Methods of making recombinant disulfide-stabilized polypeptide fragments having binding specificity

• US 6,558,672 B1
• Filed: 09/07/2000
• Issued: 05/06/2003
• Est. Priority Date: 06/14/1993
• Status: Expired due to Term

• Current Assignee: United States Department Of Health And Human Services
• Original Assignee: United States Department Of Health And Human Services
• Inventors: Brinkmann, Ulrich, Lee, Byungkook, Jung, Sun-Hee, Pastan, Ira H.
• Primary Examiner(s): Nolan, Patrick J.
• Assistant Examiner(s): Ewoldt, Gerald R
• Application Number:
  US09/657,274
• Time in Patent Office: 971 Days
• Field of Search: 424/184.1, 424/185.1, 435/69.1, 435/71.2, 536/23.1, 536/23.53
• US Class Current: 424/185.1
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61K 47/6829 Bacterial toxins, e.g. diphteria toxins or Pseudomonas exotoxin A
• CPC Class Codes: A61K 47/6835 the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: C07K 14/7051 T-cell receptor (TcR)-CD3 complex
• 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 2319/00 Fusion polypeptide
• CPC Class Codes: C07K 2319/33 fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
• CPC Class Codes: C07K 2319/55 containing a fusion with a toxin, e.g. diphteria toxin
• CPC Class Codes: Y10S 530/866 involving immunoglobulin or antibody fragment, e.g. fab', fab, fv, fc, heavy chain or light chain
• CPC Class Codes: Y10S 530/867 involving immunoglobulin or antibody produced via recombinant dna technology

Abstract

The present invention relates to disulfide-stabilized recombinant polypeptide molecules which have the binding ability and specificity for another peptide, such as the variable region of an antibody molecule. Methods of producing these molecules and nucleic acid sequences encoding these molecules are also described. In particular, the invention discloses Fv antibody fragments stabilized by a disulfide bond connecting the V_H and V_L regions of the Fv fragment. The α and β chains of T cell receptors may be similarly stabilized by means described in the invention.

Claims

1. A method of producing a polypeptide specifically binding a ligand, the polypeptide comprising a first variable region of an antibody connected through a disulfide body to a second variable region of the antibody in framework regions of the two variable regions, the method comprising the steps of:

(a) mutating a nucleic acid encoding the first variable region so that cysteine is encoded at position 42, 43, 44, 45 or 46, and mutating a nucleic acid sequence encoding the second variable region so that cysteine is encoded at position 103, 104, 105, or 106, such positions being determined in accordance with the numbering scheme published by Kabat and Wu, corresponding to a light chain and a heavy chain region, respectively, of an antibody;

or

(b) mutating a nucleic acid encoding the first variable region so that cysteine is encoded at position 43, 44, 45, 46 or 47 and mutating a nucleic acid encoding the second variable region so that cysteine is encoded at position 98, 99, 100, or 101 such positions being determined in accordance with the numbering scheme published by Kabat and Wu, corresponding to a heavy chain or a light chain region respectively of an antibody;

then

(c) expressing the nucleic acid for the first variable region and the nucleic acid for the second variable region in an expression system; and

(d) recovering the polypeptide having a binding affinity for the antigen.

2. The method of claim 1, wherein the method further comprises purifying the polypeptide.