Method of producing biospecific molecules by protein trans-splicing

• US 20040077842A1
• Filed: 11/03/2003
• Published: 04/22/2004
• Est. Priority Date: 11/01/2000
• Status: Active Grant

• Current Assignee: Elusys Therapeutics, Inc.
• Original Assignee: Elusys Therapeutics, Inc.
• Inventors: Himawan, Jeff
• Granted Patent: US 7,405,276 B2
• Time in Patent Office: Days
• US Class Current: 530/388.22
• CPC Class Codes: A61K 2039/505 comprising antibodies
• CPC Class Codes: A61P 31/04 Antibacterial agents
• CPC Class Codes: A61P 31/10 Antimycotics
• CPC Class Codes: A61P 31/12 Antivirals
• CPC Class Codes: A61P 31/18 for HIV
• CPC Class Codes: A61P 33/00 Antiparasitic agents
• CPC Class Codes: A61P 33/02 Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
• CPC Class Codes: A61P 37/06 Immunosuppressants, e.g. drugs for graft rejection
• CPC Class Codes: A61P 39/02 Antidotes
• CPC Class Codes: C07K 14/70535 Fc-receptors, e.g. CD16, CD32, CD64 (CD2314/705F)
• CPC Class Codes: C07K 16/283 against Fc-receptors, e.g. CD16, CD32, CD64 CD23 C07K16/2851
• CPC Class Codes: C07K 16/2896 against molecules with a "CD"-designation, not provided for elsewhere
• CPC Class Codes: C07K 16/468 Immunoglobulins having two or more different antigen binding sites, e.g. multifunctional antibodies
• CPC Class Codes: C07K 2317/31 multispecific
• CPC Class Codes: C07K 2317/52 Constant or Fc region; Isotype
• CPC Class Codes: C07K 2319/22 containing a Strep-tag
• 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: C07K 2319/92 containing an intein ("protein splicing")domain
• CPC Class Codes: C12N 15/62 DNA sequences coding for fusion proteins

Abstract

The invention provides methods of using protein trans-splicing for the production of bispecific molecule which has a first antigen recognition portion that binds a C3b-like receptor and a second antigen recognition portion that binds an antigenic molecule present in the circulatory system of a mammal. The invention also provides bispecific molecules produced by protein trans-splicing. The bispecific molecules of the invention can be used for the clearance of pathogenic antigenic molecules from the circulatory system of a mammal. The invention further provides methods of using protein trans-splicing for the production of polyclonal libraries of bispecific molecules, which comprise populations of bispecific molecules with different antigen recognition specificities. Such polyclonal libraries of bispecific molecules can be used for targeting multiple epitopes of a pathogenic antigenic molecule and/or multiple variants of a pathogenic antigenic molecule.

Claims

1. A method of producing a bispecific molecule having a first antigen recognition portion that binds a C3b-like receptor and a second antigen recognition portion that binds a pathogenic antigenic molecule, comprising contacting an N-intein first antigen recognition portion and a C-intein second antigen recognition portion under conditions such that protein trans-splicing occurs, wherein said N-intein first antigen recognition portion comprises said first antigen recognition portion conjugated to the amino terminus of an N-intein of a split intein, and wherein said C-intein second antigen recognition portion comprises said second antigen recognition portion conjugated to the carboxy terminus of a C-intein of said split intein, wherein the amino acid residue immediately at the C-terminal side of the splice junction of said C-intein is an amino acid residue selected from the group consisting of cysteine, serine, and threonine.

3. The method of claim 1 or 2, wherein said split intein is a naturally occurring split intein.

4. The method of claim 3, wherein the sequence of the N-intein of said split intein is SEQ ID NO:

1, and the sequence of the C-intein of said split intein is SEQ ID NO;

2.

5. The method of claim 1 or 2, wherein said split intein is an engineered split intein generated by separating a naturally occurring non-split intein into an amino terminal fragment and a carboxy terminal fragment such that said amino terminal fragment and said carboxy terminal fragment can reconstitute and undergo trans-splicing.

6. The method of claim 5, wherein said engineered split intein is generated from the Mycobacterium tuberculosis RecA intein.

7. The method of claim 1 or 2, wherein said N-intein further comprises a sequence of 1 or more native proximal N-extein amino acid residue(s) attached to the amino terminus of said N-intein.

8. The method of claim 7, wherein said N-intein comprises a sequence of 3 to 5 native proximal N-extein amino acid residue(s) attached to the amino terminus of said N-intein.

9. The method of claim 1 or 2, wherein said C-intein further comprises an amino acid residue selected from the group consisting of cysteine, serine, and threonine at the carboxy terminus of the splice junction.

10. The method of claim 9, wherein said C-intein further comprises a sequence of 1 or more native proximal C-extein amino acid residue(s) attached to the carboxy terminus of said C-intein.

11. The method of claim 10, wherein said C-intein comprises a sequence of 3 to 5 more native proximal C-extein amino acid residue(s) attached to the carboxy terminus of said C-intein.

12. The method of claim 1 or 2, wherein said N-intein comprises an unnatural amino acid residue such that upon conversion of said unnatural amino acid residue into normal amino acid residues by a suitable means said N-intein becomes functional for trans-splicing.

13. The method of claim 12, wherein said unnatural amino acid residue is O-(2-nitrobenzyl)serine, and said suitable means comprises irradiating using light having a wavelength in the range of 300-350 nm.

14. The method of claim 1 or 2, wherein said first antigen recognition portion comprises an antigen binding domain that binds a C3b-like receptor on a mammalian blood cell and an effector domain that facilitates transfer of said pathogenic antigen molecule from said mammalian blood cell to a phagocytic cell.

