AU2008345674A1 - Alternative scaffold protein fusions phage display via fusion to pIX of M13 phage - Google Patents

Description

WO 2009/086116 PCT/US2008/087696 NON-ANTIBODY SCAFFOLD PROTEIN FUSIONS PHAGE DISPLAY VIA FUSION TO pIX OF M13 PHAGE CROSS-REFERENCE TO RELATED APPLICATIONS 5 [1] This application claims the benefit of U.S. Provisional Application No. 61/014,778, filed December 19, 2007, which is incorporated by reference in its entirety. FIELD OF THE INVENTION [2] The invention relates to a compositions and methods for generating and using pIX phage display libraries for producing non-antibody scaffold protein fusions using pIX of 10 M13 phage. BACKGROUND OF THE INVENTION [3] Filamentous phage display using pill, pVII, pVIII, pIX, and combinations thereof as fusion partners in phage or phagemid systems have been used as a technology for protein engineering, notably for de novo protein and peptide isolation and affinity 15 maturation (see, for example, Janda et al, U.S. 7,078,166). Random peptides and proteins can be generated and isolated from phage libraries displaying variations of the peptide sequences via panning against a protein target of interest. Previously used human protein de novo libraries have been created synthetically. In a synthetic library, putatively useful DNA sequences encoding potential proteins are designed 20 and synthesized based on known sequences or motifs. In addition to the synthetic peptide library, libraries can also be created by combinatorial cloning of protein encoding DNA derived from human tissues. Such libraries have been used for providing potentially useful peptides and for running successive rounds of panning and maturation or modification to attempt to find non-antibody peptide or protein 25 peptides that have desired properties such as inhibitory biological activity of a selected target protein. [4] Human peptides and proteins, such as mimetic proteins or known protein muteins that bind or mimic target proteins have been isolated from phage display piII or pVIII peptide libraries. Although successful at isolating some proteins that bind to specific 30 targets, such phage display library approaches suffer from the problems of having to repeat the process of library generation, requirement of panning and maturation several times over to isolate peptides or proteins having the desired characteristics, and other know limitations. Such phage libraries also suffer from the problem that they do not fully encompass or mimic the range of peptide diversity present in 1 WO 2009/086116 PCT/US2008/087696 humans, the position and extent of amino acid variation, and the relative abundance of biologically active proteins from different human genes. Deviation of synthetic proteins from the natural repertoire may increase the risk of unfavorable biochemical properties and of immunogenecity if used as therapeutics in man, and this issue 5 could potentially be addressed by how the sequences are generated and screened for selection from such libraries. [5] Monoclonal antibodies are the most widely used class of therapeutic proteins when high affinity and specificity for a target molecule are desired. However, non-antibody antigen-binding peptides or proteins can be engineered to bind to such targets that 10 are also of high interest for use in therapeutics or diagnostics. Such proteins or peptides that are capable of binding to biomolecules may have several potential advantages over traditional antibodies such as, but not limited to, smaller size, lack of disulphide bonds, ability to be expressed in prokaryotic hosts, novel methods of purification, high stability, ease of conjugation to drugs/toxins, and intellectual 15 property advantages, among others. [6] One type of scaffold is the immunoglobulin (Ig) fold. This fold is found in the variable regions of antibodies, as well as thousands of non-antibody peptide or protein proteins. It has been shown that one such Ig protein, the tenth fibronectin type III repeat from human fibronectin, can tolerate a number of mutations in surface 20 exposed loops while retaining the overall Ig-fold structure. Thus, libraries of amino acid variants have been built into these loops and specific binders selected to a number of different targets. Such engineered Fn3 domains have been found to bind to targets with reasonably high affinity, while retaining important biophysical properties 25 [7] Prior use of phage libraries has included antibody-based protein fusion libraries. However, there is a need for synthetic alternative scaffold or non-antibody putative antigen-binding peptide or protein fusion libraries and methods that simultaneously deliver the critical elements of human therapeutic antibodies of high affinity and activity, high productivity, good solution properties, and a propensity of low immune 30 response when administered in man. There is a further need to increase the efficiency of non-antibody peptide or protein isolation from synthetic libraries, relative to current methods, to reduce the resource costs of non-antibody peptide or protein discovery and accelerate delivery of antibodies for biological evaluation. The libraries and methods of this invention meet these needs by coupling comprehensive 35 design, assembly technologies, and phage pIX non-antibody peptide or protein display. 2 WO 2009/086116 PCT/US2008/087696 SUMMARY OF THE INVENTION [8] In contrast to the teaching of the prior art, it has now been discovered that pVII and pIX can successfully be used for generating high affinity non-antibody peptide or protein libraries using pIX from M13 phage, e.g., using mutagenesis or other diversity producing 5 techniques, optionally with in line maturation, to provide an efficient and fast platform for non-antibody peptide or protein and non-antibody peptide or protein fragment generation and selection of therapeutic antibodies. According to the present invention, non-antibody peptide or proteins that are capable of binding to a desired biomolecule or antigen are fused to pVII and pIX engage in a dynamic interaction on the phage surface to display a 10 functional non-antibody peptide or protein, optionally in a representative heterodimeric motif. The display on phage of non-antibody peptide or protein binding agents