Polyalkylene oxide-modified single chain polypeptides

• US 20020061307A1
• Filed: 02/26/2001
• Published: 05/23/2002
• Est. Priority Date: 04/30/1997
• Status: Active Grant

• Current Assignee: Enzon Pharmaceuticals Inc. (Icahn Associates Holding LLC)
• Original Assignee: Enzon Pharmaceuticals Inc. (Icahn Associates Holding LLC)
• Inventors: Filpula, David R., Whitlow, Marc, Lee, Lihsyng Standford, Shorr, Robert G.L.
• Granted Patent: US 6,872,393 B2
• Time in Patent Office: Days
• US Class Current: 424/178.1
• CPC Class Codes: A61K 2039/505 comprising antibodies
• CPC Class Codes: A61K 47/60 the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: C07K 16/30 from tumour cells
• CPC Class Codes: C07K 19/00 Hybrid peptides , i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
• CPC Class Codes: C07K 2317/41 Glycosylation, sialylation, or fucosylation
• CPC Class Codes: C07K 2317/622 Single chain antibody (scFv)
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: G01N 33/6857 Antibody fragments

Abstract

The present invention relates to the chemical modification of single chain polypeptides by means of covalent attachment of strands of poly(ethylene glycol) PEG and similar poly(alkylene oxides) to single chain polypeptide binding molecules that have the three dimensional folding and, thus, the binding ability and specificity, of the variable region of an antibody. Such preparations of modified single chain polypeptide binding molecules have reduced immugenicity and antigenicity as well as having a longer halflife in the bloodstream as compared to the parent polypeptide. These beneficial properties of the modified single chain polypeptide binding molecules make them very useful in a variety of therapeutic applications. The invention also relates to multivalent antigen-binding molecules capable of PEGylation. Compositions of, genetic constructions for, methods of use, and methods for producing PEGylated antigen-binding proteins are disclosed.

Claims

1. A single-chain antigen-binding polypeptide-polyalkylene oxide conjugate, comprising a single-chain antigen-binding polypeptide comprising:

(a) a first polypeptide comprising the antigen binding portion of the variable region of an antibody heavy or light chain;

(b) a second polypeptide comprising the antigen binding portion of the variable region of an antibody heavy or light chain; and

(c) a peptide linker linking the first and second polypeptides (a) and (b) into a single chain polypeptide having an antigen binding site, wherein said single-chain antigen-binding polypeptide is conjugated to polyalkylene oxide and

wherein said single-chain antigen-binding polypeptide-polyalkylene oxide conjugate has an antigen binding affinity within a range of about one-fold to about ten-fold of the antigen binding affinity of said single-chain antigen-binding polypeptide in its unconjugated form.

37. A method of detecting an antigen suspected of being in a sample, comprising:

(a) contacting said sample with the polyalkylene oxide conjugated polypeptide or protein of claim 1 or 11, wherein said polyalkylene oxide conjugated polypeptide or protein is conjugated to one or plurality of detectable label molecule(s), or conjugated to a carrier having one or plurality of detectable label molecule(s) bound to said carrier; and

(b) detecting whether said polyalkylene oxide conjugated single-chain antigen-binding polypeptide or protein has bound to said antigen.

38. A method of imaging the internal structure of an animal, comprising administering to said animal an effective amount of the polyalkylene oxide conjugated polypeptide or protein of claim 1 or 11, wherein said polyalkylene oxide conjugated polypeptide or protein is conjugated to one or plurality of detectable label or chelator molecule(s), or conjugated to a carrier having one or plurality of detectable label or chelator molecule(s) bound to said carrier, and measuring detectable radiation associated with said animal.

40. A method for treating a targeted disease, comprising administering an effective amount of a composition comprising the polyalkylene oxide conjugated polypeptide or protein of claim 1 or 11 and a pharmaceutically acceptable carrier vehicle, wherein said polyalkylene oxide conjugated polypeptide or protein is conjugated to one or plurality of peptide, lipid, nucleic acid, drug, toxin, boron addend or radioisotope molecule(s), or conjugated to a carrier having one or plurality of peptide, lipid, nucleic acid, drug, toxin, boron addend or radioisotope molecule(s) bound to said carrier.