15. The method of claim 1 or 2, wherein said first antigen recognition portion is an monoclonal antibody.

16. The method of claim 15, wherein said N-intein first antigen recognition portion is generated by conjugating the carboxy terminus of the heavy chain of said monoclonal antibody to said amino terminus of said N-intein.

17. The method of claim 15, wherein said N-intein first antigen recognition portion fragment is generated by conjugating the carboxy terminus of the light chain of said monoclonal antibody to the amino terminus of said N-intein.

18. The method of claim 1 or 2, wherein said first antigen recognition portion is an ScFv that binds a C3b-like receptor fused to the N-terminus of an immunoglobulin Fc domain.

19. The method of claim 1 or 2, wherein said second antigen recognition portion comprises a polypeptide.

20. The method of claim 1 or 2, wherein said second antigen recognition portion comprises a nucleic acid.

21. The method of claim 1 or 2, wherein said N-intein first antigen recognition portion is produced recombinantly.

22. The method of claim 1 or 2, wherein said C-intein second antigen recognition portion is produced recombinantly.

23. The method of claim 1 or 2, wherein said C-intein second antigen recognition portion is produced by peptide synthesis.

24. The method of claim 1 or 2, wherein N-intein first antigen recognition portion and said C-intein second antigen recognition portion are recombinantly co-expressed in a cell, and wherein said N-intein first antigen recognition portion and said C-intein second antigen recognition portion reconstitute and undergo trans-splicing in said cell to produce said bispecific molecule.

25. The method of claim 1 or 2, wherein said N-intein first antigen recognition portion is recombinantly expressed in a first cell and said C-intein second antigen recognition portion is recombinantly expressed in a second cell, and wherein said N-intein first antigen recognition portion and said C-intein second antigen recognition portion are mixed in vitro to produce said bispecific molecule.

26. The method of claim 25, wherein said N-intein first antigen recognition portion and said C-intein second antigen recognition portion are expressed by separate expression vectors.

27. The method of claim 25, wherein said N-intein first antigen recognition portion and said C-intein second antigen recognition portion are expressed by one expression vector.

28. The method of claim 15, wherein the heavy and light chains of said anti-CR1 mAb are recombinantly expressed using same vector.

29. The method of claim 15, wherein the heavy and light chains of said anti-CR1 mAb are recombinantly expressed using separate vectors.

30. The method of claim 29, wherein said heavy and light chains of said anti-CR1 mAb are recombinantly expressed in same cell.

31. The method of claim 29, wherein said heavy and light chains of said anti-CR1 mAb are recombinantly expressed in separate cells using separate vectors, and wherein said heavy and light chains are assembled into said mAb in vitro.

2. A method of producing a bispecific molecule having a first antigen recognition portion that binds a C3b-like receptor and a second antigen recognition portion that binds a pathogenic antigenic molecule, comprising:

(a) obtaining an N-intein first antigen recognition portion by conjugating said first antigen recognition portion to the amino terminus of a molecule comprising an N-intein of a split intein;

(b) obtaining a C-intein second antigen recognition portion by conjugating said second antigen recognition portion to the carboxy terminus of a molecule comprising a C-intein of said split intein, wherein the amino acid residue immediately at the C-terminal side of the splice junction of said C-intein is an amino acid residue selected from the group consisting of cysteine, serine, and threonine; and

(c) contacting said N-intein first antigen recognition portion and said C-intein second antigen recognition portion under conditions such that protein trans-splicing occurs, to produce said bispecific molecule.

32. A method of producing a bispecific molecule having a first antigen recognition portion that binds a C3b-like receptor and a second antigen recognition portion that binds a pathogenic antigenic molecule, comprising

(a) contacting an N-intein first antigen recognition portion and a C-intein streptavidin under conditions such that trans-splicing occurs to produce a first antigen recognition portion-streptavidin molecule, wherein said N-intein first antigen recognition portion comprises said first antigen recognition portion conjugated to the amino terminus of an N-intein of a split intein, and wherein said C-intein streptavidin comprises a core streptavidin conjugated to the carboxy terminus of a C-intein of said split intein, wherein the amino acid residue immediately at the C-terminal side of the splice junction of said C-intein is an amino acid residue selected from the group consisting of cysteine, serine, and threonine;

and

(b) contacting said first antigen recognition portion-streptavidin molecule and a biotinylated second antigen recognition portion under conditions conducive to binding between streptavidin and biotin, to produce said bispecific molecule.

34. The method of claim 32 or 33, wherein said C-intein streptavidin is generated recombinantly.

35. The method of claim 34, further comprising in step (b) the step of dissociating multimers of C-intein streptavidin.

36. The method of claim 32 or 33, wherein said biotinylated second antigen recognition portion is a biotinylated monoclonal antibody.

37. The method of claim 32 or 33, wherein said biotinylated second antigen recognition portion is produced by biotinylating of an antibody.

38. The method of claim 36, wherein said biotinylated monoclonal antibody is biotinylated at a single site.

39. The method of claim 36, wherein said biotinylated monoclonal antibody is biotinylated at a site such that the antigen binding capability of the antigen recognition domain is not impaired.

40. The method of claim 32 or 33, wherein said biotinylated second antigen recognition portion is a biotinylated nucleic acid.

41. The method of claim 40, wherein said biotinylated nucleic acid is produced from a nucleic acid sample by

(a) amplifying said nucleic acid using a PCR primer containing a restriction digestion site for a restriction endonuclease wherein said restriction endonuclease generates an overhanging terminus upon cleavage at said restriction digestion site, and said restriction endonuclease does not cleave said nucleic acid other than within the sequence of said PCR primer;

(b) cleaving the PCR product containing said PCR primer by said restriction endonuclease to generate an overhanging terminus; and

(c) adding a biotinylated nucleotide at said overhanging terminus using a DNA polymerase to produce said biotinylated nucleic acid.