is therefore a suitable and preferred method for display and assay of diverse libraries of combinatorial heterodimeric arrays in which members can function as monomeric or dimeric artificial non-antibody peptide or protein species and allow for selection of novel 15 or desired biological activities. [9] The present invention provides designs and display of non-antibody peptide or protein de novo libraries fused to the pIX protein of filamentous phage, a phage surface protein that is different from the widely used the pill and pVIII proteins. We constructed the individual scaffold libraries separately and developed phage selection processes to 20 systematically examine the activity of each of the scaffold libraries, and evaluate the structural topologies for antigen recognition. [10]The present invention provides various improved and new pIX and pVII phage display de novo library generation methods and components, such as but not limited to, one or more of (i) designed and displayed non-antibody peptide or protein de novo libraries 25 fused to the pIX or pVII phage proteins; (ii) the use of a phage surface protein different from the widely used pill and pVIII of M13 phage; (iii) the use of the pIX phage display system to screen a library of peptides fused to a non-antibody protein scaffold; (iv) non antibody peptide or protein selection processes that allow systematic examination of the effect of the designed sequences and structural topologies for antigen recognition; (v) a 30 streamlined affinity maturation and in line maturation process as a part of the library selection. Such a new system of library design, selection, optimization and maturation of individual or groups of libraries provide a reproducible and reliable system for successful non-antibody peptide or protein de novo discovery and also facilitate understanding the structure function relation of non-antibody peptide or protein to antigen interaction. 35 [11]The human non-antibody peptide or protein de novo library described above is distinct from current antibody library state-of-the-art by its display via the pIX gene of M1 3 phage. Non-antibody peptides or proteins can be successfully displayed on the surface of M13 3 WO 2009/086116 PCT/US2008/087696 phage as pIX fusion proteins according to the present invention. Both scaffold proteins can be engineered to bind a specific protein target that is not bound by the corresponding scaffold proteins in their native state. These engineered scaffolds retained binding to this specific target while displayed on phage. In addition, libraries of amino acid variants 5 can be made according to the invention in each scaffold, and their ability to bind a specific target addressed by displaying library members on phage as pIX fusions and panning against a target protein. [12]Artificial antibodies or scaffold proteins as used in the present invention are herein defined as protein motifs of large diversity that use the functional strategy of the non 10 antibody peptide or protein molecule, but can be generated free of in vivo constraints, including (1) sequence homology and toxicity of target antigens; (2) biological impact of the generated non-antibody peptide or protein in the host or in hybridoma cultures used to recover the non-antibody peptide or protein; and (3) screening versus selection for desired activity. 15 [13]Thus the invention describes a combinatorial phage display format for construction of highly diverse monomeric or heterodimeric polypeptide arrays. In particular, the invention describes a filamentous phage particle encapsulating a genome encoding a fusion polypeptide, wherein the fusion polypeptide comprises a non-antibody scaffold protein fused to the amino terminus of a filamentous phage pVII or pIX protein. Preferably, the 20 phage particle comprises the expressed fusion protein on the surface of the phage particle. [14] In a related embodiment, the invention describes a vector for expressing a fusion protein on the surface of a filamentous phage comprising a cassette for expressing the fusion protein. The cassette includes upstream and downstream translatable DNA sequences 25 operatively linked via a sequence of nucleotides adapted for directional ligation of an insert DNA, i.e., a polylinker, where the upstream sequence encodes a prokaryotic secretion signal, the downstream sequence encodes a pVII or pIX filamentous phage protein. The translatable DNA sequences are operatively linked to a set of DNA expression signals for expression of the translatable DNA sequences as portions of the 30 fusion polypeptide. In a preferred variation, the vector further comprises a second cassette for expressing a second fusion protein on the surface of the filamentous phage, wherein the second cassette has the structure of the first cassette with the proviso that the first fusion protein expression cassette encodes pVII protein and the second fusion protein expression cassette encodes pIX protein. The vector is used as a phage genome 35 to express heterodimeric protein complexes on the surface of the phage particle in which the two polypeptides of the heterodimer are anchored on the phage particle by the fusion to the first and second phage proteins, pVII and pIX, respectively. 4 WO 2009/086116 PCT/US2008/087696 [15]In another embodiment, the invention contemplates a library of phage particles according to the present invention, i.e., a combinatorial library, in which representative particles in the library each display a different fusion protein. Where the particle displays a heterodimeric protein complex, the library comprises a combinatorial library of 5 heterodimers. Preferred libraries have a combinatorial diversity of at least 10 3 , 10 4 , 10 5 , 6 7 8 9 10 11 12 13 10 , 10 , 10 , 10 , 10 , 10 , 10 , 10 , or any range or value therein, of different or similar species of fusion proteins. [16]A related embodiment describes a fusion protein comprising first and second polypeptides wherein the first polypeptide is a non-antibody scaffold protein and the 10 second polypeptide is a filamentous phage pVII or pIX protein, wherein the non-antibody scaffold protein is fused to the amino terminus of the filamentous phage protein. [17]Still further, the invention contemplates a variety of