Grafting of a Murine Antibody Onto a Human Framework
Description of Invention:
This invention relates to humanization of antibodies specifically providing novel biophysically stable human framework sequences that can be used to humanize antibody single chain Fv (scFv) fragments. An exemplary RFB4 humanized scFv antibody was constructed using the new sequences. The novel sequences were obtained after stringent panning of a human phage display library on (irrelevant) antigen. These antibody variable domain frameworks were subsequently used as human acceptor scaffolds for grafting the murine antibody specificity. The general approach described here differs from other humanization procedures wherein appropriate human acceptor scaffolds are selected from either antibodies with solved crystal structures or (germline) sequence databases. In the current invention, human acceptor frameworks were first pre-selected for stability. Appropriate framework sequences with high sequence identities to the murine antibody to be humanized were then chosen from the pre-selected pool of stable scaffolds. As a result, humanized scFv fragments with low immunogenic potential and high biophysical stability were generated.
In contrast to other methodologies, unusual human framework residues were identified by aligning the human variable domain sequences to several sequence reference templates from antibody repertoires. The structural role of each identified unusual residue was further examined on the basis of information of antibodies with known crystal structures. Several residues were considered critical for interfering with the structural integrity of the antigen binding site and were successively back-mutated to the murine donor sequence. As a result, a panel of three humanized scFv antibodies with nanomolar affinity constants were generated. Importantly, the introduced back-mutations did not alter the biophysical properties of the constructs.
Inventors:
S. Rybak (NCI) J. Krauss (NCI) M. Arndt (NCI) et al.
Patent Status:
DHHS Reference No. E-056-2002/0 --
U.S. Provisional Application No. 60/390,033 filed 17 Jun 2002
PCT Application No. PCT/US03/19333 filed 17 Jun 2003, which published as WO 2003/105782 on 24 Dec 2003
U.S. Patent Application No. 10/515,149 filed 18 Nov 2004, allowed
Portfolios: Cancer
Cancer -Therapeutics
For Additional Information Please Contact: David A. Lambertson Ph.D.
NIH Office of Technology Transfer
6011 Executive Blvd, Suite 325
Rockville, MD 20852-3804
Phone: (301)435-4632
Email: lambertsond@mail.nih.gov
Fax: (301) 402-0220