33. A method of producing a bispecific molecule having a first antigen recognition portion that binds a C3b-like receptor and a second antigen recognition portion that binds a pathogenic antigenic molecule, comprising:

(a) obtaining an N-intein first antigen recognition portion by conjugating said first antigen recognition portion to the amino terminus of an N-intein of a split intein;

(b) obtaining a C-intein streptavidin by conjugating a core streptavidin to the carboxy terminus of the C-intein of said split intein, wherein the amino acid residue immediately at the C-terminal side of the splice junction of said C-intein is an amino acid residue selected from the group consisting of cysteine, serine, and threonine;

(c) contacting said N-intein first antigen recognition portion and said C-intein streptavidin under conditions such that protein trans-splicing occurs, to produce a first antigen recognition portion-streptavidin molecule, wherein said first antigen recognition portion is conjugated to said streptavidin;

(d) obtaining a biotinylated second antigen recognition portion; and

(e) contacting said first antigen recognition portion-streptavidin molecule and said biotinylated second antigen recognition portion under conditions conducive to binding between streptavidin and biotin, to produce said bispecific molecule.

42. A method of producing a polyclonal library of bispecific molecules, each member of said polyclonal library having a first antigen recognition portion that binds a C3b-like receptor and a second antigen recognition portion that binds a pathogenic antigenic molecule, comprising contacting a plurality of N-intein first antigen recognition portions and a plurality of different C-intein second antigen recognition portions under conditions such that protein trans-splicing occurs to produce a polyclonal library of bispecific molecules, wherein each of said plurality of N-intein first antigen recognition portions comprises said first antigen recognition portion conjugated to the amino terminus of an N-intein of a split intein, and wherein each of said plurality of C-intein second antigen recognition portions comprises a second antigen recognition portion having a different binding specificity conjugated to the carboxy terminus of a C-intein of said split intein, wherein the amino acid residue immediately at the C-terminal side of the splice junction of said C-intein is an amino acid residue selected from the group consisting of cysteine, serine, and threonine.

47. The method of claim 42, 43, 44, 45, or 46, wherein said plurality of antigen recognition portions has binding specificities for a plurality of epitopes of an antigen.

48. The method of claim 42, 43, 44, 45, or 46, wherein said plurality of antigen recognition portions has binding specificities for a plurality of variants of an antigen.

43. A method of producing a polyclonal library of bispecific molecules, each member of said polyclonal library having a first antigen recognition portion that binds a C3b-like receptor and a second antigen recognition portion that binds a pathogenic antigenic molecule, comprising:

(a) obtaining a plurality of N-intein first antigen recognition portions by a method comprising conjugating each of a plurality of first antigen recognition portions to the amino terminus of an N-intein of a split intein;

(b) selecting from a phage display library a plurality of phage that display antigen recognition polypeptides, each having a different respective binding specificity using affinity screening;

(c) obtaining a plurality of nucleic acids encoding said plurality of antigen recognition polypeptides, respectively;

(d) fusing each nucleic acid of said plurality of nucleic acids to the amino terminus of a C-intein of said split intein to obtain a plurality of nucleic acids encoding a plurality of C-intein antigen recognition portions, wherein the amino acid residue immediately at the C-terminal side of the splice junction of said C-intein is an amino acid residue selected from the group consisting of cysteine, serine, and threonine;

(e) expressing said plurality of nucleic acids encoding said plurality of C-intein antigen recognition portion fragments in a host;

(f) obtaining said plurality of C-intein antigen recognition portions from said host; and

(g) contacting said plurality of C-intein antigen recognition portions and a plurality of said N-intein first antigen recognition portions under conditions such that protein trans-splicing occurs, to produce said polyclonal library of bispecific molecules.

49. The method of claim 43 or 46, wherein said plurality of nucleic acids encoding said plurality of antigen recognition polypeptides is fused to said C-intein without clonal isolation.

50. The method of claim 43 or 46, wherein step (a) further comprising obtaining a plurality of different N-intein first antigen recognition portions by conjugating each of a plurality of different first antigen recognition portions to the amino terminus of a plurality of N-inteins of said split intein.

44. A method of producing a polyclonal library of bispecific molecules, each member of said polyclonal library having a first antigen recognition portion that binds a C3b-like receptor and a second antigen recognition portion that binds a pathogenic antigenic molecule, comprising:

(a) obtaining a plurality of N-intein first antigen recognition portions by a method comprising conjugating each of a plurality of first antigen recognition portions to the amino terminus of an N-intein of a split intein;

(b) fusing the amino terminus of the nucleic acid encoding a polypeptide antibody in each member of said phage display library to the carboxy terminus of a C-intein of said split intein to obtain a phage display library displaying a repertoire of C-intein antigen recognition portions, wherein the amino acid residue immediately at the C-terminal side of the splice junction of said C-intein is an amino acid residue selected from the group consisting of cysteine, serine, and threonine;

(c) selecting from said phage display library displaying a repertoire of C-intein antigen recognition portions a plurality of phage that display C-intein antigen recognition portions, each having a different respective binding specificity using affinity screening;

(d) obtaining a plurality of nucleic acids encoding said plurality of C-intein antigen recognition portions, respectively;

(e) expressing said plurality of nucleic acids encoding said plurality of C-intein antigen recognition portions in a host;

(g) obtaining said plurality of C-intein antigen recognition portions from said host; and

(h) contacting said plurality of C-intein antigen recognition portions and a plurality of said N-intein first antigen recognition portions under conditions such that protein trans-splicing occurs, to produce said polyclonal library of bispecific molecules.

