, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 290, 291, 292, 293, 294, 295, 296, 298, 299, 300, 301, 302, 303, 304 or 305; and

ii) at least one combination of mutations selected from 3307A/315D/330V/382V/389T/434Y, 256N/378V/383N/434Y, 259I/315D/434Y, 230S/315D/428L/434Y, 294del/307P/434Y and 315D/330V/361D/378V/434Y, it being understood that mutation A can not take place on the same amino acid as one of the amino acids of mutation ii), and

that the amino acid position numbering of the Fc fragment is that of the EU index or equivalent in Kabat.

6 . Variant according to claim 2 , wherein the mutation A is del294 or 264E, it being understood that the amino acid position numbering of the Fc fragment is that of the EU index or equivalent in Kabat.

7 . Variant according to claim 2 , wherein the parent polypeptide consists of an Fc fragment.

8 . Variant according to claim 2 , wherein the parent polypeptide is an immunoglobulin or an antibody.

9 . Variant according to claim 2 , wherein the Fc fragment of the parent polypeptide is an Fc fragment of an IgG.

10 . Variant according to claim 2 , wherein the half-life of the variant is increased by a factor at least equal to 2 relative to the parent polypeptide.

11 . Method of increasing the half-life of a polypeptide comprising an Fc fragment, comprising the following steps:

i) insertion of a mutation A of at least one amino acid selected from amino acids in position 240, 241, 242, 243, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 290, 291, 292, 293, 294, 295, 296, 298, 299, 300, 301, 302, 303, 304 or 305;

ii) insertion of a mutation B selected from the group consisting of 378V, 378T, 434Y and 434S and insertion of at least one mutation C selected from the group consisting of 226G, 228L, 228R, 230S, 230T, 230L, 241L, 264E, 307P, 315D, 330V, 362R, 378V, 378T, 389T, 389K 434Y and 434S;

it being understood that:

the mutations are carried out on the Fc fragment of the polypeptide,

the mutations A, B and C do not take place on the same amino acid,

and the amino acid position numbering of the Fc fragment is that of the EU index or equivalent in Kabat.

12 . Method for producing a variant of a parent polypeptide comprising an Fc fragment, the variant having an improved half-life relative to the parent polypeptide, comprising the steps of:

a) providing a nucleic sequence encoding the parent polypeptide comprising the Fc fragment;

b) modifying the nucleic sequence provided in a) to obtain a nucleic sequence encoding the variant; and

c) expressing the nucleic sequence obtained in b) in a host cell YB2/0, and recovering the variant,

wherein step b) comprises:

i) insertion of a mutation A of at least one amino acid selected from amino acids in position 240, 241, 242, 243, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 290, 291, 292, 293, 294, 295, 296, 298, 299, 300, 301, 302, 303, 304 or 305;

ii) insertion of a mutation B selected from the group consisting of 378V, 378T, 434Y and 434S and

iii) insertion of at least one mutation C selected from the group consisting of 226G, 228L, 228R, 230S, 230T, 230L, 241L, 264E, 307P, 315D, 330V, 362R, 378T, 389T, 389K and 434S;

it being understood that:

the mutations are carried out on the Fc fragment of the polypeptide,

the mutations A, B and C do not take place on the same amino acid, and the amino acid position numbering of the Fc fragment is that of the EU index or equivalent in Kabat.

13 . Method for producing a variant of a parent polypeptide comprising an Fc fragment, the variant having an improved half-life relative to the parent polypeptide, comprising the steps of:

a) providing a nucleic sequence encoding the parent polypeptide comprising the Fc fragment;

b) modifying the nucleic sequence provided in a) to obtain a nucleic sequence encoding the variant; and

c) expressing the nucleic sequence obtained in b) in a host cell selected from modified transgenic animal cells to produce the polypeptide in the milk, and recovering the variant,

wherein step b) comprises:

i) insertion of a mutation A of at least one amino acid selected from amino acids in position 240, 241, 242, 243, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 290, 291, 292, 293, 294, 295, 296, 298, 299, 300, 301, 302, 303, 304 or 305;

ii) insertion of a mutation B selected from the group consisting of 378V, 378T, 434Y and 434S and iii) insertion of at least one mutation C selected from the group consisting of 226G, 228L, 228R, 230S, 230T, 230L, 241L, 264E, 307P, 315D, 330V, 362R, 378T, 389T, 389K and 434S;

it being understood that: that:

the mutations are carried out on the Fc fragment of the polypeptide,

the mutations A, B and C do not take place on the same amino acid, and the amino acid position numbering of the Fc fragment is that of the EU index or equivalent in Kabat.

14 . Method according to claim 11 , wherein the step i) is a deletion step of the amino acid in position 294 or a step of insertion of the mutation 264E.

15 . Method according to claim 11 , wherein the step ii) consists of the insertion of a combination of mutations selected from the group consisting of 226G/315D/434Y, 230S/315D/434Y, 230T/315D/434Y, 230T/264E/434S, 230T/389T/434S, 241L/264E/378V, 241L/264E/434S, 250A/389K/434Y, 259I/315D/434Y, 3781/396L, 378V/416K, 378V/434S, 396L/434S, 307P/434Y, 307P/378V/434Y, 315D/330V/434Y, 315D/382V/434Y and 378V/383N/434Y, the amino acid position numbering of the Fc fragment being that of the EU index or equivalent in Kabat.

16 . Method according to claim 14 , wherein the step ii) consists of inserting a combination of mutations selected from the group consisting of 307A/315D/330V/382V/389T/434Y, 256N/378V/383N/434Y, 315D/330V/361D/378V/434Y, 259I/315D/434Y, 230S/315D/428L/434Y, 294del/307P/434Y, the amino acid position numbering of the Fc fragment being that of the EU index or equivalent in Kabat.

17 . Method according to claim 14 , wherein the step i) is a deletion step of the amino acid in position 294, and the step ii) consists of the insertion of a combination of mutations 315D/330V/361D/378V/434Y.

18 . Method according to claim 14 , wherein the half-life of the polypeptide comprising an Fc fragment is increased by a factor of at least 2 with respect to the half-life of the polypeptide comprising an Fc fragment before the mutation steps of the method.

19 . Variant according to claim 2 , wherein the half-life of the variant is increased by a factor at least equal to 25 relative to the parent polypeptide.

20 . Method according to claim 14 , wherein the half-life of the polypeptide comprising an Fc fragment is increased by a factor of at least 30 with respect to the half-life of the polypeptide comprising an Fc fragment before the mutation steps of the method.