Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
2000-02-29
2004-11-16
Tate, Christopher R. (Department: 1654)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S007100
Reexamination Certificate
active
06818418
ABSTRACT:
BACKGROUND OF THE INVENTION
This invention relates to protein scaffolds useful, for example, for the generation of products having novel binding characteristics.
Proteins having relatively defined three-dimensional structures, commonly referred to as protein scaffolds, may be used as reagents for the design of engineered products. These scaffolds typically contain one or more regions which are amenable to specific or random sequence variation, and such sequence randomization is often carried out to produce libraries of proteins from which desired products may be selected. One particular area in which such scaffolds are useful is the field of antibody design.
A number of previous approaches to the manipulation of the mammalian immune system to obtain reagents or drugs have been attempted. These have included injecting animals with antigens of interest to obtain mixtures of polyclonal antibodies reactive against specific antigens, production of monoclonal antibodies in hybridoma cell culture (Koehler and Milstein, Nature 256:495, 1975), modification of existing monoclonal antibodies to obtain new or optimized recognition properties, creation of novel antibody fragments with desirable binding characteristics, and randomization of single chain antibodies (created by connecting the variable regions of the heavy and light chains of antibody molecules with a flexible peptide linker) followed by selection for antigen binding by phage display (Clackson et al., Nature 352:624, 1991).
In addition, several non-immunoglobulin protein scaffolds have been proposed for obtaining proteins with novel binding properties. For example, a “minibody” scaffold, which is related to the immunoglobulin fold, has been designed by deleting three beta strands from a heavy chain variable domain of a monoclonal antibody (Tramontano et al., J. Mol. Recognit. 7:9, 1994). This protein includes 61 residues and can be used to present two hypervariable loops. These two loops have been randomized and products selected for antigen binding, but thus far the framework appears to have somewhat limited utility due to solubility problems. Another framework used to display loops has been tendamistat, a 74 residue, six-strand beta sheet sandwich held together by two disulfide bonds (McConnell and Hoess, J. Mol. Biol. 250:460, 1995). This scaffold includes three loops, but, to date, only two of these loops have been examined for randomization potential.
Other proteins have been tested as frameworks and have been used to display randomized residues on alpha helical surfaces (Nord et al., Nat. Biotechnol. 15:772, 1997; Nord et al., Protein Eng. 8:601, 1995), loops between alpha helices in alpha helix bundles (Ku and Schultz, Proc. Natl. Acad. Sci. USA 92:6552, 1995), and loops constrained by disulfide bridges, such as those of the small protease inhibitors (Markland et al., Biochemistry 35:8045, 1996; Markland et al., Biochemistry 35:8058, 1996; Rottgen and Collins, Gene 164:243, 1995; Wang et al., J. Biol. Chem. 270:12250, 1995).
SUMMARY OF THE INVENTION
The present invention provides a new family of proteins capable of evolving to bind any compound of interest. These proteins, which make use of a fibronectin or fibronectin-like scaffold, function in a manner characteristic of natural or engineered antibodies (that is, polyclonal, monoclonal, or single-chain antibodies) and, in addition, possess structural advantages. Specifically, the structure of these antibody mimics has been designed for optimal folding, stability, and solubility, even under conditions which normally lead to the loss of structure and function in antibodies.
These antibody mimics may be utilized for the purpose of designing proteins which are capable of binding to virtually any compound (for example, any protein) of interest. In particular, the fibronectin-based molecules described herein may be used as scaffolds which are subjected to directed evolution designed to randomize one or more of the three fibronectin loops which are analogous to the complementarity-determining regions (CDRs) of an antibody variable region. Such a directed evolution approach results in the production of antibody-like molecules with high affinities for antigens of interest. In addition, the scaffolds described herein may be used to display defined exposed loops (for example, loops previously randomized and selected on the basis of antigen binding) in order to direct the evolution of molecules that bind to such introduced loops. A selection of this type may be carried out to identify recognition molecules for any individual CDR-like loop or, alternatively, for the recognition of two or all three CDR-like loops combined into a non-linear epitope.
Accordingly, the present invention features a protein that includes a fibronectin type III domain having at least one randomized loop, the protein being characterized by its ability to bind to a compound that is not bound by the corresponding naturally-occurring fibronectin.
In preferred embodiments, the fibronectin type III domain is a mammalian (for example, a human) fibronectin type III domain; and the protein includes the tenth module of the fibronectin type III (
10
Fn3) domain. In such proteins, compound binding is preferably mediated by either one, two, or three
10
Fn3 loops. In other preferred embodiments, the second loop of
10
Fn3 may be extended in length relative to the naturally-occurring module, or the
10
Fn3 may lack an integrin-binding motif. In these molecules, the integrin-binding motif may be replaced by an amino acid sequence in which a basic amino acid-neutral amino acid-acidic amino acid sequence (in the N-terminal to C-terminal direction) replaces the integrin-binding motif; one preferred sequence is serine-glycine-glutamate. In another preferred embodiment, the fibronectin type III domain-containing proteins of the invention lack disulfide bonds.
Any of the fibronectin type III domain-containing proteins described herein may be formulated as part of a fusion protein (for example, a fusion protein which further includes an immunoglobulin F
c
domain, a complement protein, a toxin protein, or an albumin protein). In addition, any of the fibronectin type III domain proteins may be covalently bound to a nucleic acid (for example, an RNA), and the nucleic acid may encode the protein. Moreover, the protein may be a multimer, or, particularly if it lacks an integrin-binding motif, it may be formulated in a physiologically-acceptable carrier.
The present invention also features proteins that include a fibronectin type III domain having at least one mutation in a &bgr;-sheet sequence which changes the scaffold structure. Again, these proteins are characterized by their ability to bind to compounds that are not bound by the corresponding naturally-occurring fibronectin.
In addition, any of the fibronectin scaffolds of the invention may be immobilized on a solid support (for example, a bead or chip), and these scaffolds may be arranged in any configuration on the solid support, including an array.
In a related aspect, the invention further features nucleic acids encoding any of the proteins of the invention. In preferred embodiments, the nucleic acid is DNA or RNA.
In another related aspect, the invention also features a method for generating a protein which includes a fibronectin type III domain and which is pharmaceutically acceptable to a mammal, involving removing the integrin-binding domain of said fibronectin type III domain. This method may be applied to any of the fibronectin type III domain-containing proteins described above and is particularly useful for generating proteins for human therapeutic applications. The invention also features such fibronectin type III domain-containing proteins which lack integrin-binding domains.
In yet other related aspects, the invention features screening methods which may be used to obtain or evolve randomized fibronectin type III proteins capable of binding to compounds of interest, or to obtain or evolve compounds (for example, proteins) capable of binding to a particular prote
Kuimelis Robert G.
Lipovsek Dasa
Wagner Richard W.
Audet Maury
Compound Therapeutics, Inc.
Ropes & Gray LLP
Tate Christopher R.
LandOfFree
Protein scaffolds for antibody mimics and other binding... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Protein scaffolds for antibody mimics and other binding..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Protein scaffolds for antibody mimics and other binding... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3360036