2. A single-chain antigen-binding polypeptide-polyalkylene oxide conjugate, comprising a single-chain antigen-binding polypeptide comprising:

(a) a first polypeptide comprising the antigen binding portion of the variable region of an antibody heavy or light chain;

(b) a second polypeptide comprising the antigen binding portion of the variable region of an antibody heavy or light chain; and

(c) a peptide linker linking the first and second polypeptides (a) and (b) into a single chain polypeptide having an antigen binding site, wherein said single-chain antigen-binding polypeptide is conjugated to polyalkylene oxide and

wherein said single-chain antigen-binding polypeptide-polyalkylene oxide conjugate has an antigen binding affinity within about ten-fold of the antigen binding affinity of said single-chain antigen-binding polypeptide in its unconjugated form.

3. A single-chain antigen-binding polypeptide-polyalkylene oxide conjugate, comprising a single-chain antigen-binding polypeptide comprising:

(a) a first polypeptide comprising the antigen binding portion of the variable region of an antibody heavy or light chain;

(b) a second polypeptide comprising the antigen binding portion of the variable region of an antibody heavy or light chain; and

(c) a peptide linker linking the first and second polypeptides (a) and (b) into a single chain polypeptide having an antigen binding site, wherein said single-chain antigen-binding polypeptide is conjugated to polyalkylene oxide and

wherein said single-chain antigen-binding polypeptide-polyalkylene oxide conjugate has an antigen binding affinity within about five-fold of the antigen binding affinity of said single-chain antigen-binding polypeptide in its unconjugated form.

4. A single-chain antigen-binding polypeptide-polyalkylene oxide conjugate, comprising a single-chain antigen-binding polypeptide comprising:

(a) a first polypeptide comprising the antigen binding portion of the variable region of an antibody heavy or light chain;

(b) a second polypeptide comprising the antigen binding portion of the variable region of an antibody heavy or light chain; and

(c) a peptide linker linking the first and second polypeptides (a) and (b) into a single chain polypeptide having an antigen binding site, wherein said single-chain antigen-binding polypeptide is conjugated to polyalkylene oxide and

wherein said single-chain antigen-binding polypeptide-polyalkylene oxide conjugate has an antigen binding affinity within about two-fold of the antigen binding affinity of said single-chain antigen-binding polypeptide in its unconjugated form.

5. A single-chain antigen-binding polypeptide capable of polyalkylene oxide conjugation, comprising:

(a) a first polypeptide comprising the antigen binding portion of the variable region of an antibody heavy or light chain;

(b) a second polypeptide comprising the antigen binding portion of the variable region of an antibody heavy or light chain; and

(c) a peptide linker linking said first and second polypeptides (a) and (b) into a single chain polypeptide having an antigen binding site,

wherein the single-chain antigen-binding polypeptide has at least one Cys residue which is capable of polyalkylene oxide conjugation, wherein said Cys residue is located at a position selected from the group consisting of;

(i) the amino acid position 11, 12, 13, 14 or 15 of the light chain variable region;

(ii) the amino acid position 77, 78 or 79 of the light chain variable region;

(iii) the amino acid position 11, 12, 13, 14 or 15 of the heavy chain variable region;

(iv) the amino acid position 82B, 82C or 83 of the heavy chain variable region;

(v) any amino acid position of the peptide linker;

(vi) adjacent to the C-terminus of polypeptide (a) or (b); and

(vii) combinations thereof,

wherein the polyalkylene oxide conjugated single-chain antigen-binding polypeptide is capable of binding an antigen.

6. The single-chain antigen-binding polypeptide of claim 5, wherein said Cys residue capable of polyalkylene oxide conjugation is located at a position selected from the group consisting of:

(i′

) the amino acid position 77 of the light chain variable region;

(ii′

) the amino acid position 82B of the heavy chain variable region;

(iii′

) the amino acid position 3 of the peptide linker;

(iv′

) adjacent to the C-terminus of said polypeptide (a) or (b); and

(v′

) combinations thereof.