45. A method of producing a polyclonal library of bispecific molecules, each member of said polyclonal library having a first antigen recognition portion that binds a C3b-like receptor and a second antigen recognition portion that binds a pathogenic antigenic molecule, comprising

(a) contacting a population of N-intein first antigen recognition portions and a population of C-intein streptavidin under conditions such that protein trans-splicing occurs to produce a population of first antigen recognition portion-streptavidin molecules, wherein each of said plurality of N-intein first antigen recognition portions comprises said first antigen recognition portion conjugated to the amino terminus of an N-intein of a split intein, and wherein each of said plurality of C-intein streptavidin comprises a core streptavidin conjugated to the carboxy terminus of a C-intein of said split intein, wherein the amino acid residue immediately at the C-terminal side of the splice junction of said C-intein is an amino acid residue selected from the group consisting of cysteine, serine, and threonine;

and

(b) contacting said population of first antigen recognition portion streptavidin molecule and a plurality of biotinylated second antigen recognition portions, each having a different antigen recognition specificity under conditions conducive to binding between streptavidin and biotin, to produce said polyclonal library of bispecific molecules.

46. A method of producing a polyclonal library of bispecific molecules, each member of said polyclonal library having a first antigen recognition portion that binds a C3b-like receptor and a second antigen recognition portion that binds a pathogenic antigenic molecule, comprising:

(a) obtaining a population of N-intein first antigen recognition portions by a method comprising conjugating each of a population of first antigen recognition portions to the amino terminus of an N-intein of a split intein;

(b) obtaining a population of C-intein streptavidin by a method comprising conjugating each of a population of core streptavidins to a C-intein of said split intein, wherein the amino acid residue immediately at the C-terminal side of the splice junction of said C-intein is an amino acid residue selected from the group consisting of cysteine, serine, and threonine;

(c) contacting said population of N-intein first antigen recognition portions with said population of C-intein streptavidins under conditions such that protein trans-splicing occurs to produce a population of first antigen recognition portion streptavidin molecules;

(d) selecting from a phage display library a plurality of phage that display antigen recognition polypeptides, each having a different respective binding specificity using affinity screening;

(f) obtaining a plurality of nucleic acids encoding said plurality of antigen recognition polypeptides, respectively;

(g) expressing said plurality of nucleic acids encoding said plurality of antigen recognition polypeptides in a host;

(h) obtaining said plurality of antigen recognition polypeptides from said host;

(i) obtaining a plurality of biotinylated antigen recognition polypeptides by conjugating each member of said plurality of antigen recognition polypeptides with biotin using an activated biotin agent; and

(j) contacting said plurality of biotinylated antigen recognition polypeptides and said population of first antigen recognition portion streptavidin molecules under conditions conducive to binding between streptavidin and biotin, to produce said polyclonal library of bispecific molecules.

51. A method of producing a C-intein antigen recognition portion phage display library, each phage in said C-intein antigen recognition portion phage display library displaying on its surface a different C-intein antigen recognition portion, comprising fusing the amino terminus of the nucleic acid encoding a polypeptide antibody in each of a plurality of members of a phage display library to the carboxy terminus of a C-intein of a split intein to obtain a phage display library displaying a repertoire of C-intein antigen recognition portions, wherein the amino acid residue immediately at the C-terminal side of the splice junction of said C-intein is an amino acid residue selected from the group consisting of cysteine, serine, and threonine.

52. A bispecific molecule, comprising:

(a) a first antigen recognition portion that binds a C3b-like receptor; and

(b) a second antigen recognition portion that binds a pathogenic antigenic molecule;

wherein said first antigen recognition portion comprises a monoclonal antibody, and wherein said second antigen recognition portion is conjugated to a carboxy terminus of a heavy or light chain of said first antigen recognition portion.

59. The bispecific molecule of claim 52 or 58, wherein said second antigen recognition portion is conjugated to the C-terminus of a heavy chain of said first antigen recognition portion.

60. The bispecific molecule of claim 52 or 58, wherein said second antigen recognition portion is conjugated to the C-terminus of a light chain of said first antigen recognition portion.

61. The bispecific molecule of claim 52 or 58, wherein said second antigen recognition portion comprises two antigen recognition moieties each conjugated to the C-terminus of a different heavy chain of said first antigen recognition portion.

62. The bispecific molecule of claim 52 or 58, wherein said first antigen recognition portion is an anti-CR1 monoclonal antibody produced by the hybridoma cell line ATCC HB 8592 from the American Type Culture Collection (ATCC), and wherein the second antigen recognition portion comprises the α

-subunit of FceRI receptor.

63. The bispecific molecule of claim 52 or 58, wherein said second antigen recognition portion comprises two antigen recognition moieties each conjugated to the C-terminus of a different light chain of said first antigen recognition portion.

64. The bispecific molecule of claim 61, wherein said two antigen recognition moieties are the same antigen recognition moieties.

65. The bispecific molecule of claim 61, wherein said two antigen recognition moieties are different antigen recognition moieties.

66. The bispecific molecule of claim 65, wherein said different antigen recognition moieties target said pathogenic antigen cooperatively.

67. The bispecific molecule of claim 65, wherein the action of one of said different antigen recognition moieties enhances the binding affinity of the other one of said different antigen recognition moieties.

68. The bispecific molecule of claim 52 or 53, wherein said second antigen recognition portion comprises a peptide or polypeptide.

69. The bispecific molecule of claim 68, wherein said peptide or polypeptide is an antibody.

70. The bispecific molecule of claim 69, wherein said peptide or polypeptide is an monoclonal antibody.

71. The bispecific molecule of claim 68, wherein said peptide or polypeptide comprises an epitope.

72. The bispecific molecule of claim 52 or 53, wherein said second antigen recognition portion comprises a nucleic acid.

53. A bispecific molecule, comprising:

(a) a first antigen recognition portion that binds a C3b-like receptor;

(b) a linker; and

(c) a second antigen recognition portion that binds a pathogenic antigenic molecule;

wherein said second antigen recognition portion is conjugated to a carboxy terminus of said first antigen recognition portion via said linker.

