IMMUNOGLOBULIN FC POLYPEPTIDES

• US 20100330076A1
• Filed: 06/30/2010
• Published: 12/30/2010
• Est. Priority Date: 06/30/2009
• Status: Active Grant

• Current Assignee: Research & Development Foundation
• Original Assignee: Research & Development Foundation
• Inventors: Reddy, Sai, Jung, Sang Taek, Georgiou, George
• Granted Patent: US 8,679,493 B2
• Time in Patent Office: Days
• US Class Current: 424/131.1
• CPC Class Codes: A61P 37/04 Immunostimulants
• CPC Class Codes: C07K 16/00 Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies antibodies with enzymatic activity, e.g. abzymes C12N9/0002
• CPC Class Codes: C07K 16/005 constructed by phage libraries
• CPC Class Codes: C07K 16/32 against translation products of oncogenes
• CPC Class Codes: C07K 2317/24 containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
• CPC Class Codes: C07K 2317/41 Glycosylation, sialylation, or fucosylation
• CPC Class Codes: C07K 2317/72 Increased effector function due to an Fc-modification
• CPC Class Codes: C07K 2317/732 Antibody-dependent cellular cytotoxicity [ADCC]
• CPC Class Codes: C07K 2317/75 Agonist effect on antigen
• CPC Class Codes: C07K 2317/76 Antagonist effect on antigen, e.g. neutralization or inhibition of binding
• CPC Class Codes: G01N 2500/10 involving cells
• CPC Class Codes: G01N 33/56911 Bacteria
• CPC Class Codes: G01N 33/6857 Antibody fragments

Abstract

Methods and compositions involving polypeptides having an aglycosylated antibody Fc domain. In certain embodiments, polypeptides have an aglycosylated Fc domain that contains one or more substitutions compared to a native Fc domain. Additionally, some embodiments involve an Fc domain that is binds some Fc receptors but not others. For example, polypeptides are provided with an aglycosylated Fe domain that selectively binds FcγRI at a level within 2-fold of a glycosylated Fc domain, but that is significantly reduced for binding to other Fc receptors. Furthermore, methods and compositions are provided for promoting antibody-dependent cell-mediated toxicity (ADCC) using a polypeptide having a modified aglycosylated Fc domain and a second non-Fc binding domain, which can be an antigen binding region of an antibody or a non-antigen binding region. Some embodiments concern antibodies with such polypeptides, which may have the same or different non-Fc binding domain.

Claims

1. A polypeptide comprising an aglycosylated antibody Fc domain capable of binding a human FcR polypeptide, wherein the Fc domain comprises an amino acid substitution at amino acids 382 and 428 and at least one additional substitution of any of the following amino acids:

224, 241, 251, 266, 269, 276, 279, 286, 295, 297, 300, 315, 325, 328, 330, 331, 332, 338, 340, 341, 348, 369, 378, 382, 392, 424, 426, 428 and/or 434.

2. The polypeptide of claim 1, wherein the Fc domain is capable of binding a human Fcγ

RI polypeptide.

3. The polypeptide of claim 1, wherein at least one additional substitution is of an amino acid in the upper CH2 region.

4. The polypeptide of claim 3, wherein at least one additional substitution is of an amino acid in the following part of the upper CH2 region:

234L-239S;

264V-268H;

297N-299T;

or 328L-332I.

5. The polypeptide of claim 1, wherein the substitution at amino acid 382 is a valine (E382V).

6. The polypeptide of claim 1, wherein the substitution at amino acid 428 is an isoleucine (M428I).

7. The polypeptide of claim 6, wherein the substitution at amino acid 382 is a valine (E382V) and the substitution at amino acid 428 is an isoleucine (M428I).

8. The polypeptide of claim 1, wherein the substitution at amino acid 328 is a tryptophan (L328W).

9. The polypeptide of claim 1, wherein the substitution at amino acid 332 is a tyrosine (I332Y).

10. The polypeptide of claim 1 having another substitution at least at amino acids 328 and 332.

11. The polypeptide of claim 10, wherein the substitution at amino acid 328 is a tryptophan (L328W) and the substitution at amino acid 332 is a tyrosine (I332Y).

12. The polypeptide of claim 1 having another substitution at amino acid 341.

13. The polypeptide of claim 12, wherein the substitution at amino acid 341 is a valine (G341V).

14. The polypeptide of claim 1, wherein the at least one additional substitution is:

H224R/Y, F241L, K251F, V266M, E269K, N276D, V279M, N286D, Q295R, N297D, Y300C, N315D, N325S, L328W, A330V/E/I, P331A/S/E, I332Y, K338I/R, K340N/Q, G341V, V348M, V369A, A378D, K392E, S424L, S4261, or N434S/D.

15. The polypeptide of claim 1, wherein polypeptide has at least a 2-fold reduction in pH-dependent FcRn binding compared to polypeptide with an aglycosylated wild-type antibody Fc domain.

16. The polypeptide of claim 14, wherein the polypeptide does not have the additional substitution of G341V.

17. The polypeptide of claim 14, wherein the polypeptide does not have the additional substitution of K338R.

18. The polypeptide of claim 17, wherein the polypeptide does not have the additional substitutions of K338R and G341V.

19. The polypeptide of claim 14, wherein the Fc domain has a E382V and M428I substitution and an additional substitution of G341V and/or K340N/Q.

20. The polypeptide of claim 19, wherein the Fc domain has an additional substitution of G341V.

21. The polypeptide of claim 20, wherein the Fc domain has at least one other substitution selected from the group consisting of:

H224Y, F241L, E269K, N276D, N286D, Y300C, N325S, K338R, V348M, V369A, K392E, S424L, and N434D/S.