7. The single-chain antigen-binding polypeptide of claim 5, wherein said first polypeptide (a) comprises the antigen binding portion of the variable region of an antibody light chain and said second polypeptide (b) comprises the antigen binding portion of the variable region of an antibody heavy chain.

8. The single-chain antigen-binding polypeptide of claim 5, wherein the C-terminus of said second polypeptide (b) is the native C-terminus.

9. The single-chain antigen-binding polypeptide of claim 5, wherein the C-terminus of said second polypeptide (b) comprises a deletion of one or plurality of amino acid residue(s), such that the remaining N-terminus amino acid residues of the second polypeptide are sufficient for the polyalkylene oxide conjugated single-chain antigen-binding polypeptide to be capable of binding an antigen.

10. The single-chain antigen-binding polypeptide of claim 5, wherein the C-terminus of said second polypeptide (b) comprises an addition of one or plurality of amino acid residue(s), such that the polyalkylene oxide conjugated single-chain antigen-binding polypeptide is capable of binding an antigen.

11. The single-chain antigen-binding polypeptide of claim 5, wherein said wherein said Cys residue capable of polyalkylene oxide conjugation is attached to a polyalkylene oxide moiety.

12. The polyalkylene oxide conjugated single-chain antigen-binding polypeptide of claim 11, wherein said polyalkylene oxide conjugated single-chain antigen-binding polypeptide is conjugated to one or plurality of peptide, lipid, nucleic acid, drug, toxin, chelator, boron addend or detectable label molecules.

13. The polyalkylene oxide conjugated single-chain antigen-binding polypeptide of claim 11, wherein said polyalkylene oxide conjugated single-chain antigen-binding polypeptide is conjugated to a carrier having one or plurality of peptide, lipid, nucleic acid, drug, toxin, chelator, boron addend or detectable label molecules bound to said carrier.

15. The polynucleotide sequence of claim 14, wherein said Cys residue capable of polyalkylene oxide conjugation is located at a position selected from the group consisting of:

(i′

) the amino acid position 77 of the light chain variable region;

(ii′

) the amino acid position 82B of the heavy chain variable region;

(iii′

) the amino acid position 3 of the peptide linker;

(iv′

) adjacent to the C-terminus of said polypeptide (a) or (b); and

(v′

) combinations thereof.

16. A replicable cloning or expression vehicle comprising the polynucleotide sequence of claim 14.

17. The vehicle of claim 16 which is a plasmid.

18. A host cell transformed with the polynucleotide of claim 17.

19. The host cell of claim 18 which is a bacterial cell, a yeast cell or other fungal cell, an insect cell or a mammalian cell line.

20. The host cell of claim 19 which is Pichia pastoris.

14. A polynucleotide sequence encoding a single-chain antigen-binding polypeptide capable of polyalkylene oxide conjugation, comprising:

(a) a first polypeptide comprising the antigen binding portion of the variable region of an antibody heavy or light chain;

(b) a second polypeptide comprising the antigen binding portion of the variable region of an antibody heavy or light chain; and

(c) a peptide linker linking said first and second polypeptides (a) and (b) into a single chain polypeptide having an antigen binding site,

wherein the single-chain antigen-binding polypeptide has at least one Cys residue which is capable of polyalkylene oxide conjugation, wherein said Cys residue is located at a position selected from the group consisting of;

(i) the amino acid position 11, 12, 13, 14 or 15 of said light chain variable region;

(ii) the amino acid position 77, 78 or 79 of said light chain variable region;

(iii) the amino acid position 11, 12, 13, 14 or 15 of said heavy chain variable region;

(iv) the amino acid position 82B, 82C or 83 of said heavy chain variable region;

(v) any amino acid position of said peptide linker;

(vi) adjacent to the C-terminus of said polypeptide (a) or (b); and

(vii) combinations thereof, and

wherein the polyalkylene oxide conjugated single-chain antigen-binding polypeptide is capable of binding an antigen.

