# US 7,405,276 B2 · Method of producing bispecific molecules by protein trans-splicing > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 7,405,276 B2 - Document ID: US7405276B2 - Status: Granted - Kind: B2 - Application number: 10/415,840 - Filed: 2001-11-01 - Published: 2004-04-22 - Granted: 2008-07-29 - Earliest claimed: 2000-11-01 - Assignees: EluSys Therapeutics, Inc. - Inventors: Jeff Himawan - Web page: https://patentgrep.com/patent/US7405276B2 ## CPC - C07K 14/70535: {Fc-receptors, e.g. CD16, CD32, CD64 (CD2314/705F)} - A61P 31/04: Antibacterial agents - A61P 31/10: Antimycotics - A61P 31/12: Antivirals - A61P 31/18: for HIV - A61P 33/00: Antiparasitic agents - A61P 33/02: Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis - A61P 37/06: Immunosuppressants, e.g. drugs for graft rejection - A61P 39/02: Antidotes - C07K 16/283: {against Fc-receptors, e.g. CD16, CD32, CD64 (CD23 C07K16/2851)} - C07K 16/2896: {against molecules with a "CD"-designation, not provided for elsewhere} - C07K 16/468: {Immunoglobulins having two or more different antigen binding sites, e.g. multifunctional antibodies} - C12N 15/62: DNA sequences coding for fusion proteins - A61K 2039/505: {comprising antibodies} - C07K 2317/31: multispecific - C07K 2317/52: Constant or Fc region; Isotype - C07K 2319/22: containing a Strep-tag - C07K 2319/30: Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto - C07K 2319/92: containing an intein ("protein splicing")domain - C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00) - A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D) ## 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 ### Claim 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 containing a splice junction 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. ### Claim 2 (depends on 1) The method of claim 1, further comprising: (a) obtaining said N-intein first antigen recognition portion by conjugating said first antigen recognition portion to the amino terminus of a molecule comprising said N-intein of a split intein before contacting an N-intein first antigen recognition portion and a C-intein second antigen recognition portion under conditions such that protein trans-splicing occurs; and (b) obtaining said C-intein second antigen recognition portion by conjugating said second antigen recognition portion to the carboxy terminus of a molecule comprising said C-intein of said split intein before contacting an N-intein first antigen recognition portion and a C-intein second antigen recognition portion under conditions such that protein trans-splicing occurs. ### Claim 3 (depends on 1, 2) The method of claim 1 or 2, wherein said split intein is a naturally occurring split intein. ### Claim 4 (depends on 3) 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. ### Claim 5 (depends on 1, 2) 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. ### Claim 6 (depends on 5) The method of claim 5, wherein said engineered split intein is generated from the Mycobacterium tuberculosis RecA intein. ### Claim 7 (depends on 1, 2) 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 residues attached to the amino terminus of the splice junction of said N-intein. ### Claim 8 (depends on 7) The method of claim 7, wherein said N-intein comprises a sequence of 3 to 5 native proximal N-extein amino acid residues attached to the amino terminus of the splice junction of said N-intein. ### Claim 9 (depends on 1, 2) 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. ### Claim 10 (depends on 9) The method of claim 9, wherein said C-intein further comprises a sequence of 1 or more native proximal C-extein amino acid residues attached to the carboxy terminus of the splice junction of said C-intein. ### Claim 11 (depends on 10) The method of claim 10, wherein said C-intein comprises a sequence of 3 to 5 native proximal C-extein amino acid residues attached to the carboxy terminus of the splice junction of said C-intein. ### Claim 12 (depends on 1, 2) 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 a normal amino acid residue by a suitable means said N-intein becomes functional for trans-splicing. ### Claim 13 (depends on 12) 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. ### Claim 14 (depends on 1, 2) The method of claim 1 or 2, wherein said first antigen recognition portion comprises an antigen binding domain that binds a C3-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. ### Claim 15 (depends on 1, 2) The method of claim 1 or 2, wherein said first antigen recognition portion is an monoclonal antibody. ### Claim 16 (depends on 15) 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. ### Claim 17 (depends on 15) 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. ### Claim 18 (depends on 15) The method of claim 15, wherein the heavy and light chains of said monoclonal antibody are recombinantly expressed using same vector. ### Claim 19 (depends on 15) The method of claim 15, wherein the heavy and light chains of said monoclonal antibody are recombinantly expressed using separate vectors. ### Claim 20 (depends on 19) The method of claim 19, wherein said heavy and light chains of said monoclonal antibody are recombinantly expressed in same cell. ### Claim 21 (depends on 19) The method of claim 19, wherein said heavy and light chains of said monoclonal antibody are recombinantly expressed in separate cells using separate vectors, and wherein said heavy and light chains are assembled into said monoclonal antibody in vitro. ### Claim 22 (depends on 1, 2) The method of claim 1 or 2, wherein said first antigen recognition portion is an ScFv that binds a C3-like receptor fused to the N-terminus of an immunoglobulin Fc domain. ### Claim 23 (depends on 1, 2) The method of claim 1 or 2, wherein said second antigen recognition portion comprises a polypeptide. ### Claim 24 (depends on 1, 2) The method of claim 1 or 2, wherein said N-intein first antigen recognition portion is produced recombinantly. ### Claim 25 (depends on 1, 2) The method of claim 1 or 2, wherein said C-intein second antigen recognition portion is produced recombinantly. ### Claim 26 (depends on 1, 2) The method of claim 1 or 2, wherein said C-intein second antigen recognition portion is produced by peptide synthesis. ### Claim 27 (depends on 1, 2) The method of claim 1 or 2, wherein said 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. ### Claim 28 (depends on 27) The method of claim 27, wherein said N-intein first antigen recognition portion and said C-intein second antigen recognition portion are expressed by separate expression vectors. ### Claim 29 (depends on 27) The method of claim 27, wherein said N-intein first antigen recognition portion and said C-intein second antigen recognition portion are expressed by one expression vector. ### Claim 30 (depends on 1, 2) 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.