22. The polypeptide of claim 21, wherein the Fc domain has multiple other substitution selected from the group.

23. The polypeptide of claim 21, wherein the Fc domain comprises a K338R substitution.

24. The polypeptide of claim 14, wherein the Fc domain comprises a substitution set selected from the group consisting of a) K338R and G341V;

b) N297D, N315D, and K340N, c) K340N, d) K338I and K340N, e) K340Q and A378D;

f) N325S and K340N, g) H224Y, E269K, N325S, and G341V, h) G341V and K392E, i) K338R, G341V, S424L and N434D, j) F241L and G341V, k) G341V,

     1) N276D and G341V, m) G341V and V369A, n) N286D, G341V and N434S, o) N325S and G341V, p) Y300C and G341V, q) G341V and V348M, r) E382V and M428I, s) V266M;

t) A330V, P331A and Q295R, u) A330E, P331E and V279M, v) A330E and P331E, w) A330E, P331V and S426T, x) A330E and P331V, y) A330I and P331E, z) A330E, aa) P331S, and bb)A330V, P331S, H224R and L251F

25. The polypeptide of claim 20, further comprising a conjugatable linker.

26. The polypeptide of claim 20 further comprising a non-FcR binding domain.

27. The polypeptide of claim 26, wherein the non-FcR binding domain is an antigen binding site of an antibody.

28. The polypeptide of claim 26, wherein the non-Fc binding region is not an antigen binding site of an antibody

29. The polypeptide of claim 28, wherein the non-Fc binding region binds a cell-surface protein.

30. The polypeptide of claim 28, wherein the cell-surface protein is a receptor.

31. The polypeptide of claim 30, wherein the receptor is a tyrosine kinase.

32. The polypeptide of claim 31, wherein the non-Fc binding region binds multiple tyrosine kinase receptors.

33. A nucleic acid encoding any of the polypeptides of claim 1.

36. A host cell comprising the nucleic acid of claims 33.

34. (canceled)

35. (canceled)

37-41. -41. (canceled)

42. A polypeptide comprising an aglycosylated Fc domain capable of binding an FcRγ

I polypeptide and a second binding domain, wherein the second binding domain is capable of binding a cell-surface molecule.

43-54. -54. (canceled)

55. A method for preparing an aglycosylated polypeptide comprising:

a) obtaining a host cell capable of expressing an aglycosylated antibody comprising an Fc domain capable of binding an FcR polypeptide, wherein the Fc domain comprises an amino acid substitution at amino acids 382 and 428 and at least one additional substitution of any of the following amino acids;

224, 241, 251, 266, 269, 276, 279, 286, 295, 297, 300, 315, 325, 328, 330, 331, 332, 338, 340, 341, 348, 369, 378, 382, 392, 424, 426, 428 and/or 434;

b) incubating the host cell in culture under conditions to promote expression of the aglycosylated antibody; and

,

c) purifying expressed antibody from the host cell.

56-60. -60. (canceled)

61. A method of inducing an immune response in a subject with an antibody comprising providing to the subject with the antibody, wherein the antibody is aglycosylated and comprises an Fc domain capable of binding an Fcγ

RI polypeptide, wherein the Fc domain comprises an amino acid substitution at amino acids 382 and 428 and at least one additional substitution of any of the following amino acids;

224, 241, 251, 266, 269, 276, 279, 286, 295, 297, 300, 315, 325, 328, 330, 331, 332, 338, 340, 341, 348, 369, 378, 382, 392, 424, 426, 428 and/or 434.

62-64. -64. (canceled)

65. A method of inducing dendritic cell—

(DC) mediated cell killing against a target cell expressing a targeted cell surface polypeptide comprising;

a) contacting the target cell with a polypeptide comprising a mutated and aglycosylated Fc domain capable of specifically binding at least an Fcγ

RI polypeptide and second binding domain that binds the targeted cell surface polypeptide, wherein the aglycosylated Fc domain specifically binds to an Fcγ

RIIB polypeptide at a level that is at least 50-fold less than a glycosylated Fc domain; and

b) exposing the target cell to dendritic cells under conditions that promote killing of the target cell.

66. (canceled)

67. (canceled)

68. A method for screening for an aglycosylated polypeptide having an Fc domain that binds a specific Fcγ

R polypeptides comprising;

a) obtaining a population of Gram negative bacterial cells, cells of which population express a aglycosylated polypeptide comprising an Fc domain in their periplasm, wherein the population expresses a plurality of different Fc domains;

b) contacting the bacterial cells with a first FcR polypeptide under conditions to allow contact between the Fcγ

R polypeptide and the aglycosylated Fc domains, wherein the Fcγ

R polypeptide is Fcγ

RIa, Fcγ

RIIa, Fcγ

RIIb, Fcγ

RIIc, Fcγ

RIIIa, Fcγ

RIIIb, or Fcα

RI;

c) selecting at least one bacterial cell based on binding of the aglycosylated Fc domain to the first FcR polypeptide.

69-115. -115. (canceled)

116. A method for optimizing Fc binding to one or more specific FcR polypeptides of an aglycosylated polypeptide having an Fc domain comprising:

a) obtaining a population of Gram negative bacterial cells, cells of which population express a aglycosylated polypeptide comprising an Fc domain in their periplasm, wherein the population expresses a plurality of different polypeptides expressing different mutated Fc domains;

b) contacting the bacterial cells with a first FcR polypeptide under conditions to allow contact between the FcR polypeptide and the aglycosylated Fc domains, wherein the FcR polypeptide is Fcγ

RIa, Fcγ

RIIa, Fcγ

RIIb, Fcγ

RIIc, Fcγ

RIIIa, Fcγ

RIIIb, or Fcα

RI;

c) selecting at least one bacterial cell based on binding of the aglycosylated Fc domain to the first FcR polypeptide.