21. A method of a producing single-chain antigen-binding polypeptide-polyalkylene oxide conjugate, comprising a single-chain antigen-binding polypeptide comprising:

(a) providing a first genetic sequence encoding a first polypeptide comprising the antigen binding portion of the variable region of an antibody heavy or light chain;

(b) providing a second genetic sequence encoding a second polypeptide comprising the antigen binding portion of the variable region of an antibody heavy or light chain; and

(c) linking the first and second genetic sequences (a) and (b) with a third genetic sequence encoding a linker into a fourth genetic sequence encoding a single chain polypeptide having an antigen binding site, wherein said single-chain antigen-binding polypeptide is capable of polyalkylene oxide conjugation and

wherein said single-chain antigen-binding polypeptide-polyalkylene oxide conjugate retains antigen binding affinity within a range of about one-fold to about ten-fold of the antigen binding affinity of said single-chain antigen-binding polypeptide in its unconjugated form.

22. A method of producing a single-chain antigen-binding polypeptide capable of polyalkylene oxide conjugation, comprising:

(a) providing a first genetic sequence encoding a first polypeptide comprising the antigen binding portion of the variable region of an antibody heavy or light chain;

(b) providing a second genetic sequence encoding a second polypeptide comprising the antigen binding portion of the variable region of an antibody heavy or light chain; and

(c) linking said first and second genetic sequences (a) and (b) with a third genetic sequence encoding a peptide linker into a fourth genetic sequence encoding a single chain polypeptide having an antigen binding site,

wherein the single-chain antigen-binding polypeptide has at least one Cys residue which is capable of polyalkylene oxide conjugation, wherein said Cys residue is located at a position selected from the group consisting of;

(i) the amino acid position 11, 12, 13, 14 or 15 of said light chain variable region;

(ii) the amino acid position 77, 78 or 79 of said light chain variable region;

(iii) the amino acid position 11, 12, 13, 14 or 15 of said heavy chain variable region;

(iv) the amino acid position 82B, 82C or 83 of said heavy chain variable region;

(v) any amino acid position of said peptide linker;

(vi) adjacent to the C-terminus of said polypeptide (a) or (b); and

(vii) combinations thereof, and

wherein the polyalkylene oxide conjugated single-chain antigen-binding polypeptide is capable of binding an antigen;

(d) transforming a host cell with said fourth genetic sequence encoding said single-chain antigen-binding polypeptide of step (c); and

(e) expressing said single-chain antigen-binding polypeptide of step (c) in said host, thereby producing a single-chain antigen-binding polypeptide capable of polyalkylene oxide conjugation.

23. The method of claim 22, wherein said Cys residue capable of polyalkylene oxide conjugation is located at a position selected from the group consisting of:

(i′

) the amino acid position 77 of the light chain variable region;

(ii′

) the amino acid position 82B of the heavy chain variable region;

(iii′

) the amino acid position 3 of the peptide linker;

(iv′

) adjacent to the C-terminus of said polypeptide (a) or (b); and

(v′

) combinations thereof.

24. The method of claim 22, said first genetic sequence encoding a first polypeptide (a) comprises the antigen binding portion of the variable region of an antibody light chain and said second genetic sequence encoding a second polypeptide (b) comprises the antigen binding portion of the variable region of an antibody heavy chain.

25. The method of claim 22, wherein the C-terminus of said second polypeptide (b) is the native C-terminus.

26. The method of claim 22, wherein the C-terminus of said second polypeptide (b) comprises a deletion of one or plurality of amino acid residue(s), such that the remaining N-terminus amino acid residues of the second polypeptide are sufficient for the polyalkylene oxide conjugated single-chain antigen-binding polypeptide to be capable of binding an antigen.

27. The method of claim 22, wherein the C-terminus of said second polypeptide (b) comprises an addition of one or plurality of amino acid residue(s), such that the polyalkylene oxide conjugated single-chain antigen-binding polypeptide is capable of binding an antigen.

