Recombinant production of immunoglobulin-like domains in prokaryotic cells

• US 6,165,745 A
• Filed: 11/17/1994
• Issued: 12/26/2000
• Est. Priority Date: 04/24/1992
• Status: Expired due to Fees

• Current Assignee: Board of Regents of the University of Texas System (University of Texas System)
• Original Assignee: Board of Regents of the University of Texas System (University of Texas System)
• Inventors: Kim, Jin-Kyoo, Ward, E. Sally
• Primary Examiner(s): SCHEINER, LAURIE A
• Application Number:
  US08/341,560
• Time in Patent Office: 2,231 Days
• Field of Search: 536/25.3, 435/69.1, 435/71.1, 435/172.3, 435/252.3, 435/317.1, 435/320.1, 436/547
• US Class Current: 435/69.1
• CPC Class Codes: A61K 47/6827 Ricin A
• CPC Class Codes: A61K 47/6849 the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
• CPC Class Codes: C07K 14/70503 Immunoglobulin superfamily
• CPC Class Codes: C07K 14/7051 T-cell receptor (TcR)-CD3 complex
• CPC Class Codes: C07K 14/70514 CD4
• CPC Class Codes: C07K 14/70535 Fc-receptors, e.g. CD16, CD32, CD64 (CD2314/705F)
• CPC Class Codes: C07K 14/70539 MHC-molecules, e.g. HLA-molecules
• CPC Class Codes: C07K 16/00 Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies antibodies with enzymatic activity, e.g. abzymes C12N9/0002
• CPC Class Codes: C07K 2317/52 Constant or Fc region; Isotype
• CPC Class Codes: C07K 2317/53 Hinge
• CPC Class Codes: C07K 2319/00 Fusion polypeptide

Abstract

Disclosed are recombinant vectors encoding immunoglobulin-like domains and portions thereof, such as T-cell variable domains, antibody Fc-hinge fragments, subfragments and mutant domains with reduced biological half lives. Methods of producing large quantities of such domains, heterodimers, and fusion proteins following expression and secretion by prokaryotic host cells are also reported. Described are single chain T-cell receptors, which are folded into β-pleated sheet structures similar to those of immunoglobulin variable domains; antibody Fc and Fc-hinge domains, which have the same in vivo stability as intact antibodies; and domains engineered to have reduced half lives.

Claims

1. A method for producing an antibody with a decreased biological half life, comprising the steps of:

a) obtaining a DNA segment that encodes an antibody with an Fc-hinge domain;

b) introducing a mutation into said DNA segment encoding said antibody wherein expression of said DNA segment results in an antibody in which the Fc-hinge domain comprises an amino acid mutation in the CH2-CH3 domain interface region of the Fc-hinge fragment which results in impaired SpA binding relative to native Fc-hinge domain SpA binding; and

c) expressing said antibody.

2. The method of claim 1, wherein the antibody is a recombinant antibody and the mutation is introduced into a DNA segment encoding the antibody by site-specific mutagenesis.

3. The method of claim 1, wherein the amino acid mutation lies in the region between about residue 253 and about residue 434 of SEQ ID NO:

17.

4. The method of claim 3, wherein the mutation is an amino acid substitution at isoleucine 253, histidine 310, glutamine 311, histidine 433 or asparagine 434 of SEQ ID NO:

17.

5. The method of claim 4, wherein alanine is substituted for histidine 310 of SEQ ID NO:

17.

6. The method of claim 4, wherein asparagine is substituted for glutamine 311 of SEQ ID NO:

17.

7. The method of claim 4, wherein alanine is substituted for histidine 433 of SEQ ID NO:

17.

8. The method of claim 4, wherein glutamine is substituted for asparagine 434 of SEQ ID NO:

17.

9. The method of claim 1, wherein said antibody is conjugated to an imaging agent.

10. The method of claim 9, wherein said imaging agent comprises a paramagnetic ion, a radioactive agent or a fluorogenic agent.

11. The method of claim 1, wherein said antibody is conjugated to a toxin.

12. The method of claim 11, wherein said toxin is a ricin A toxin.