methods for producing a combinatorial library of phage, including by cloning repertoires of genes encoding a non antibody scaffold protein into a vector of the present invention, modifying the structure of 15 the a non-antibody scaffold protein in a library by mutagenesis, by random combination of populations of first and second fusion protein libraries, by target and affinity selection ("panning") to alter the diversity of a library, and the like. Such an embodiment can include a fusion polypeptide having an alterable non-antibody scaffold protein fused to a second polypeptide, as exemplified herein. For example, in one embodiment, the 20 alterable non-antibody scaffold protein can be TeFN3, and the second polypeptide can be pIX, where the F:G loop of TeFN3 is altered by mutagenesis or targeted substitution of the native F:G loop amino acid residues with a non-native polypeptide, such as the cysteine-constrained EGFR binding peptide, PHPEP190. In alternative embodiments, the alterable non-antibody scaffold protein can have an F:G loop made of at least one 25 polypeptide identified by SEQ ID NOs: 2 - 23, 25, or 28. Disclosed herein are also embodiments where the non-antibody scaffold protein is encoded by an engineered nucleic acid phage vector and binds to a biomolecule, such as epidermal growth factor receptor or a biologically active ligand. [18]The design of proteins with improved or novel functions is an important goal with a 30 variety of medical, industrial, environmental, and basic research applications. Following the development of combinatorial non-antibody peptide or protein libraries, a powerful next step is the evolution toward artificial non-antibody peptide or protein constructs as well as other protein motifs in which dimeric species are native or might be functional. [19] The present invention addresses these challenges by providing a phage-display format 35 for the construction of combinatorial non-antibody polypeptide arrays in which pVII and pIX are utilized for the display of fusion proteins that form monomeric or dimeric species. It is important to note that this is an entirely new methodology because one can 5 WO 2009/086116 PCT/US2008/087696 independently display one or two protein motifs in close proximity to generate a library of functional interactions using expression on pIX or pVII. [20] Furthermore, sequence randomizations to form libraries and chain-shuffling protocols to form hybrid species can lead to subsets of novel proteins. For instance, the display and 5 modification of arrays of zinc-finger domains in homodimeric or heterodimeric form produces structures that possess specific DNA interactions. In addition, entirely new constructs are possible via the insertion of a desired encoding fragment within a preformed scaffold such as a non-antibody peptide or protein chain. Possible insertions include an enzyme signature sequence or a repressor binding protein. 10 [21] It should be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention as claimed. DESCRIPTION OF THE FIGURES [22] Figure 1 shows structures of the third Fn3 domain of tenascin (residues 802-893, PDB = 15 1TEN, (Leahy, et al., Science 258: 987-91, 1992)) and the 127 domain of titin (residues 5253-5341, PDB = 1TIT, (Improta, et al., Structure 4: 323-37, 1996)). Arrows point to the location of the F:G loop ( residues 876-881 and 5258-5259 of tenascin and titin respectively), which was modified to accommodate the EGFR binding sequence. [23] Figure 2 shows graphical results for the display of tenascin and titin scaffold proteins on 20 pIX. [24] Figure 3 shows graphical results of panning for EGFR binding constructs from TeFn3 190 and Ti27-190 libraries displayed on pIX. DETAILED DESCRIPTION OF THE INVENTION [25] The present invention provides various new phage display de novo library generation 25 methods and components, such as but not limited to (i) designed and displayed non antibody peptide or protein libraries fused to the pIX or other phage proteins; (ii) the use of a phage surface protein different from the widely used pill and pVIII of M13 phage; (iii) use of such phage components as the library scaffold to provide improved designed, combinatorial diversities in the non-antibody peptide or protein selection processes that 30 allow systematical examination of the effect of the designed sequences and structural topologies for antigen recognition; (iv) a streamlined affinity maturation process as a part of the library selection. Such a new system of library design, selection, optimization and maturation of individual or groups of libraries provide a reproducible and reliable system for successful non-antibody peptide or protein de novo discovery and also facilitate 6 WO 2009/086116 PCT/US2008/087696 understanding the structure function relation of non-antibody peptide or protein to antigen interaction. [26]The human non-antibody peptide or protein de novo library described above is distinct from current antibody library state-of-the-art by its displaying via pIX or pVII gene of M13 5 phage. Definitions: [27] Fusion Polypeptide: A polypeptide comprised of at least two polypeptides and a linking sequence to operatively link the two polypeptides into one continuous polypeptide. The two polypeptides linked in a fusion polypeptide are typically derived from two 10 independent sources, and therefore a fusion polypeptide comprises two linked polypeptides not normally found linked in nature. [28]Cistron: Sequence of nucleotides in a DNA molecule coding for an amino acid residue sequence and including upstream and downstream DNA expression control elements. [29]Biomolecule: Any organic molecule produced by a living organism, or any organic 15 molecule made in vitro by processes used by living organisms to produce biomolecules, such processes include, for example, transcription, translation, bio-organic chemical reactions and the like. [30]Non-antibody scaffold protein: Any non-antibody protein, protein segment, or peptide having six or more beta strands connected by surface-exposed loops that form at least 20 two beta sheets. Examples of such non-antibody protein scaffolds include proteins that include a fibronectin type III domain or an Ig domain, such as the third fibronectin type III domain of human tenascin (TeFN3) or the 127 domain of human titin (Ti27). Filamentous Phage [31]The present invention contemplates a filamentous phage comprising a matrix of proteins 25 encapsulating a genome encoding a fusion protein (protein). The fusion protein comprises a non-antibody scaffold protein portion fused to the amino terminus of a filamentous phage pVII or pIX protein. [32] The filamentous phage will further contain the fusion protein(s) displayed on the surface of the phage particle, as described in the Examples. 