54. The bispecific molecule of claim 53, wherein said linker is a peptide linker.

55. The bispecific molecule of claim 53, wherein said linker is a streptavidin-biotin linker.

56. The bispecific molecule of claim 53, wherein said linker is a Poly(ethylene glycol) linker.

57. The bispecific molecule of claim 53, wherein said first antigen recognition portion comprises an antigen binding domain that binds a C3b-like receptor on a mammalian blood cell and an effector domain that facilitates transfer of said pathogenic antigenic molecule from said mammalian blood cell to a phagocytic cell.

58. The bispecific molecule of claim 53, wherein said first antigen recognition portion is a monoclonal antibody.

73. A polyclonal library of bispecific molecules comprising a plurality of bispecific molecules each comprising (a) a first antigen recognition portion that binds a C3b-like receptor, and (b) a different second antigen recognition portion, said different second antigen recognition portions having different binding specificities, wherein said first antigen recognition portion comprises a monoclonal antibody, and wherein said second antigen recognition portion is conjugated to a carboxy terminus of said first antigen recognition portion.

80. The polyclonal library of bispecific molecules of claim 73 or 79, wherein for each bispecific molecule said second antigen recognition portion is conjugated to the C-terminus of a heavy chain of said first antigen recognition portion.

81. The polyclonal library of bispecific molecules of claim 73 or 79, wherein for each bispecific molecule said second antigen recognition portion is conjugated to the C-terminus of a light chain of said first antigen recognition portion.

82. The polyclonal library of bispecific molecules of claim 73 or 79, wherein for each bispecific molecule said second antigen recognition portion comprises two antigen recognition moieties each conjugated to the C-terminus of a different heavy chain of said first antigen recognition portion.

83. The polyclonal library of bispecific molecules of claim 73 or 79, wherein for each bispecific molecule said second antigen recognition portion comprises two antigen recognition moieties each conjugated to the C-terminus of a different light chain of said first antigen recognition portion.

84. The polyclonal library of bispecific molecules of claim 82, wherein said two antigen recognition moieties are same antigen recognition moieties.

85. The polyclonal library of bispecific molecules of claim 82, wherein said two antigen recognition moieties are different antigen recognition moieties.

86. The polyclonal library of bispecific molecules of claim 85, wherein for each bispecific molecule said different antigen recognition moieties target said pathogenic antigen cooperatively.

87. The polyclonal library of bispecific molecules of claim 85, wherein the action of one of said different antigen recognition moieties enhances the binding affinity of the other one of said different antigen recognition moieties.

88. The polyclonal library of bispecific molecules of claim 73 or 74, wherein for each bispecific molecule said second antigen recognition portion comprises a peptide or polypeptide.

89. The polyclonal library of bispecific molecules of claim 88, wherein for each bispecific molecule said peptide or polypeptide is an antibody.

90. The polyclonal library of bispecific molecules of claim 88, wherein said peptide or polypeptide is an monoclonal antibody.

91. The polyclonal library of bispecific molecules of claim 88, wherein said peptide or polypeptide comprises an epitope.

92. The polyclonal library of bispecific molecules of claim 73 or 74, wherein for each bispecific molecule said second antigen recognition portion comprises a nucleic acid.

74. A polyclonal library of bispecific molecules comprising a plurality of bispecific molecules, each comprising:

(a) a first antigen recognition portion that binds a C3b-like receptor;

(b) a linker; and

(c) a different second antigen recognition portion having a different binding specificity;

wherein said second antigen recognition portion is conjugated to a carboxy terminus of said first antigen recognition portion via said linker.

75. The bispecific molecule of claim 74, wherein said linker is a peptide linker.

76. The bispecific molecule of claim 74, wherein said linker is a streptavidin-biotin linker.

77. The bispecific molecule of claim 74, wherein said linker is a Poly (ethylene glycol) linker.

78. The polyclonal library of bispecific molecules of claim 74, wherein the first antigen recognition portion of each bispecific molecule comprises an antigen binding domain that binds a C3b-like receptor on mammalian blood cell and an effector domain that facilitates transfer of said pathogenic antigenic molecule from said mammalian blood cell to a phagocytic cell.

79. The polyclonal library of bispecific molecules of claim 74, wherein the first antigen recognition portion of each bispecific molecule is a monoclonal antibody.

93. A composition comprising a plurality of purified bispecific molecules, wherein each bispecific molecule of said plurality of purified bispecific molecules comprises a first antigen recognition portion that binds a C3b-like receptor and a second antigen recognition portion that binds a pathogenic antigenic molecule, wherein said first antigen recognition portion comprises a monoclonal antibody, and wherein said second antigen recognition portion is conjugated to a carboxy terminus of a heavy or light chain of said first antigen recognition portion, said plurality of purified bispecific molecules each comprising a different second antigen recognition portions that has a different binding specificity.

94. A composition comprising a plurality of purified bispecific molecules, wherein each bispecific molecule of said plurality of purified bispecific molecules comprises:

(a) a first antigen recognition portion that binds a C3b-like receptor; and

(b) a second antigen recognition portion that binds a pathogenic antigenic molecule;

wherein said first antigen recognition portion comprises a monoclonal antibody, and wherein said second antigen recognition portion is conjugated to a carboxy terminus of a heavy or light chain of said first antigen recognition portion, said plurality of purified bispecific molecules each comprising a different second antigen recognition portions that has a different binding specificity.

95. A molecule, comprising:

(a) an antigen recognition portion that binds a C3b-like receptor; and

(b) an N-intein moiety comprising an N-intein of a split intein;

wherein said antigen recognition portion is conjugated to the amino terminal end of said N-intein moiety.