30. The multivalent protein of claim 29, wherein said Cys residue capable of polyalkylene oxide conjugation is located at a position selected from the group consisting of:

(i′

) the amino acid position 77 of the light chain variable region;

(ii′

) the amino acid position 82B of the heavy chain variable region;

(iii′

) the amino acid position 3 of the peptide linker;

(iv′

) adjacent to the C-terminus of said polypeptide (a) or (b); and

(v′

) combinations thereof.

31. The multivalent protein of claim 29, wherein said first polypeptide (a) comprises the antigen binding portion of the variable region of an antibody light chain and said second polypeptide (b) comprises the antigen binding portion of the variable region of an antibody heavy chain.

32. The multivalent protein of claim 29, wherein the C-terminus of said second polypeptide (b) is the native C-terminus.

33. The multivalent protein of claim 29, wherein the C-terminus of said second polypeptide (b) comprises a deletion of one or plurality of amino acid residue(s), such that the remaining N-terminus amino acid residues of the second polypeptide are sufficient for the polyalkylene oxide conjugated single-chain antigen-binding polypeptide to be capable of binding an antigen.

34. The multivalent protein of claim 29, wherein the C-terminus of said second polypeptide comprises an addition of one or plurality of amino acid residue(s), such that the polyalkylene oxide conjugated single-chain antigen-binding polypeptide is capable of binding an antigen.

35. The multivalent protein of claim 29, wherein said wherein said Cys residue capable of polyalkylene oxide conjugation is attached to a polyalkylene oxide moiety.

36. The polyalkylene oxide conjugated multivalent protein of claim 35, wherein said polyalkylene oxide conjugated multivalent protein is conjugated to one or plurality of peptide, lipid, nucleic acid, drug, toxin, chelator, boron addend or detectable label molecule(s).

39. The method of claim 38, wherein said animal includes a human.

46. The host cell of claim 39 which is a bacterial cell, a yeast cell or other fungal cell, an insect cell or a mammalian cell line.

28. A multivalent single-chain antigen-binding polypeptide-polyalkylene oxide conjugate, comprising two or more single-chain antigen binding polypeptides, each single-chain antigen-binding polypeptide comprising:

(a) a first polypeptide comprising the antigen binding portion of the variable region of an antibody heavy or light chain;

(b) a second polypeptide comprising the antigen binding portion of the variable region of an antibody heavy or light chain; and

(c) a peptide linker linking the first and second polypeptides (a) and (b) into a single chain polypeptide having an antigen binding site, wherein said single-chain antigen-binding polypeptide is conjugated to polyalkylene oxide and

wherein said single-chain antigen-binding polypeptide-polyalkylene oxide conjugate retains antigen binding affinity within a range of about one-fold to about ten-fold of the antigen binding affinity of said single-chain antigen-binding polypeptide in its unconjugated form.

29. A multivalent single-chain antigen-binding protein, comprising two or more single-chain antigen-binding polypeptides, each single-chain antigen-binding polypeptide comprising:

(a) a first polypeptide comprising the antigen binding portion of the variable region of an antibody heavy or light chain;

(b) a second polypeptide comprising the antigen binding portion of the variable region of an antibody heavy or light chain; and

(c) a peptide linker linking said first and second polypeptides wherein one of the two single-chain antigen-binding polypeptide has at least one Cys residue which is capable of polyalkylene oxide conjugation, wherein said Cys residue is located at a position selected from the group consisting of;

(i) the amino acid position 11, 12, 13, 14 or 15 of said light chain variable region;

(ii) the amino acid position 77, 78 or 79 of said light chain variable region;

(iii) the amino acid position 11, 12, 13, 14 or 15 of said heavy chain variable region;

(iv) the amino acid position 82B, 82C or 83 of said heavy chain variable region;

(v) any amino acid position of said peptide linker;

(vi) adjacent to the C-terminus of said polypeptide (a) or (b); and

(vii) combinations thereof, and

wherein the polyalkylene oxide conjugated single-chain antigen-binding polypeptide is capable of binding an antigen.

