Enrichment method for variant proteins with altered binding properties

Abstract

A method for selecting novel proteins such as growth hormone and antibody fragment variants having altered binding properties for their respective receptor molecules is provided. The method comprises fusing a gene encoding a protein of interest to the carboxy terminal domain of the gene III coat protein of the filamentous phage M13. The gene fusion is mutated to form a library of structurally related fusion proteins that are expressed in low quantity on the surface of a phagemid particle. Biological selection and screening are employed to identify novel ligands useful as drug candidates. Disclosed are preferred phagemid expression vectors and selected human growth hormone variants.

Description

RELATED APPLICATIONS [0001] This application is a continuation of application Ser. No. 09 / 717,641 filed Nov. 21, 2000; which is a continuation of application Ser. No. 08 / 922,345 filed Sep. 3, 1997; which is a continuation of application Ser. No. 08 / 463,587 filed Jun. 5, 1995, now issued as U.S. Pat. No. 5,821,047; which is a divisional of application Ser. No. 08 / 050,058 filed Apr. 30, 1993, now issued as U.S. Pat. No. 5,750,373; which is a 371 of International Application No. PCT / US91 / 09133 filed Dec. 3, 1991; which is a continuation-in-part of application Ser. No. 07 / 743,614 filed Aug. 9, 1991, now abandoned; which is a continuation-in-part of application Ser. No. 07 / 715,300 filed Jun. 14, 1991, now abandoned; which is a continuation-in-part of application Ser. No. 07 / 683,400 filed Apr. 10, 1991, now abandoned; which is a continuation-in-part of application Ser. No. 07 / 621,667 filed Dec. 3, 1990, now abandoned, the contents of these applications are incorporated herein by reference...

AI Technical Summary

Benefits of technology

[0028] Preferably the first gene will encode a polypeptide of one or more subunits containing more than about 100 amino acid residues and will be folded to form a plurality of rigid secondary structures displaying a plurality of amino acids capable of interacting with the target. Preferably the first gene will be mutated at codons corresponding to only the amino acids capable of interacting with the target so that the integrity of the rigid secondary structures will be preserved.

Problems solved by technology

The problem with prior art methods is that naturally occurring ligands may not have proper characteristics for all therapeutic applications.

Additionally, polypeptide ligands may not even be available for some target substances.

Furthermore, methods for making non-naturally occurring synthetic binding partners are often expensive and difficult, usually requiring complex synthetic methods to produce each candidate.

The inability to characterize the structure of the resulting candidate so that rational drug design methods can be applied for further optimization of candidate molecules further hampers these methods.

The Geysen et al. method suffers from the disadvantage that the chemistry upon which it is based, peptide synthesis, produces molecules with ill-defined or variable secondary and tertiary structure.

Furthermore, sequences that do not facilitate conformationally stable secondary structures provide complex peptide-sidechain interactions which may prevent sidechain interactions of a given amino acid with the target molecule.

Additionally, candidates may exist in numerous conformations making it difficult to identify the conformer that interacts or binds to the target with greatest affinity or specificity complicating rational drug design.

A final problem with the iterative polypeptide method of Geysen is that, at present, there are no practical methods with which a great diversity of different peptides can be produced, screened and analyzed.

Even having prepared such a diversity of peptides, there are no methods available with which mixtures of such a diversity of peptides can be rapidly screened to select those peptides having a high affinity for the target molecule.

There are, however, several important limitations in using such “fusion phage” to identify altered peptides or proteins with new or enhanced binding properties.

Second, prior art methods have been unable to select peptides from a library having the highest binding affinity for a target molecule.

Ladner does not address the problem of successfully panning high affinity peptides from the random peptide library which plagues the biological selection and screening methods of the prior art.

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