96. The molecule of claim 95, wherein said antigen recognition portion is conjugated to said N-intein via a linker.

97. The molecule of claim 95, wherein said N-intein moiety is an N-intein.

98. The molecule of claim 95 or 96, wherein said antigen recognition portion is a monoclonal antibody.

99. The molecule of claim 95 or 96, wherein said antigen recognition portion is an ScFv fused to an Fc domain.

100. The molecule of claim 95 or 96, wherein said antigen recognition portion is a chimeric antibody comprising variable domains or complementarity determining regions (CDRs) from one species and constant domain from another species.

101. The molecule of claim 100, wherein said variable domains or complementarity determining regions (CDRs) are murine and said constant domain is human.

102. The molecule of claim 95 or 96, wherein said N-intein further comprises a sequence of 1 or more native proximal N-extein amino acid residue.

103. The molecule of claim 102, wherein said N-intein comprises a sequence of 3 to 5 native proximal N-extein amino acid residue.

104. The molecule of claim 95 or 96, wherein said N-intein comprises at the N-terminus an O-(2-nitrobenzyl)serine.

105. The molecule of claim 95 or 96, wherein said N-intein is the N-intein of the split intein encoded in the DnaE gene of Ssp.

106. The molecule of claim 102, wherein said N-intein is the N-intein of the split intein encoded in the DnaE gene of Ssp.

107. The molecule of claim 105, wherein said antigen recognition portion is a monoclonal antibody as produced by the hybridoma cell line ATCC HB 8592 from the American Type Culture Collection (ATCC).

108. The molecule of claim 106, wherein said antigen recognition portion is a monoclonal antibody as produced by the hybridoma cell line ATCC HB 8592 from the American Type Culture Collection (ATCC).

109. The molecule of claim 95 or 96, wherein said N-intein is the N-intein of an engineered split intein generated from the Mtu RecA intein.

110. A molecule, comprising:

(a) an antigen recognition portion; and

(b) a C-intein moiety comprising a C-intein of a split intein;

wherein said antigen recognition portion is conjugated to the carboxy terminal end of said C-intein moiety, and wherein the amino acid residue immediately at the C-terminal side of the splice junction of said C-intein is an amino acid residue selected from the group consisting of cysteine, serine, and threonine.

111. The molecule of claim 110, wherein said antigen recognition portion is conjugated to said C-intein via a linker.

112. The molecule of claim 110, wherein said C-intein moiety is a C-intein.

113. The molecule of claim 110 or 111, wherein said antigen recognition portion comprises a peptide or polypeptide.

114. The molecule of claim 110 or 111, wherein said antigen recognition portion comprises a nucleic acid.

115. The molecule of claim 110 or 111, wherein said C-intein further comprises, after said amino acid residue selected from the group consisting of cysteine, serine, and threonine, a sequence of 1 or more native proximal C-extein amino acid residues.

116. The molecule of claim 113, wherein said C-intein comprises, after said amino acid residue selected from the group consisting of cysteine, serine, and threonine, a sequence of 3 to 5 native proximal C-extein amino acid residues.

117. The molecule of claim 110 or 111, wherein said C-intein is the C-intein of the split intein encoded in the DnaE gene of Ssp.

118. The molecule of claim 115, wherein said C-intein is the C-intein of the split intein encoded in the DnaE gene of Ssp.

119. The C-intein antigen recognition portion of claim 117, wherein said antigen recognition portion comprises the α

-subunit of the Fcε

RI receptor.

120. The molecule of claim 110 or 111, wherein said C-intein is the C-intein of an engineered split intein generated from the Mtu RecA intein.

121. The molecule of claim 115, wherein said C-intein is the C-intein of an engineered split intein generated from the Mtu RecA intein.

122. A molecule, comprising:

(a) a streptavidin; and

(b) a C-intein of a split intein comprising, immediately at the C-terminal side of the splice junction, an amino acid residue selected from the group consisting of cysteine, serine, and threonine;

wherein said streptavidin is conjugated to the carboxy terminus of said C-intein.

123. The molecule of claim 122, wherein said streptavidin is conjugated to said C-intein via a linker.

124. The molecule of claim 122 or 123, wherein said C-intein further comprises a sequence of 1 or more native proximal C-extein amino acid residues.

125. The molecule of claim 124, wherein said C-intein further comprises a sequence of 3 to 5 native proximal C-extein amino acid residues.

126. The molecule of claim 122 or 123, wherein said C-intein is the C-intein of the split intein encoded in the DnaE gene of Ssp.

127. The molecule of claim 124, wherein said C-intein is the C-intein of the split intein encoded in the DnaE gene of Ssp.

128. The molecule of claim 122 or 123, wherein said C-intein is the C-intein of an engineered split intein generated from the Mtu RecA intein.

129. A C-intein antigen recognition portion phage display library, comprising a plurality of phage, each phage in said plurality displaying on its surface a different C-intein polypeptide fusion protein, wherein each polypeptide has a different binding specificity.

130. An expression vector, comprising a transcriptional regulatory element operably linked to a nucleotide sequence encoding a first antigen recognition portion that binds a C3b-like receptor fused to the amino terminus of the N-intein of a split intein.

131. An expression vector, comprising a transcriptional regulatory element operably linked to a nucleotide sequence encoding a second antigen recognition portion fused to the carboxy terminus of a C-intein of a split intein wherein the amino acid residue immediately at the C-terminal side of the splice junction of said C-intein is an amino acid residue selected from the group consisting of cysteine, serine, and threonine.

132. An expression vector, comprising a transcriptional regulatory element operably linked to a nucleotide sequence encoding a core streptavidin fused to the carboxy terminus of a C-intein of a split intein wherein the amino acid residue immediately at the C-terminal side of the splice junction of said C-intein is an amino acid residue selected from the group consisting of cysteine, serine, and threonine.

133. A nucleic acid encoding an N-intein fused to the carboxy terminus of a heavy chain of an anti-CR1 monoclonal antibody.

