Binding polypeptides Number:7,119,171 from the United States Patent and Trademark Office (PTO) owispatent The present invention provides modified fibronectin type III (Fn3) molecules, nucleic acid molecules encoding the modified Fn3 molecules, and related vectors and host cells.<H polypeptides, Binding, Binding, polypep, 7119171.html, Patent, pto, 7119171

Title: Binding polypeptides

Abstract: The present invention provides modified fibronectin type III (Fn3) molecules, nucleic acid molecules encoding the modified Fn3 molecules, and related vectors and host cells.

Patent Number: 7,119,171 Issued on 10/10/2006 to Koide

Inventors: Koide; Shohei (Rochester, NY)
Assignee: Research Corporation Technologies, Inc. (Tucson, AZ)

Appl. No.: 10/165,155

Filed: June 6, 2002

Related U.S. Patent Documents


Application Number	Filing Date	Patent Number	Issue Date
09096749	Jun., 1998	6673901
60049410	Jun., 1997

Current U.S. Class: 530/387.1 ; 530/300; 530/350; 530/387.3

Current International Class: C07K 16/00 (20060101); C07K 2/00 (20060101); C07K 4/00 (20060101); C07K 5/00 (20060101); C07K 7/00 (20060101)

Field of Search: 530/387.1,387.3,300,350,380,381 424/130.1 514/2

References Cited [Referenced By]

U.S. Patent Documents


6348584	February 2002	Hodgson et al.
6391855	May 2002	Blaschuk et al.
6462189	October 2002	Koide
6673901	January 2004	Koide
6703199	March 2004	Koide
6818418	November 2004	Lipovsek et al.
2003/0027319	February 2003	Koide
2003/0108948	June 2003	Koide
2003/0134386	July 2003	Koide
2003/0170753	September 2003	Koide
2003/0186385	October 2003	Koide
2004/0259155	December 2004	Chan et al.
2005/0038229	February 2005	Lipovsek et al.

Foreign Patent Documents


8-511417	Mar., 1996	JP
WO 94/18221	Aug., 1994	WO
WO 94/24278	Oct., 1994	WO
WO-94/24278	Oct., 1994	WO
WO 95/27045	Oct., 1995	WO
WO 98/56915	Dec., 1998	WO
WO 00/34784	Jun., 2000	WO
WO 01/64942	Sep., 2001	WO
WO 02/04523	Jan., 2002	WO
WO 03/104418	Dec., 2003	WO
WO 04/019878	Mar., 2004	WO
WO 05/056764	Jun., 2005	WO

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Primary Examiner: Harris; Alana M.
Attorney, Agent or Firm: Viksnins Harris & Padys PLLP

Parent Case Text

RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 09/096,749, filed Jun. 12, 1998 (which issued as U.S. Pat. No. 6.673,901), which claims priority under 35 U.S.C. 119(e) to provisional application U.S. Ser. No. 60/049,410, filed Jun. 12, 1997. These applications are incorporated herein by reference.

Claims

What is claimed is:

1. A fibronectin type III (Fn3) monobody polypeptide comprising at least two Fn3 .beta.-strand domain sequences with a ioop region linked between each Fn3 .beta.-strand domain sequence, wherein at least one monobody loop region sequence varies as compared to the wild-type (SEQ ID NO:110, FIG. 2) loop region sequence by deletion of two to twelve amino acids in the loop region sequence, insertion of at least two to 25 amino acids, or replacement of at least two amino acids in the loop region sequence, and wherein the monobody polypeptide is capable of binding to a target molecule with a dissociation constant of less than 10.sup.-6 moles/liter.

2. The monobody of claim 1, wherein at least one loop region binds to the target molecule.

3. The monobody of claim 1, wherein a loop region comprises amino acid residues: i) from 15 to 16 inclusive in an AB loop; ii) from 22 to 30 inclusive in a BC loop; iii) from 39 to 45 inclusive in a CD loop; iv) from 51 to 55 inclusive in a DE loop; v) from 60 to 66 inclusive in an EF loop; or vi) from 76 to 87 inclusive in an FG loop.

4. The monobody of claim 1, wherein the monobody loop region sequence varies from a corresponding wild-type Fn3 loop region sequence by the replacement of at least two amino acids in the loop region or the deletion of two to twelve amino acids in the loop region.

5. The monobody of claim 1, wherein the monobody loop region sequence varies from a corresponding wild-type Fn3 loop region sequence by the insertion of from two to 25 amino acids.