30 [33] In a fusion protein present on a phage of this invention, the "fusion" between the non antibody scaffold protein and the filamentous phage pVII or pIX protein may comprise a typical amide linkage, or may comprise a linker polypeptide (i.e., a "linker") as described in the Examples. Any of a variety of linkers may be used which are typically a stretch of about 5 to 50 amino acids in length. Particularly preferred linkers provide a high degree 35 of mobility to the fusion protein at the point of the linker. 7 WO 2009/086116 PCT/US2008/087696 [34] Library design: prior synthetic libraries have incorporated some of the following, but none have included all in a comprehensive manner. [35] Expression, biochemical, and biophysical properties. Preferred non-antibody peptides or proteins not only have desired biological and binding activities, but also are efficiently 5 produced from a variety of hosts, are stable, and have good solution properties. It is understood that the disclosed scaffold proteins, either expressed as a fusion protein by an engineered recombinant nucleic acid phage vector or expressed as a scaffold alone, have the ability to bind biomolecules such as antigens, receptors, ligands, cell surface protein markers and the like. In preferred embodiments the scaffold proteins described 10 herein bind epidermal growth factor receptor. High-frequency germline gene usage also indicates good expression in mammalian systems. In addition, such fusion proteins recovered from libraries by bacterial phage display methods of selection or screening should be expressed well in the bacterial host. The libraries of the invention are based on human germline derived templates that are well-expressed and purified from standard 15 recombinant mammalian hosts (e.g. HEK 293 and CHO cells) as well as bacterial hosts, and have high stability and good solution properties. [36] Maturation. The large number of positions in the V-region mimicking alternative scaffold sequences that can impact recognition of antigen or ligand binding, coupled with potential variation of up to 20 different amino acids at each position preclude the 20 practicality of including all variations in a single library. Human antibodies achieve high affinity and specificity by the progressive process of somatic mutation. The libraries of the invention are designed and ordered to permit parallel selection and targeted variation while maintaining the sequence integrity of each non-antibody peptide or protein chain such that they reflect features and characteristics similar to those of human antibodies. 25 [37]Alternatives design. The above design simulates characteristics of natural human antibodies. The modular nature of the system is amenable to incorporation of any collection of amino acids at any collection of positions. [38] Library assembly technologies. Preferred non-antibody peptide or protein libraries are of low or high diversity (> 1010), amenable to alteration, and easy to assemble and have 30 a low background of undesired sequences. These background sequences include parental template and low-targeted diversity. Coupling the following methods accelerates library assembly and leads to low background. (a) Kunkle-based single stranded mutagenesis; (b) Palindromic loop with restriction site; (c) Megaprimer-based PCR. 35 [39]pIX non-antibody peptide or protein phage display. The combination of pIX with the selected non-antibody peptide or protein templates is an efficient selection system for 8 WO 2009/086116 PCT/US2008/087696 recovering non-antibody peptides or proteins that retain their selected properties upon conversion into other related molecules. [40]Phagemid display. The expressed molecule is large relative to the phage pIX coat protein and thus may interfere with assembly of recombinant phage particles if linked to 5 all pIX proteins produced in the bacterial cell. One approach to by-pass this interference is to use a pIX phagemid system, such as those known in the art are as described herein, whereby non-antibody peptide or protein-linked pIX or pVII proteins can be incorporated into the recombinant phage particle. In a preferred application, libraries of the current invention are displayed by pIX in a phagmid system. 10 [41]Phage coat protein pIX for display. Like pill, pIX is present at low copy number on the phage and is amenable to affinity selection of displayed non-antibody peptides or proteins. However, the piII protein is critically involved in the infection process and proteins displayed on this protein can interfere with the efficiency of infection. The libraries of the current invention displayed on the pIX protein are predicted to be 15 efficiently replicated and presented for selection and/or screening. [42] Non-antibody peptide or protein-pIX expression. One approach to screening non antibody peptides or proteins recovered from phage libraries is to remove the phage coat protein that is linked to the non-antibody peptide or protein molecule for display. The small size of the pIX protein provides the option of production of screening of non 20 antibody peptide or proteins directly without this step. [43]Therapeutic uses. As described herein, the disclosed scaffolds may be used as alternatives to antibodies. Accordingly, the disclosed scaffolds can have therapeutic applications. As such, it is contemplated that the scaffold proteins described herein may be used to form therapeutic compositions. One such composition can include a scaffold 25 as described herein and a pharmaceutically acceptable carrier. In another embodiment the composition includes an isolated EGFR-specific, non-antibody protein scaffold such as TeFN3, having its F:G loop replaced with a non-native polypeptide, and a pharmaceutically acceptable carrier. In alternative embodiments, the composition includes an alterable non-antibody scaffold protein having an F:G loop made of at least 30 one polypeptide identified by SEQ ID NOs: 2 - 23, 25, or 28 and a pharmaceutically acceptable carrier. Disclosed herein are also embodiments where the non-antibody scaffold protein is encoded by an engineered nucleic acid phage vector and binds to a biomolecule, such as epidermal growth factor receptor and is combined with a pharmaceutically acceptable carrier. 