Artificial antibody polypeptides

Abstract

The present invention provides a fibronectin type III (Fn3) molecule, wherein the Fn3 contains a stabilizing mutation. The present invention also provides Fn3 polypeptide monobodies, nucleic acid molecules encoding monobodies, and variegated nucleic acid libraries encoding such monobodies. Also provided are methods of preparing a Fn3 polypeptide monobody, and kits to perform the methods.

Description

[0001]Portions of the present invention were made with support of the United States Government via a grant from the National Institutes of Health under grant number GM 55042 The U.S. Government therefore may have certain rights in the invention.FIELD OF THE INVENTION[0002]The present invention relates generally to the field of the production and selection of binding and catalytic polypeptides by the methods of molecular biology. The invention specifically relates to the generation of both nucleic acid and polypeptide libraries encoding the molecular scaffolding of a modified Fibronectin Type III (Fn3) molecule. The invention also relates to “artificial mini-antibodies” or “monobodies,” i.e., polypeptides containing an Fn3 scaffold onto which loop regions capable of binding to a variety of different molecular structures (such as antibody binding sites) have been grafted.BACKGROUND OF THE INVENTIONAntibody Structure[0003]A standard antibody (Ab) is a tetrameric structure consisting of...

AI Technical Summary

Problems solved by technology

However, the size of the antibodies and the complexity of six loops represents a major design hurdle if the end result is to be a relatively small peptide ligand.

These antibody fragments are not suitable for structural analysis using NMR spectroscopy due to their size, low solubility or low conformational stability.

However, the short persistence of scFvs in the circulation limits the exposure of tumor cells to the scFvs, placing limits on the level of uptake.

Since X-ray crystallography is not suited for characterizing flexible parts of molecules, structural studies in the solution state have not been possible to provide dynamic pictures of the conformation of antigen-binding sites.

However, as expected, they are too small and too flexible to maintain full affinity and specificity.

Mouse CDRs have been grafted onto the human Ig framework without the loss of affinity (Jones et al., 1986; Riechmann et al., 1988), though this “humanization” does not solve the above-mentioned problems specific to solution studies.

Thus, while Tendamistat may be antigenic in humans, its small size may reduce or inhibit its antigenicity.

Also, Tendamistat's stability is uncertain.

Disulfide bonds, however, are a significant disadvantage to such molecules in that they can be broken under reducing conditions and must be properly formed in order to have a useful protein structure.

Further, the size of the loops in Tendamistat are relatively small, thus limiting the size of the inserts that can be accommodated in the scaffold.

Moreover, it is well known that forming correct disulfide bonds in newly synthesized peptides is not straightforward.

When a protein is expressed in the cytoplasmic space of E. coli, the most common host bacterium for protein overexpression, disulfide bonds are usually not formed, potentially making it difficult to prepare large quantities of engineered molecules.

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