Description

FIELD OF THE INVENTION

The present invention relates generally to the field of the production and selection of binding and catalytic polypeptides by the methods of molecular biology, using both combinatorial chemistry and recombinant DNA. The invention specifically relates to the generation of both nucleic acid and polypeptide libraries derived therefrom encoding the molecular scaffolding of Fibronectin Type III (Fn3) modified in one or more of its loop regions. The invention also relates to the "artificial mini-antibodies" or "monobodies," i.e., the polypeptides comprising 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 INVENTION

Antibody structure

A standard antibody (Ab) is a tetrameric structure consisting of two identical immunoglobulin (Ig) heavy chains and two identical light chains. The heavy and light chains of an Ab consist of different domains. Each light chain has one variable domain (VL) and one constant domain (CL), while each heavy chain has one variable domain (VH) and three or four constant domains (CH) (Alzari et al., 1988). Each domain, consisting of .about.110 amino acid residues, is folded into a characteristic .beta.-sandwich structure formed from two .beta.-sheets packed against each other, the immunoglobulin fold. The VH and VL domains each have three complementarity determining regions (CDR1 3) that are loops, or turns, connecting .beta.-strands at one end of the domains (FIG. 1: A, C). The variable regions of both the light and heavy chains generally contribute to antigen specificity, although the contribution of the individual chains to specificity is not always equal. Antibody molecules have evolved to bind to a large number of molecules by using six randomized loops (CDRs). 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.

Antibody Substructures

Functional substructures of Abs can be prepared by proteolysis and by recombinant methods. They include the Fab fragment, which comprises the VH-CH1 domains of the heavy chain and the VL-CL1 domains of the light chain joined by a single interchain disulfide bond, and the Fv fragment, which comprises only the VH and VL domains. In some cases, a single VH domain retains significant affinity (Ward et al., 1989). It has also been shown that a certain monomeric .kappa. light chain will specifically bind to its cognate antigen. (L. Masat et al., 1994). Separated light or heavy chains have sometimes been found to retain some antigen-binding activity (Ward et al., 1989). These antibody fragments are not suitable for structural analysis using NMR spectroscopy due to their size, low solubility or low conformational stability.

Another functional substructure is a single chain Fv (scFv), comprised of the variable regions of the immunoglobulin heavy and light chain, covalently connected by a peptide linker (S-z Hu et al., 1996). These small (M, 25,000) proteins generally retain specificity and affinity for antigen in a single polypeptide and can provide a convenient building block for larger, antigen-specific molecules. Several groups have reported biodistribution studies in xenografted athymic mice using scFv reactive against a variety of tumor antigens, in which specific tumor localization has been observed. 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. As a result, tumor uptake by scFvs in animal studies has generally been only 1 5% ID/g as opposed to intact antibodies that can localize in tumors ad 30 40% ID/g and have reached levels as high as 60 70% ID/g.

A small protein scaffold called a "minibody" was designed using a part of the Ig VH domain as the template (Pessi et al., 1993). Minibodies with high affinity (dissociation constant (K.sub.d).about.10.sup.-7 M) to interleukin-6 were identified by randomizing loops corresponding to CDR1 and CDR2 of VH and then selecting mutants using the phage display method (Martin et al., 1994). These experiments demonstrated that the essence of the Ab function could be transferred to a smaller system. However, the minibody had inherited the limited solubility of the VH domain (Bianchi et al., 1994).

It has been reported that camels (Camelus dromedarius) often lack variable light chain domains when IgG-like material from their serum is analyzed, suggesting that sufficient antibody specificity and affinity can be derived form VH domains (three CDR loops) alone. Davies and Riechmann recently demonstrated that "camelized" VH domains with high affinity (K.sub.d.about.10.sup.-7 M) and high specificity can be generated by randomizing only the CDR3. To improve the solubility and suppress nonspecific binding, three mutations were introduced to the framework region (Davies & Riechmann, 1995). It has not been definitively shown, however, that camelization can be used, in general, to improve the solubility and stability of VHs.

An alternative to the "minibody" is the "diabody." Diabodies are small bivalent and bispecific antibody fragments, i.e., they have two antigen-binding sites. The fragments comprise a heavy-chain variable domain (V.sub.H) connected to a light-chain variable domain (V.sub.L) on the same polypeptide chain (V.sub.H V.sub.L). Diabodies are similar in size to an Fab fragment. By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. These dimeric antibody fragments, or "diabodies," are bivalent and bispecific. P. Holliger et al., PNAS 90:6444 6448 (1993).

Since the development of the monoclonal antibody technology, a large number of 3D structures of Ab fragments in the complexed and/or free states have been solved by X-ray crystallography (Webster et al., 1994; Wilson & Stanfield, 1994). Analysis of Ab structures has revealed that five out of the six CDRs have limited numbers of peptide backbone conformations, thereby permitting one to predict the backbone conformation of CDRs using the so-called canonical structures (Lesk & Tramontano, 1992; Rees et al., 1994). The analysis also has revealed that the CDR3 of the VH domain (VH-CDR3) usually has the largest contact surface and that its conformation is too diverse for canonical structures to be defined; VH-CDR3 is also known to have a large variation in length (Wu et al., 1993). Therefore, the structures of crucial regions of the Ab-antigen interface still need to be experimentally determined.

Comparison of crystal structures between the free and complexed states has revealed several types of conformational rearrangements. They include side-chain rearrangements, segmental movemen