35 [44]Design library scaffolds. The library scaffold is made of a set of human protein sequences that mimic the structure and function of germline VH and VL genes. 9 WO 2009/086116 PCT/US2008/087696 [45] Expression and display ability of the library scaffolds. Good expression and display ability of the library scaffold non-antibody peptide or proteins directly relate to the quality of the library to be developed over the scaffold genes. The library scaffold non-antibody peptide or protein expression and display ability was examined before the library 5 construction. A few scaffold non-antibody peptide or proteins that expressed but unable or poorly displayed were excluded from the library construction. The well expressed and displayed library scaffolds ensures that high proportion of the non-antibody peptide or proteins in the library are functional, more superior than the libraries derived from combinatorial cloning of VH and VL genes genetically amplified from a natural sources. 10 [46] Methods for library generation. A modified Kunkel mutagenesis method can be used according to the present invention, which efficiently generates billions of E. coli colonies each harboring a different non-antibody peptide or protein sequence, which can be used for the generation of the non-antibody peptide or protein libraries. While efficient, the percentage of non-mutagenized parental DNA increases when adapted in generation of 15 a highly sequence complex library. In addition, technical limitations of synthesis of long oligonucleotides cab reduce the effectiveness of the method of making libraries containing sequence diversities in distant regions. To overcome such limitations, additional techniques of generating oligonucleotide >350 bases (mega-primer) and of creation of a stem-loop sequence containing a restriction enzyme recognition site in the 20 mutagenesis template can optionally be used in combination with techniques such as the Kunkel mutagenesis method. As compared to other known library technologies, such as restriction cloning, phage recombination and sequence specific recombination, as used by others to for library generation, the improved Kunkel based method can be more effective in generation of >10 9 sequences per library and is more versatile in introducing 25 sequence diversity in any location on the targeted DNA. [47]In-line affinity maturation. An integrated affinity maturation process, or in-line affinity maturation, can be used according to the present invention for design and improvement of binding affinity of non-antibody peptides or proteins selected from the library. Improving binding affinity of desired non-antibody peptides or proteins after panning can 30 increase the success of identifying therapeutic non-antibody peptide or protein leads. The use of Kunkel method for library generation can improve the effective execution of sequence diversification strategies in a simple and continuous process. The design strategy and the technical advantages of using the improved Kunkel mutagenesis method can provide a superior approach over other pooled maturation strategies, where 35 tedious library generation methods are employed that reduce the efficiency and effectiveness of the results. 10 WO 2009/086116 PCT/US2008/087696 [48]Parallel library panning. A parallel panning process using a automated or semi automated equipment can be used to process the individually made sub-libraries. Parallel panning can maximize the potential of discovering a suitably diverse set of non antibody peptides or proteins to provide the desired libraries having peptides or proteins 5 of desired characteristics. Effective use of parallel panning in in-line affinity maturation also enables such proteins to have several simultaneously improved characteristics, such as improved affinities or biological activities. Development of a machine based panning system also allows systematic monitoring and adjustment of different panning conditions to more quickly screen and isolate non-antibody peptides or proteins having 10 desired properties. [49]Affinity ranking. Affinity based binding assays are applied to the large, diverse and high affinity ligand or antigen specific binding peptides or proteins to select the best binding for further characterization. Standard biochemical methods like ELISA as well as affinity measuring equipments, for example, BlAcore, Octet and BIND that are suitable for 15 processing large number of samples are used alone or in combination for this purpose. [50] While having described the invention in general terms, the embodiments of the invention will be further disclosed in the following examples that should not be construed as limiting the scope of the claims. [51]EXAMPLE 1: DISPLAY OF NON-ANTIBODY PROTEINS: Two non-antibody peptides 20 or proteins were successfully displayed on the surface of M13 phage as pIX fusion proteins. Both scaffold proteins were engineered to bind a specific protein target that is not bound by the scaffold proteins in their native state. These engineered scaffolds retained binding to this specific target while displayed on phage. In addition, libraries of amino acid variants were made in each scaffold, and their ability to bind a specific target 25 addressed by displaying library members on phage as pIX fusions and panning against a target protein. Both libraries produced a number of positive hits. [52]Two non-antibody peptide or protein, immunoglobulin (Ig) domain proteins were selected as candidates to be engineered to present an EGFR binding peptide on the surface of M13 phage as a pIX fusion protein. The following criteria were used for 30 selection of candidates: [53] High resolution atomic structure available in the PDB database a. Structure deposited