IMMUNOGLOBULIN FC LIBRARIES

• US 20090136936A1
• Filed: 04/30/2008
• Published: 05/28/2009
• Est. Priority Date: 05/01/2007
• Status: Active Grant

• Current Assignee: Research & Development Foundation
• Original Assignee: Research & Development Foundation
• Inventors: Georgiou, George, Jung, Sang Taek
• Granted Patent: US 8,629,245 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• 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 16/32 against translation products of oncogenes
• CPC Class Codes: C07K 2317/52 Constant or Fc region; Isotype
• CPC Class Codes: C07K 2317/72 Increased effector function due to an Fc-modification
• CPC Class Codes: C07K 2319/035 containing a signal for targeting to the external surface of a cell, e.g. to the outer membrane of Gram negative bacteria, GPI- anchored eukaryote proteins
• CPC Class Codes: C07K 2319/30 Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
• CPC Class Codes: G01N 33/56911 Bacteria

Abstract

Methods and composition for the screening and isolation of aglycosylated antibody Fc domain polypeptides. For example, in certain aspects methods for identifying aglycosylated Fc domains that bind to Fc receptors or preferentially bind to particular Fc receptors are described. Furthermore, the invention provides aglycosylated Fc domains that bind to Fc receptors with high affinity. Enhanced methods and media for prokaryotic based interaction screening are also provided.

Claims

1. A method of selecting a bacterial cell comprising an aglycosylated antibody Fc domain having specific affinity for an Fc receptor (FcR) polypeptide comprising the steps of:

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

b) contacting the bacterial cells with an FcR polypeptide under conditions wherein the FcR polypeptide contacts the aglycosylated Fc domains; and

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

2. The method of claim 1, wherein the bacterial cells are E. coli cells.

3. The method of claim 1, wherein the Fc domain is an IgG, IgA or IgE Fc domain.

4. The method of claim 3, wherein the IgG Fc domain is an IgG1 Fc domain.

5. The method of claim 4, wherein the IgG1 Fc domain is the Fc domain of an anti-HER2 antibody.

6. The method of claim 5, wherein the IgG1 Fc domain is the Fc domain of the Fc domain of trastuzumab.

7. The method of claim 1, wherein the population of Gram negative bacterial cells comprise a plurality of nucleic acids encoding said plurality of aglycosylated Fc domains.

8. The method of claim 7, wherein the plurality of nucleic acids further encodes a membrane secretion signal fused to said plurality of aglycosylated Fc domains.

9. The method of claim 8, wherein the membrane secretion signal is PelB.

10. The method of claim 8, wherein the membrane secretion signal is DsbA.

11. The method of claim 1, wherein the aglycosylated Fc domain comprises a hinge, CH2 and CH3 region.

12. The method of claim 1, wherein the FcR polypeptide comprises an eukaryotic FcR domain.

13. The method of claim 1, wherein the FcR polypeptide comprises a prokaryotic or synthetic FcR domain.

14. The method of claim 12, wherein the FcR polypeptide comprises an antibody-binding domain from one of the polypeptides of Table 1.

15. The method of claim 14, wherein the FcR polypeptide comprises an antibody-binding domain from human Fcγ

RIa, Fcγ

RIIa, Fcγ

RIIb, Fcγ

RIIc, Fcγ

RIIIa, Fcγ

RIIIb, Fcα

RI or C1q.

16. The method of claim 15, wherein the FcR polypeptide comprises an antibody-binding domain from human Fcγ

RIa.

17. The method of claim 1, wherein the FcR polypeptide is labeled.

18. The method of claim 17, wherein the FcR polypeptide is labeled with a fluorophore, a radioisotope or an enzyme.

19. The method of claim 18, wherein the FcR polypeptide is labeled with a fluorophore.

20. The method of claim 17, wherein the FcR polypeptide is fused to a fluorescence protein or an enzyme.

21. The method of claim 20, wherein the FcR polypeptide is fused to a green fluorescence protein GFP.

22. The method of claim 1, wherein the FcR polypeptide is immobilized.

23. The method of claim 1, wherein the selecting of step (c) is further defined as comprising at least two rounds of selection wherein the sub-population of bacterial cells obtained in the first round of selection is subjected to at least a second round of selection based on the binding of the Fc polypeptide to FcR polypeptide.