41. A single-chain antigen-binding polypeptide capable of polyalkylene oxide conjugation, comprising:

(a) a first polypeptide comprising the antigen binding portion of the variable region of an antibody heavy or light chain;

(b) a second polypeptide comprising the antigen binding portion of the variable region of an antibody heavy or light chain; and

(c) a peptide linker linking the first and second polypeptides (a) and (b) into a single chain polypeptide having an antigen binding site, wherein the single-chain antigen-binding polypeptide has at least three consecutive Lys residues which are capable of polyalkylene oxide conjugation and wherein any one of said consecutive Lys residues is located at a position selected from the group consisting of

(i) any amino acid position of the peptide linker;

(ii) adjacent to the C-terminus of the second polypeptide (b); and

(iii) combinations thereof,

wherein the polyalkylene oxide conjugated single-chain antigen-binding polypeptide is capable of binding an antigen.

49. The single-chain antigen-binding polypeptide of claim 41, wherein said wherein said wherein any one of said consecutive Lys residues capable of polyalkylene oxide conjugation is attached to a polyalkylene oxide moiety.

42. A polynucleotide sequence encoding a single-chain antigen-binding polypeptide capable of polyalkylene oxide conjugation, comprising:

(a) a first polypeptide comprising the antigen binding portion of the variable region of an antibody heavy or light chain;

(b) a second polypeptide comprising the antigen binding portion of the variable region of an antibody heavy or light chain; and

(c) a peptide linker linking the first and second polypeptides (a) and (b) into a single chain polypeptide having an antigen binding site, wherein the single-chain antigen-binding polypeptide has at least three consecutive Lys residues which are capable of polyalkylene oxide conjugation and wherein any one of said consecutive Lys residues is located at a position selected from the group consisting of

(i) any amino acid position of the peptide linker;

(ii) adjacent to the C-terminus of the second polypeptide (b); and

(iii) combinations thereof,

wherein the polyalkylene oxide conjugated single-chain antigen-binding polypeptide is capable of binding an antigen.

43. A replicable cloning or expression vehicle comprising the polynucleotide sequence of claim 42.

44. The vehicle of claim 43 which is a plasmid.

45. A host cell transformed with the polynucleotide of claim 42.

47. The host cell of claim 46 which is Pichia pastoris.

50. The polyalkylene oxide conjugated single-chain antigen-binding polypeptide of claim 49, wherein said polyalkylene oxide conjugated single-chain antigen-binding polypeptide is conjugated to one or plurality of peptide, lipid, nucleic acid, drug, toxin, chelator, boron addend or detectable label molecules.

51. The polyalkylene oxide conjugated single-chain antigen-binding polypeptide of claim 49, wherein said polyalkylene oxide conjugated single-chain antigen-binding polypeptide is conjugated to a carrier having one or plurality of peptide, lipid, nucleic acid, drug, toxin, chelator, boron addend or detectable label molecules bound to said carrier.

48. A method of producing a single-chain antigen-binding polypeptide capable of polyalkylene oxide conjugation, comprising:

(a) a first polypeptide comprising the antigen binding portion of the variable region of an antibody heavy or light chain;

(b) a second polypeptide comprising the antigen binding portion of the variable region of an antibody heavy or light chain; and

(c) a peptide linker linking the first and second polypeptides (a) and (b) into a single chain polypeptide having an antigen binding site, wherein the single-chain antigen-binding polypeptide has at least three consecutive Lys residues which are capable of polyalkylene oxide conjugation and wherein any one of said consecutive Lys residues is located at a position selected from the group consisting of

(i) any amino acid position of the peptide linker;

(ii) adjacent to the C-terminus of the second polypeptide (b); and

(iii) combinations thereof,

wherein the polyalkylene oxide conjugated single-chain antigen-binding polypeptide is capable of binding an antigen.