represents a single, isolated Ig domain b. Entirely human sequence c. Sequences contain no disulphide bonded residues 35 d. Expression and purification conditions published 11 WO 2009/086116 PCT/US2008/087696 e. Demonstrated to be successfully expressed and purified from E. coli without the need of a refolding step and with a high yield. [54] The PDB database was searched manually, as well as with the aid of structure based alignment programs in order to investigate all proteins and protein fragments containing a 5 single Ig domain. The scientific literature was then surveyed in order to determine published expression and purification conditions. [55]Although the criteria above were used to select molecules with the greatest chance for success, it is envisioned that other Ig molecules that do not strictly meet these criteria could also be used as successful scaffolds for peptide display. It is also envisioned that 10 scaffolds composed of multiple Ig domains or repeats of a single Ig domain could be used as successful scaffolds. The analysis above resulted in the selection of two Ig molecules, the third Fn3 domain from human tenascin (residues 802-893, TeFn3) and the 127 domain of human titin (residues 5253-5341, Ti27) for further study. The genes encoding these protein fragments were synthesized by Blue Heron, and subcloned into 15 the plasmid pPEP9-BbsI by standard PCR and restriction digest methods. The mutations N5331G in Ti27 and S881G in TeFn3 were included in order to introduce restriction sites for insertion of peptides (see below). [56]The F:G loops (residues 876-881 of TeFn3 and 5258-5259 of Ti27) (Figure 1) of both scaffolds were then replaced with a portion of the cysteine-constrained EGFR binding 20 peptide, PHPEP190 (sequence DPCTWEVWGRECLQ) (Wang) using standard restriction site cloning methods to make the Ti27-190 and TeFn3-190 constructs. A number of control constructs were also created, including those containing non-EGFR binding F:G loop sequences (referred to as Ti27 and TeFn3) and containing six stop codons in the F:G loop (Ti27-stop and TeFn3-stop). Table 1 summarizes these 25 constructs. 12 WO 2009/086116 PCT/US2008/087696 Construct F:G loop sequence Description TeFn3 RRGDMGS (SEQ ID NO:24) Non EGFR binding sequence TeFn3-190 DPCTWEVWGRECLQ EGFR binding sequence (SEQ ID NO:25) TeFn3-stop ***TWEVWGRE*** Negative control (SEQ ID NO:26) Ti27 AG (SEQ ID NO:27) Non EGFR binding sequence Ti27-190 DPCTWEVWGRECLQ EGFR binding sequence (SEQ ID NO:28) Ti27-stop ***TWEVWGRE*** Negative control (SEQ ID NO:29) Table 1. Tenascin and titin scaffold constructs. * Denotes stop codon. SEQ ID NOs. 24 29 indicate the particular polypeptide inserted into the scaffold F:G loop segment, the amino acid sequences for the corresponding full-length scaffolds are provided as SEQ ID NOs. 54-59, respectively. 5 [57]Each construct was expressed as fusion to the minor coat protein pIX of filamentous phage from a phagemid vector with an EDSGGSGG (SEQ ID NO:30) linker sequence between the C-terminus of the scaffold protein and the N-terminus of pIX. All constructs were expressed with an N-terminal Myc tag in order to assay for protein expression and 10 display in the absence of EGFR binding. Successful display on the surface of M13 phage and specific binding activity of TeFn3-190 and Ti27-190 proteins were verified by phage ELISA. 0.5 pg of an Fc-fusion form of the EGFR ectodomain (sEGFR-MMB) (Wang), an anti-Myc non-antibody peptide or protein, or a control Fc molecule (CNTO 360) were immobilized on 96 well MaxiSorop T M plates and blocked with StartingBlock TM 15 T-20. Phage displaying the constructs described in Table 1 were serially diluted before addition of 300 pL of the phage containing supernatant to each plate followed by incubation at room temperature for 1 hour. Each plate was subsequently washed with TBST and bound phage detected with an anti-M13 HRP-conjugated non-antibody peptide or protein using a fluorescent plate reader. Figure 2 shows that TeFn3-190 and 20 Ti27-190 selectively bound the plate-bound Fc-EGFR with a low level of background binding as demonstrated by the lack of appreciable binding to CNTO 360. TeFn3 and Ti27 were also displayed on the phage surface as indicated by binding to the anti-Myc coated plate. These proteins however did not bind to EGFR. For reasons unknown, TeFn3-190 did not bind to the anti-Myc coated plate, although it is clearly expressed as 25 shown by EGFR binding. As expected, TeFn3-stop and Ti27-stop were not displayed on phage. 13 WO 2009/086116 PCT/US2008/087696 [58]A library was constructed in order to determine the necessity of the cysteine constraints of PHPEP190 in the context of TeFn3 and Ti27 and to confirm that such a library can be efficiently displayed within these scaffolds on pIX and panned against a target protein. This library was constructed by randomizing all six positions containing stop codons in 5 Ti27-stop and TeFn3-stop constructs (Table 1). Sequencing of 100 randomly picked colonies following mutagenesis by the Kunkel method (Kunkel, et al., Methods Enzymol 154: 367-82, 1987) confirmed that mutations were found in 51% and 64% of colonies from the Ti27-190 and TeFn3-190 libraries respectively. These libraries were then panned for three rounds against the EGFR-Fc fusion by incubating 100 pL of a phage 10 solution with 10 pg (round 1) or 5 pg (rounds 2 and 3) of biotinylated EGFR for 1 hour at room temperature. Bound complexes were pulled down with streptavidin coated magnetic beads and washed 3 times with TBST. Bound phage were then used to re infect TG1 cells, resulting in the phage titers described in table 2. Library Round 1 Round 2 Round 3 Ti27-190 2.5E5 cfu 1.5E5 cfu 2.2E8 cfu TeFn3-190 2.5E5 cfu 1.5E5 cfu 3.5E7 cfu Table 2. Phage titers obtained during library panning. 