137. The nucleic acid of claim 133 or 135, wherein said N-intein is the N-intein of the split intein encoded in the DnaE gene of Ssp.

138. The nucleic acid of claim 133 or 135, wherein said N-intein is the N-intein of an engineered split intein generated from the Mtu RecA intein.

139. The nucleic acid of claim 133 or 135, wherein said anti-CR1 monoclonal antibody is as produced by the hybridoma cell line ATCC HB 8592 from the American Type Culture Collection (ATCC).

134. An expression vector, comprising a transcriptional regulatory element operably linked to a nucleotide sequence encoding an N-intein fused to the carboxy terminus of a heavy chain of an anti-CR1 monoclonal antibody.

135. A nucleic acid encoding an N-intein fused to the carboxy terminus of the light chain of an anti-CR1 monoclonal antibody.

136. An expression vector, comprising a transcriptional regulatory element operably linked to a nucleotide sequence encoding an N-intein fused to the carboxy terminus of a light chain of an anti-CR1 monoclonal antibody.

140. A nucleic acid encoding an N-intein fused to the carboxy terminus of the Fc domain that is fused to a scFv.

141. An expression vector, comprising a transcriptional regulatory element operably linked to a nucleotide sequence encoding an N-intein fused to the carboxy terminus of the Fc domain that is fused to a scFv.

142. A nucleic acid encoding a C-intein fused to the amino terminus of a polypeptide that binds an antigen.

143. An expression vector, comprising a transcriptional regulatory element operably linked to a nucleotide sequence encoding a C-intein fused to the amino terminus of polypeptide that binds an antigen.

144. A nucleic acid encoding a C-intein fused to the amino terminus of a core streptavidin via an optional linker.

145. An expression vector, comprising a transcriptional regulatory element operably linked to a nucleotide sequence encoding a C-intein fused to the amino terminus of core streptavidin via an optional linker.

146. A plurality of nucleic acids encoding a plurality of C-intein antigen recognition portions.

147. A plurality of expression vectors, each comprising a transcriptional regulatory element operably linked to a nucleotide sequence encoding a C-intein antigen recognition portion.

148. A kit comprising in a container a bispecific molecule having a first antigen recognition portion that binds a C3b-like receptor and a second antigen recognition portion that binds a pathogenic antigenic molecule, wherein said first antigen recognition portion comprises a monoclonal antibody, and wherein said second antigen recognition portion is conjugated to a carboxy terminus of a heavy or light chain of said first antigen recognition portion.

149. A kit comprising in a container a bispecific molecule comprising:

(a) a first antigen recognition portion that binds a C3b-like receptor;

(b) a linker; and

(c) a second antigen recognition portion that binds a pathogenic antigenic molecule;

wherein said second antigen recognition portion is conjugated to a carboxy terminus of said first antigen recognition portion via said linker.

150. A kit comprising in a container a molecule comprising:

(a) an antigen recognition portion that binds a C3b-like receptor; and

(b) an N-intein moiety comprising an N-intein of a split intein;

wherein said antigen recognition portion is conjugated to the amino terminal end of said N-intein moiety.

151. A kit comprising in a container a molecule comprising:

(a) an antigen recognition portion; and

(b) a C-intein moiety comprising a C-intein of a split intein;

wherein said antigen recognition portion is conjugated to the carboxy terminal end of said C-intein moiety, and wherein the amino acid residue immediately at the C-terminal side of the splice junction of said C-intein is an amino acid residue selected from the group consisting of cysteine, serine, and threonine.

152. A kit comprising in two containers a first molecule comprising an antigen recognition portion that binds a C3b-like receptor and an N-intein moiety comprising an N-intein of a split intein, wherein said antigen recognition portion is conjugated to the amino terminal end of said N-intein moiety;

and a second molecule comprising an antigen recognition portion and a C-intein moiety comprising a C-intein of a split intein, wherein said antigen recognition portion is conjugated to the carboxy terminal end of said C-intein peptide, and wherein the amino acid residue immediately at the C-terminal side of the splice junction of said C-intein is an amino acid residue selected from the group consisting of cysteine, serine, and threonine.

153. A kit comprising in one or more containers a first vector and a second vector, said first vector comprising a first DNA sequence encoding a first antigen recognition portion that binds a C3b-like receptor fused to the amino terminus of an N-intein peptide comprising an N-intein of a split intein, said second vector comprising a second DNA sequence encoding a second antigen recognition portion fused to the carboxy terminus of a C-intein peptide comprising a C-intein of said split intein, wherein the amino acid residue immediately at the C-terminal side of the splice junction of said C-intein peptide is an amino acid residue selected from the group consisting of cysteine, serine, and threonine.

156. The kit of claim 153 or 154, wherein said first vector further comprises a first promoter operably linked to said first DNA sequence, and said second vector further comprises a second promoter operably linked to said second DNA sequence.

154. A kit comprising in one or more containers a first vector and a second vector, said first vector comprising a first DNA sequence encoding a first antigen recognition portion that binds a C3b-like receptor fused to the amino terminus of an N-intein peptide comprising an N-intein of a split intein, said second vector comprising a second DNA sequence encoding a core streptavidin fused to the carboxy terminus of a C-intein peptide comprising a C-intein of said split intein wherein the amino acid residue immediately at the C-terminal side of the splice junction of said C-intein peptide is an amino acid residue selected from the group consisting of cysteine, serine, and threonine, and in a separate container a biotinylated second antigen recognition portion.

155. A kit comprising a phage display library, wherein each phage displays a different C-intein polypeptide fusion protein on its surface.

157. A cell transformed with a vector comprising a DNA sequence encoding a first antigen recognition portion that binds a C3b-like receptor fused to the amino terminus of the N-intein of a split intein.