24. The method of claim 23, comprising two to ten rounds of selection.

25. The method of claim 24, wherein the selecting is carried out by FACS or magnetic separation.

26. The method of claim 1, further comprising contacting the bacterial cells with at least two FcR polypeptides.

27. The method of claim 26, wherein the at least two FcR polypeptides comprise distinct labels.

28. The method of claim 27, further comprising selecting bacterial cells based on binding of the aglycosylated Fc domain to the at least two FcR polypeptides.

29. The method of claim 27, further comprising selecting bacterial cells based on binding of the aglycosylated Fc domain to at least one FcR polypeptide and based on the aglycosylated Fc domain not binding to at least one other FcR polypeptide.

30. The method of claim 1, further comprising disrupting the outer membrane of the bacterial cells before contacting the bacterial cells with an FcR polypeptide.

31. The method of claim 30, wherein disrupting the outer membrane of the bacterial cell comprises treatment with hyperosmotic conditions, treatment with physical stress, infecting the bacterium with a phage, treatment with lysozyme, treatment with EDTA, treatment with a digestive enzyme or treatment with a chemical that disrupts the outer membrane.

32. The method of claim 31, wherein disrupting the outer membrane of the bacterial cell comprises a combination of said methods.

33. The method of claim 30, wherein disrupting the outer membrane of the bacterial cell comprises heating the bacterial cell with a combination of physical, chemical and enzyme disruption of the outer membrane.

34. The method of claim 30, wherein disrupting the outer membrane of the bacterial cell further comprises removing the outer membrane of said bacterium.

35. The method of claim 1, further comprising removing FcR polypeptide not bound to the aglycosylated Fc domain.

36. The method of claim 1, wherein the bacteria are grown in a media comprising sucrose, sorbitol, mannitol or trehalose.

37. The method of claim 1, wherein the bacteria are grown in a media comprising trehalose.

38. The method of claim 1, further defined as a method of producing a nucleic acid sequence encoding an antibody Fc polypeptide having a specific affinity for an FcR polypeptide and further comprising the step of cloning a nucleic acid sequence encoding the Fc polypeptide from the bacterial cell to produce a nucleic acid sequence encoding an antibody Fc polypeptide having a specific affinity for an FcR polypeptide.

39. The method of claim 38, wherein cloning comprises amplification of the nucleic acid sequence.

40. The method of claim 38, further defined as a method of producing an antibody Fc polypeptide having a specific affinity for an FcR polypeptide further comprising the step of expressing a nucleic acid sequence encoding the antibody Fc polypeptide to produce an antibody Fc polypeptide having a specific affinity for an FcR polypeptide.

51. A polypeptide comprising an aglycosylated antibody Fc domain capable of binding an FcR polypeptide, wherein the Fc domain comprises an amino acid substitution or insertion identified from a bacterial cell selected in accordance with claim 1.

67. An isolated DNA segment encoding a i) polypeptide in accordance with claim 51, ii) a polypeptide comprising an aglycosylated antibody Fc domain capable of binding an FcR polypeptide, wherein the Fc domain comprises an amino acid substitution at amino acid 331, 382 and/or 428 of the Fc domain, or iii) a polypeptide comprising an aglycosylated antibody Fc domain capable of binding an FcR polypeptide, wherein the Fc domain comprises an amino acid insertion between amino acids N297 and S298.

41. A method of identifying a bacteria cell comprising a first binding partner associated with an inner membrane comprised in the bacteria cell, wherein the binding partner having specific affinity for a second binding partner, comprising the steps of:

a) obtaining a population of bacteria cells, cells of which population comprise said first binding partner associated with the inner membrane in the periplasm of the bacteria cells, wherein the population comprises a plurality of different said first binding partners;

b) contacting the bacteria cells with said second binding partner, wherein said first binding partner or said second binding partner comprises a label, wherein a signal is elicited when said first binding partner binds to said second binding partner; and

c) selecting at least one bacterial cell by detecting such a signal from at least one said first binding partner binding to at least one said second binding partner, wherein the bacteria cells are grown in a media comprising trehalose.

42-50. -50. (canceled)

52. A polypeptide comprising an aglycosylated antibody Fc domain capable of binding an FcR polypeptide, wherein the Fc domain comprises an amino acid substitution at amino acid 331, 382 and/or 428 of the Fc domain.

53-62. -62. (canceled)

63. A polypeptide comprising an aglycosylated antibody Fc domain capable of binding an FcR polypeptide, wherein the Fc domain comprises an amino acid insertion between amino acids N297 and S298.

64-66. -66. (canceled)

68-69. -69. (canceled)