15 [59]96 colonies from the third round of panning from each library were randomly selected to test binding to EGFR-Fc by phage ELISA. Figure 3 shows that 10 and 20 clones from the Ti27-190 and TeFn3-190 libraries respectively showed significant binding to EGFR (ratio of counts from EGFR:CNTO 360 greater than 5). The sequences of the F:G loops from all positive hits are displayed in Table 3. All sequenced hits contained cysteine 20 residues at the identical positions as in the original PHPEP190, indicating that the cysteine-constrained nature of this peptide is necessary for function. SEQ ID Ti27 F:G loop Sequences Frequency Colonies NO:1 PHPEP190 DPCTWEVWGRECLQ NO:2 Ti1901 PLCTWEVWGRECYA 6 Ti3, Ti4, Ti5, Ti6, Ti9, TiO NO:3 Ti1902 PLCTWEVWGRECLM 1 Ti 7 NO:4 Ti1903 ANCTWEVWGRECAH 1 Ti1 NO:5 Ti1904 SFCTWEVWGRECQN 1 Ti2 NO:6 Ti1905 EGCTWEVWGRECMS 1 Ti8 SEQ ID TeFn3 F:G loop Sequences Frequency Colonies NO:7 Te1901 PLCTWEVWGRECHT 1 Te4 NO:8 Te1902 TSCTWEVWGRECHM 2 Tel3, Tel6 NO:9 Te1903 NSCTWEVWGRECHV 1 Tel1 NO:10 Te1904 DSCTWEVWGRECTL 1 Te6 NO:l1 Te1905 DSCTWEVWGRECIL 1 Tel7 NO:12 Te1906 DTCTWEVWGRECSS 1 Te9 NO:13 Te1907 DICTWEVWGRECFG 1 Tel2 NO:14 Te1908 DLCTWEVWGRECHA 2 Te8, Tel4 NO:15 Te1909 GGCTWEVWGRECYQ 2 Tel0, Tel8 14 WO 2009/086116 PCT/US2008/087696 NO:16 Te19010 SVCTWEVWGRECNM 1 Te5 NO:17 Te19011 VVCTWEVWGRECSQ 1 Te3 NO:18 Te19012 TTCTWEVWGRECYS 1 Te20 NO:19 Te19013 HACTWEVWGRECFG 1 Te19 NO:20 Te19014 HFCTWEVWGRECQS 1 Te2 NO:21 Te19015 SHCTWEVWGRECNL 1 Te15 NO:22 Te19016 NVCTWEVWGRECMN 1 Te7 NO:23 Te19017 NNCTWEVWGRECNW 1 Tel Table 3. Positive hits from Ti27 and TeFn3 libraries. Residues randomized as part of the library are underlined. SEQ ID NOs. 2-6 indicate the particular EGFR-specific polypeptide inserted into the Ti27 F:G loop segment, the amino acid sequences for the corresponding full-length EGFR-specific scaffolds are provided as SEQ ID NOs. 32-36, 5 respectively. SEQ ID NOs. 7-23 indicate the particular EGFR-specific polypeptide inserted into the TeFN3 F:G loop segment, the amino acid sequences for the corresponding full-length EGFR-specific scaffolds are provided as SEQ ID NOs. 37-53, respectively. 10 15 WO 2009/086116 PCT/US2008/087696 SEQUENCE LISTING SEQ ID NO. 1: DPCTWEVWGRECLQ 5 SEQ ID NO. 2: PLCTWEVWGRECYA SEQ ID NO. 3: 10 PLCTWEVWGRECLM SEQ ID NO. 4: ANCTWEVWGRECAH 15 SEQ ID NO. 5: SFCTWEVWGRECQN SEQ ID NO. 6: EGCTWEVWGRECMS 20 SEQ ID NO. 7: PLCTWEVWGRECHT SEQ ID NO. 8: 25 TSCTWEVWGRECHM SEQ ID NO. 9: NSCTWEVWGRECHV 16 WO 2009/086116 PCT/US2008/087696 SEQ ID NO. 10: DSCTWEVWGRECTL SEQ ID NO. 11: 5 DSCTWEVWGRECIL SEQ ID NO. 12: DTCTWEVWGRECSS 10 SEQ ID NO. 13: DICTWEVWGRECFG SEQ ID NO. 14: DLCTWEVWGRECHA 15 SEQ ID NO. 15: GGCTWEVWGRECYQ SEQ ID NO. 16: 20 SVCTWEVWGRECNM SEQ ID NO. 17: VVCTWEVWGRECSQ 25 SEQ ID NO. 18: TTCTWEVWGRECYS 17 WO 2009/086116 PCT/US2008/087696 SEQ ID NO. 19: HACTWEVWGRECFG SEQ ID NO. 20: 5 HFCTWEVWGRECQS SEQ ID NO. 21: SHCTWEVWGRECNL 10 SEQ ID NO. 22: NVCTWEVWGRECMN SEQ ID NO. 23: NNCTWEVWGRECNW 15 SEQ ID NO. 24: RRGDMGS SEQ ID NO. 25: 20 DPCTWEVWGRECLQ SEQ ID NO. 26: ***TWEVWGRE*** 25 SEQ ID NO. 27: AG 18 WO 2009/086116 PCT/US2008/087696 SEQ ID NO. 28: DPCTWEVWGRECLQ SEQ ID NO. 29: 5 ***TWEVWGRE*** SEQ ID NO. 30: EDSGGSGG 10 SEQ ID NO. 31: MAVFNSDSECPLSHDGYCLHDGVCMYIEALDKYACNCVVGYIGERCQYRDLKWWE SEQ ID NO: 32: LIEVEKPLYGVEVFVGETAHFEIELSEPDVHGQWKLKGQPLTASPDCEIIEDGKKHILILHNC 15 QLGMTGEVSFQAPLCTWEVWGRECYAAKSAANLKVKEL SEQ ID NO: 33: LIEVEKPLYGVEVFVGETAHFEIELSEPDVHGQWKLKGQPLTASPDCEIIEDGKKHILILHNC QLGMTGEVSFQAPLCTWEVWGRECLMAKSAANLKVKEL 20 SEQ ID NO: 34: LIEVEKPLYGVEVFVGETAHFEIELSEPDVHGQWKLKGQPLTASPDCEIIEDGKKHILILHNC QLGMTGEVSFQAANCTWEVWGRECAHAKSAANLKVKEL 25 SEQ ID NO: 35: LIEVEKPLYGVEVFVGETAHFEIELSEPDVHGQWKLKGQPLTASPDCEIIEDGKKHILILHNC QLGMTGEVSFQASFCTWEVWGRECQNAKSAANLKVKEL 19 WO 2009/086116 PCT/US2008/087696 SEQ ID NO: 36: LIEVEKPLYGVEVFVGETAHFEIELSEPDVHGQWKLKGQPLTASPDCEIIEDGKKHILILHNC QLGMTGEVSFQAEGCTWEVWGRECMSAKSAANLKVKEL 5 SEQ ID NO: 37: RLDAPSQIEVKDVTDTTALITWFKPLAEIDGIELTYGIKDVPGDRTTIDLTEDENQYSIGNLKP DTEYEVSLISPLCTWEVWGRECHTSNPAKETFTTGL SEQ ID NO: 38: 10 RLDAPSQIEVKDVTDTTALITWFKPLAEIDGIELTYGIKDVPGDRTTIDLTEDENQYSIGNLKP DTEYEVSLISTSCTWEVWGRECHMSNPAKETFTTGL SEQ ID NO: 39: RLDAPSQIEVKDVTDTTALITWFKPLAEIDGIELTYGIKDVPGDRTTIDLTEDENQYSIGNLKP 15 DTEYEVSLISNSCTWEVWGRECHVSNPAKETFTTGL SEQ ID NO: 40: RLDAPSQIEVKDVTDTTALITWFKPLAEIDGIELTYGIKDVPGDRTTIDLTEDENQYSIGNLKP DTEYEVSLISDSCTWEVWGRECTLSNPAKETFTTGL 20 SEQ ID NO: 41: RLDAPSQIEVKDVTDTTALITWFKPLAEIDGIELTYGIKDVPGDRTTIDLTEDENQYSIGNLKP DTEYEVSLISDSCTWEVWGRECILSNPAKETFTTGL 25 SEQ ID NO: 42: RLDAPSQIEVKDVTDTTALITWFKPLAEIDGIELTYGIKDVPGDRTTIDLTEDENQYSIGNLKP DTEYEVSLISDTCTWEVWGRECSSSNPAKETFTTGL 30 20 WO 2009/086116 PCT/US2008/087696 SEQ ID NO: 43: RLDAPSQIEVKDVTDTTALITWFKPLAEIDGIELTYGIKDVPGDRTTIDLTEDENQYSIGNLKP DTEYEVSLISDICTWEVWGRECFGSNPAKETFTTGL 5 SEQ ID NO: 44: RLDAPSQIEVKDVTDTTALITWFKPLAEIDGIELTYGIKDVPGDRTTIDLTEDENQYSIGNLKP DTEYEVSLISDLCTWEVWGRECHASNPAKETFTTGL SEQ ID NO: 45: 10 RLDAPSQIEVKDVTDTTALITWFKPLAEIDGIELTYGIKDVPGDRTTIDLTEDENQYSIGNLKP DTEYEVSLISGGCTWEVWGRECYQSNPAKETFTTGL SEQ ID NO: 46: RLDAPSQIEVKDVTDTTALITWFKPLAEIDGIELTYGIKDVPGDRTTIDLTEDENQYSIGNLKP 15 DTEYEVSLISSVCTWEVWGRECNMSNPAKETFTTGL SEQ ID NO: 47: RLDAPSQIEVKDVTDTTALITWFKPLAEIDGIELTYGIKDVPGDRTTIDLTEDENQYSIGNLKP DTEYEVSLISVVCTWEVWGRECSQSNPAKETFTTGL 20 SEQ ID NO: 48: RLDAPSQIEVKDVTDTTALITWFKPLAEIDGIELTYGIKDVPGDRTTIDLTEDENQYSIGNLKP DTEYEVSLISTTCTWEVWGRECYSSNPAKETFTTGL 25 SEQ ID NO: 49: RLDAPSQIEVKDVTDTTALITWFKPLAEIDGIELTYGIKDVPGDRTTIDLTEDENQYSIGNLKP DTEYEVSLISHACTWEVWGRECFGSNPAKETFTTGL 30 21 WO 2009/086116 PCT/US2008/087696 SEQ ID NO: 50: RLDAPSQIEVKDVTDTTALITWFKPLAEIDGIELTYGIKDVPGDRTTIDLTEDENQYSIGNLKP DTEYEVSLISHFCTWEVWGRECQSSNPAKETFTTGL 5 SEQ ID NO: 51: RLDAPSQIEVKDVTDTTALITWFKPLAEIDGIELTYGIKDVPGDRTTIDLTEDENQYSIGNLKP DTEYEVSLISSHCTWEVWGRECNLSNPAKETFTTGL SEQ ID NO: 52 10 RLDAPSQIEVKDVTDTTALITWFKPLAEIDGIELTYGIKDVPGDRTTIDLTEDENQYSIGNLKP DTEYEVSLISNVCTWEVWGRECMNSNPAKETFTTGL SEQ ID NO: 53 RLDAPSQIEVKDVTDTTALITWFKPLAEIDGIELTYGIKDVPGDRTTIDLTEDENQYSIGNLKP 15 DTEYEVSLISNNCTWEVWGRECNWSNPAKETFTTGL SEQ ID NO. 54: RLDAPSQIEVKDVTDTTALITWFKPLAEIDGIELTYGIKDVPGDRTTIDLTEDENQYSIGNLKP DTEYEVSLISRRGDMGSSNPAKETFTTGL 20 SEQ ID NO. 55: RLDAPSQIEVKDVTDTTALITWFKPLAEIDGIELTYGIKDVPGDRTTIDLTEDENQYSIGNLKP DTEYEVSLISDPCTWEVWGRECLQSNPAKETFTTGL 25 SEQ ID NO. 56: RLDAPSQIEVKDVTDTTALITWFKPLAEIDGIELTYGIKDVPGDRTTIDLTEDENQYSIGNLKP DTEYEVSLIS***TWEVWGRE***SNPAKETFTTGL 30 22 WO 2009/086116 PCT/US2008/087696 SEQ ID NO. 57: LIEVEKPLYGVEVFVGETAHFEIELSEPDVHGQWKLKGQPLTASPDCEIIEDGKKHILILHNC QLGMTGEVSFQAAGAKSAANLKVKEL 5 SEQ ID NO. 58: LIEVEKPLYGVEVFVGETAHFEIELSEPDVHGQWKLKGQPLTASPDCEIIEDGKKHILILHNC QLGMTGEVSFQADPCTWEVWGRECLQAKSAANLKVKEL SEQ ID NO: 59: 10 LIEVEKPLYGVEVFVGETAHFEIELSEPDVHGQWKLKGQPLTASPDCEIIEDGKKHILILHNC QLGMTGEVSFQA***TWEVWGRE***AKSAANLKVKEL 23