52. A method of producing a single-chain antigen-binding polypeptide capable of polyalkylene oxide conjugation, comprising:

(a) providing a first genetic sequence encoding a first polypeptide comprising the antigen binding portion of the variable region of an antibody heavy or light chain;

(b) providing a second genetic sequence encoding a second polypeptide comprising the antigen binding portion of the variable region of an antibody heavy or light chain; and

(c) linking said first and second genetic sequences (a) and (b) with a third genetic sequence encoding a peptide linker into a fourth genetic sequence encoding a single chain polypeptide having an antigen binding site,

wherein the single-chain antigen-binding polypeptide has at least three consecutive Lys residues which are capable of polyalkylene oxide conjugation and wherein any one of said consecutive Lys residues is located at a position selected from the group consisting of

(i) any amino acid position of the peptide linker;

(ii) adjacent to the C-terminus of the second polypeptide (b); and

(iii) combinations thereof,

wherein the polyalkylene oxide conjugated single-chain antigen-binding polypeptide is capable of binding an antigen;

(d) transforming a host cell with said fourth genetic sequence encoding said single-chain antigen-binding polypeptide of step (c); and

(e) expressing said single-chain antigen-binding polypeptide of step (c) in said host, thereby producing a single-chain antigen-binding polypeptide capable of polyalkylene oxide conjugation.

53. The method of claim 52, said first genetic sequence encoding a first polypeptide (a) comprises the antigen binding portion of the variable region of an antibody light chain and said second genetic sequence encoding a second polypeptide (b) comprises the antigen binding portion of the variable region of an antibody heavy chain.

54. The method of claim 52, wherein the C-terminus of said second polypeptide (b) is the native C-terminus.

55. The method of claim 52, wherein the C-terminus of said second polypeptide (b) comprises a deletion of one or plurality of amino acid residue(s), such that the remaining N-terminus amino acid residues of the second polypeptide are sufficient for the polyalkylene oxide conjugated single-chain antigen-binding polypeptide to be capable of binding an antigen.

56. The method of claim 52, wherein the C-terminus of said second polypeptide (b) comprises an addition of one or plurality of amino acid residue(s), such that the polyalkylene oxide conjugated single-chain antigen-binding polypeptide is capable of binding an antigen.

58. The multivalent protein of claim 57, wherein said first polypeptide (a) comprises the antigen binding portion of the variable region of an antibody light chain and said second polypeptide (b) comprises the antigen binding portion of the variable region of an antibody heavy chain.

59. The multivalent protein of claim 57, wherein the C-terminus of said second polypeptide (b) is the native C-terminus.

60. The multivalent protein of claim 57, wherein the C-terminus of said second polypeptide (b) comprises a deletion of one or plurality of amino acid residue(s), such that the remaining N-terminus amino acid residues of the second polypeptide are sufficient for the polyalkylene oxide conjugated single-chain antigen-binding polypeptide to be capable of binding an antigen.

61. The multivalent protein of claim 57, wherein the C-terminus of said second polypeptide comprises an addition of one or plurality of amino acid residue(s), such that the polyalkylene oxide conjugated single-chain antigen-binding polypeptide is capable of binding an antigen.

62. The multivalent protein of claim 57, wherein any one of said consecutive Lys residues capable of polyalkylene oxide conjugation is attached to a polyalkylene oxide moiety.

63. The polyalkylene oxide conjugation multivalent protein of claim 62, wherein said polyalkylene oxide conjugated multivalent protein is conjugated to one or plurality of peptide, lipid, nucleic acid, drug, toxin, chelator, boron addend or detectable label molecule(s).

57. A multivalent single-chain antigen-binding protein, comprising two or more single-chain antigen-binding polypeptides, each single-chain antigen-binding polypeptide comprising:

(a) a first polypeptide comprising the antigen binding portion of the variable region of an antibody heavy or light chain;

(b) a second polypeptide comprising the antigen binding portion of the variable region of an antibody heavy or light chain; and

(c) a peptide linker linking said first and second polypeptides wherein one of the two single-chain antigen-binding polypeptides has at least three consecutive Lys residues which are capable of polyalkylene oxide conjugation and wherein any one of said consecutive Lys residues is located at a position selected from the group consisting of

(i) any amino acid position of the peptide linker;

(ii) adjacent to the C-terminus of the second polypeptide (b); and

(iii) combinations thereof,

wherein the polyalkylene oxide conjugated single-chain antigen-binding polypeptide is capable of binding an antigen.