158. A cell transformed with a vector comprising a DNA sequence encoding a second antigen recognition portion fused to the carboxy terminus of a C-intein of a split intein wherein the amino acid residue immediately at the C-terminal side of the splice junction of said C-intein is an amino acid residue selected from the group consisting of cysteine, serine, and threonine.

159. A cell transformed with a vector comprising a DNA sequence encoding a core streptavidin fused to the carboxy terminus of a C-intein of a split intein wherein the amino acid residue immediately at the C-terminal side of the splice junction of said C-intein is an amino acid residue selected from the group consisting of cysteine, serine, and threonine.

160. A cell transformed with a first vector and a second vector, said first vector comprising a first DNA sequence encoding a first antigen recognition portion that binds a C3b-like receptor fused to the amino terminus of an N-intein of a split intein, said second vector comprising a second DNA sequence encoding a second antigen recognition portion fused to the carboxy terminus of a C-intein of said split intein wherein the amino acid residue immediately at the C-terminal side of the splice junction of said C-intein is an amino acid residue selected from the group consisting of cysteine, serine, and threonine.

161. A cell containing a first antigen recognition portion that binds a C3b-like receptor fused to the amino terminus of the N-intein of a split intein.

162. A cell containing a second antigen recognition portion fused to the carboxy terminus of a C-intein of a split intein, wherein the amino acid residue immediately at the C-terminal side of the splice junction of said C-intein is an amino acid residue selected from the group consisting of cysteine, serine, and threonine.

163. A cell containing a first antigen recognition portion that binds a C3b-like receptor fused to the amino terminus of an N-intein of a split intein and a second antigen recognition portion fused to the carboxy terminus of a C-intein of said split intein, wherein the amino acid residue immediately at the C-terminal side of the splice junction of said C-intein is an amino acid residue selected from the group consisting of cysteine, serine, and threonine.

164. A method of treating a mammal having a disease or disorder or undesirable condition associated with the presence of a pathogen or pathogenic antigenic molecule, comprising administering to said mammal a therapeutically effective dose of a bispecific molecule comprising:

(a) a first antigen recognition portion that binds a C3b-like receptor, said first antigen recognition portion comprising a monoclonal antibody; and

(b) a second antigen recognition portion that binds an epitope of said pathogen or pathogenic antigenic molecule, said second antigen recognition portion being conjugated to a carboxy terminus of a heavy or light chain of said first antigen recognition portion.

166. The method of claim 164 or 165, wherein said second antigen recognition portion is conjugated to the C-terminus of a heavy chain of said first antigen recognition portion.

167. The method of claim 164 or 165, wherein said second antigen recognition portion is conjugated to the C-terminus of a light chain of said first antigen recognition portion.

168. The method of claim 164 or 165, wherein said second antigen recognition portion comprises two antigen recognition moieties each conjugated to the C-terminus of a different heavy chain of said first antigen recognition portion.

169. The method of claim 164 or 165, wherein said administering is intravenous.

170. The method of claim 164 or 165, wherein said mammal is a human, and said C3b-like receptor is CR1.

171. The method of claim 164 or 165, wherein said mammal is a non-human mammal.

172. The method of claim 170, wherein said pathogen is an autoimmune antigen.

173. The method of claim 170, wherein said pathogen is an infectious agent.

174. The method of claim 173, wherein said infectious agent is a virus.

175. The method of claim 174, wherein said virus is HIV-1 virus.

176. The method of claim 173, wherein said infectious agent is a bacterium.

177. The method of claim 176, wherein said bacterium is Bacillus anthracis.

178. The method of claim 173, wherein said infectious agent is a fungus.

179. The method of claim 173, wherein said infectious agent is a parasite.

180. The method of claim 179, wherein said parasite is a protozoan.

181. The method of claim 170, wherein said pathogenic antigenic molecule is a toxin.

165. A method of preventing a disease or disorder or undesirable condition associated with the presence of a pathogen or pathogenic antigenic molecule in a mammal, comprising administering to said mammal a therapeutically effective dose of a bispecific molecule comprising:

(a) a first antigen recognition portion that binds a C3b-like receptor, said first antigen recognition portion comprising a monoclonal antibody; and

(b) a second antigen recognition portion that binds an epitope of said pathogen or pathogenic antigenic molecule, said second antigen recognition portion being conjugated to a carboxy terminus of a heavy or light chain of said first antigen recognition portion.

182. A method of treating a mammal having an undesirable condition associated with the presence of a pathogen or pathogenic antigenic molecule comprising the steps of:

(a) contacting a bispecific molecule with hematopoietic cells expressing a C3b-like receptor, to form a hematopoietic cell/bispecific molecule complex, wherein the bispecific molecule comprises (i) a first antigen recognition portion that binds said C3b-like receptor, said first antigen recognition portion comprising a monoclonal antibody; and

(ii) a second antigen recognition portion that binds an epitope of said pathogen or pathogenic antigenic molecule, said second antigen recognition portion being conjugated to a carboxy terminus of a heavy or light chain of said first antigen recognition portion; and

(b) administering said hematopoietic cell/bispecific molecule complex to said mammal in a therapeutically effective amount.

183. A method of treating a mammal having an undesirable condition associated with the presence of a pathogen or pathogenic antigenic molecule, comprising the step of administering a therapeutically effective amount of a hematopoietic cell/bispecific molecule complex to said mammal, said complex consisting essentially of a hematopoietic cell expressing a C3b-like receptor bound to one or more bispecific molecules, wherein said bispecific molecule comprises (a) a first antigen recognition portion that binds said C3b-like receptor, said first antigen recognition portion comprising a monoclonal antibody;

and (b) a second antigen recognition portion that binds an epitope of said pathogen or pathogenic antigenic molecule, said second antigen recognition portion being conjugated to a carboxy terminus of a heavy or light chain of said first antigen recognition portion.