Claims (21)

1. An engineered recombinant nucleic acid phage vector for expressing a phage display fusion protein that binds selectively to a biomolecule, comprising a. a recombinant phage leader coding nucleic acid sequence; operably linked 5 to: b. a recombinant restriction site; operably linked to: c. a peptide linker encoding nucleic acid sequence; operably linked to a: d. a nucleotide sequence encoding a non-antibody scaffold protein that selectively binds to a biomolecule. 10

2. The biomolecule of claim 1, wherein said biomolecule is epidermal growth factor receptor.

3. An engineered nucleic acid phage vector according to claim 1, wherein said phage leader coding sequence is a pelB sequence.

4. A non-antibody scaffold protein encoded by the engineered nucleic acid phage 15 vector of claim 1, wherein said non-antibody scaffold protein is selected from SEQ ID NOS:32-53, 55 and 58.

5. A non-antibody scaffold protein encoded by the engineered nucleic acid phage vector of claim 1, wherein said non-antibody scaffold protein binds to epidermal growth factor receptor. 20

6. An engineered nucleic acid phage vector according to claim 1, wherein said vector encodes a second exogenous peptide fused to said non-antibody scaffold protein.

7. An engineered nucleic acid phage vector according to claim 6, wherein said second exogenous peptide binds to said biomolecule.

8. An engineered nucleic acid phage vector according to claim 7, wherein said second 25 exogenous peptide binds to epidermal growth factor receptor.

9. A bacterial host cell comprising an engineered nucleic acid phage vector according to claim 1.

10. A biologically active fusion protein expressed by a bacterial host cell according to claim 9. 30

11. A biologically active peptide or protein derived from said fusion protein according to claim 10.

12. A phage library comprising a plurality of engineered nucleic acid phage vectors according to claim 1. 24 WO 2009/086116 PCT/US2008/087696

13. A phage library according to claim 12, wherein variants of said non-antibody scaffold protein are expressed.

14. A method for screening a phage non-antibody peptide or protein library for peptides or proteins having a desired biological activity, comprising (a) expressing said 5 peptides or proteins from a phage library according to claim 13, and (b) selecting bacterial cells expressing an the peptide or protein having said desired biological activity.

15. A peptide- or protein-encoding nucleic acid, obtained from a method according to claim 14. 10

16. An isolated non-antibody scaffold protein comprising at least one portion of SEQ ID NOS: 2 - 23, 25, or 28 that mediates binding to a biomolecule.

17. An isolated non-antibody scaffold protein of claim 16, wherein said portion that mediates binding to a biomolecule binds to epidermal growth factor receptor.

18. A pharmaceutical composition comprising the non-antibody scaffold protein of claim 15 16 and a pharmaceutically acceptable carrier.

19. An isolated non-antibody scaffold protein comprising a polypeptide selected from at least one of SEQ ID NOS: 2-23, 25 and 28.

20. An isolated non-antibody scaffold protein of claim 19, wherein said polypeptide binds to epidermal growth factor receptor. 20

21. A pharmaceutical composition comprising the non-antibody scaffold protein of claim 19 and a pharmaceutically acceptable carrier. 25