AU2015271900B2 - Stabilized fibronectin domain compositions, methods and uses - Google Patents

Abstract

A protein scaffold based on a consensus sequence of fibronectin type III (FN3) proteins, such as the tenth FN3 repeat from human fibronectin (human Tenascin), including isolated nucleic acids that encode a protein scaffold, vectors, host cells, and methods of 5 making and using thereof. The protein scaffold molecules of the present invention exhibit enhanced thermal and chemical stability while presenting six modifiable loop domains which can be engineered to form a binding partner capable of binding to a target for applications in diagnostic and/or therapeutic compositions, methods and devices.

Description

STABILIZED FIBRONECTIN DOMAIN COMPOSITIONS, METHODS AND 2015271900 17 Dec 2015

USES

The present application is a divisional application of Australian Application No. 2011245225, which is incorporated in its entirety herein by reference.

5 BACKGROUND

Field of the Invention

The present invention relates to protein scaffolds with novel properties, including the ability to bind to cellular targets. More particularly, the present invention is directed to a protein scaffold based on a consensus sequence of a fibronectin type ΙΠ (FN3) 10 repeat.

Discussion of the Field

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 proteins that can be engineered to bind such targets are also of high interest in the 15 biopharmaceutical industry. These “alternative scaffold” proteins may have advantages over traditional antibodies due to their small size, lack of disulphide bonds, high stability, and ability to be expressed in prokaryotic hosts. Novel methods of purification are readily applied; they are easily conjugated to drugs/toxins, penetrate efficiently into tissues and are readily formatted into multispecific binders (Skerra 2000 J Mol Recognit 13(4): 167-87; 20 Binz and Pluckthun 2005 Curr Qpin Biotechnol 16(4): 459-69).

One such alternative scaffold is the immunoglobulin (Ig) fold. This fold is found in the variable regions of antibodies, as well as thousands of non-antibody proteins. It has been shown that one such Ig protein, the tenth fibronectin type III (FN3) repeat from human fibronectin, can tolerate a number of mutations in surface exposed loops while 25 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 (Koide etal. 1998 J Mol Biol 28414T 1141-51; Karatan et al. 2004 Chem Biol 11(61: 835-44).

Such engineered FN3 domains have been found to bind to targets with high affinity, while retaining important biophysical properties (Parker et al. 2005 Protein Eng Des Sel 18(9): 30 435-44). 1 2015271900 17 Dec 2015

Desirable physical properties of potential alternative scaffold molecules include high thermal stability and reversibility of thermal folding and unfolding. Several_ 1a 2015271900 22 Dec 2016 methods have been applied to increase the apparent thermal stability of proteins and enzymes, including rational design based on comparison to highly similar thermostable sequences, design of stabilizing disulfide bridges, mutations to increase alpha-helix propensity, engineering of salt bridges, alteration of the surface charge of the protein, directed evolution, and composition of consensus sequences (Lehmann and Wyss 2001 Curr Opin Biotechnol 12(4): 371-5). High thermal stability is a desired property of such scaffolds as it may increase the yield of recombinant protein obtained, improve solubility of the purified molecule, improve activity of intracellular scaffolds, decrease immunogenicity, and minimize the need of a cold chain in manufacturing.

Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of common general knowledge in the field.

SUMMARY OF THE INVENTION

According to a first aspect, the invention provides a protein scaffold comprising loop domains topologically similar to loop domains of a fibronectin type III (FN3) domain, the protein scaffold having an amino acid sequence based on the consensus amino acid sequence of SEQ ID NO: 16, wherein one or more specific residues of SEQ ID NO: 16 are replaced to enhance melting temperature (Tm) and chemical stability of the protein scaffold as compared to the Tm and chemical stability of a protein scaffold having the amino acid sequence of SEQ ID NO: 16, and the replacements of SEQ ID NO: 16 are selected from the group consisting of N46V, E14P, L17A, E86I, a combination of N46V and E86I, a combination of N46V and E86I, and a combination of L17A, N46V and E86I.

According to a second aspect, the invention provides a protein scaffold comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 142-144 and 147- 151.

According to a third aspect, the invention provides a method of generating a protein scaffold binding to a specific target with a predefined binding affinity, comprising the steps of: (a) contacting a library of a scaffold-based protein derived from a stability enhanced consensus sequence of a fibronectin type III domain incorporating randomized codons wherein 2 2015271900 22 Dec 2016 a polynucleotide sequence encoding a polypeptide comprising a stability enhanced consensus sequence of a fibronectin type III domain selected from the group consisting of SEQ ID NOs: 142-151 is provided, randomizing codons are introduced into the polynucleotide sequence at selected positions and copies of the polynucleotide sequence are propagated to form a library of polynucleotides encoding variant scaffold proteins, with the specific target; and (b) isolating a protein binding to the specific target with the predefined affinity.

According to a fourth aspect, the invention provides a protein scaffold when generated by the method of the invention.

Unless the context clearly requires otherwise, throughout the description and the claims, the words “comprise”, “comprising”, and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of “including, but not limited to”.

The present invention provides a protein scaffold based on a fibronectin type III (FN3) repeat protein, encoding or complementary nucleic acids, vectors, host cells, compositions, combinations, formulations, devices, and methods of making and using them. In a preferred embodiment, the protein scaffold is comprised of a consensus sequence of multiple FN3 domains from human Tenascin-C (hereinafter “Tenascin”). In a further preferred embodiment, the protein scaffold of the present invention is a consensus sequence of 15 FN3 domains (SEQ ID NO: 1-15) or a variant thereof. In a particular aspect of the invention, the protein scaffold of the invention has substitute residues which cause the scaffold protein to demonstrate enhanced ability to resist thermal and chemical denaturation. The protein scaffolds of the invention can be engineered by methods known in the art, including inserting residues at designated loop regions within the scaffold, to form a binding domain selective for a binding partner. The binding partner may be a soluble molecule or a cellularly anchored molecule, for example, the extracellular domain of a receptor protein.

In one embodiment, specific substitutions of the in the consensus-based sequence of SEQ ID NO: 16 (Tencon) selected for inherent thermal and chemical stability described herein improve the thermal stability of the Tencon scaffold by up to 11°C and shift the mid-point of GdmCl induced denaturation from 3.4 M to greater than 5 M. In one embodiment, the specific substitutions to SEQ ID NO: 16 (Tencon) are unitary, such as N46V, E14P, and E86I, and, in an alternative embodiment the substitutions are_ 2a multiple, such as N46V and E86I, all of E14P and N46V and E86I, and all of L17A and N46V and E86I. Tencon-based polypeptides with enhanced stability provide scaffolds with improved ease of purification, formulation, and increased shelf-life. Engineered binding partners with improved overall stability can be produced by introducing randomized peptides into loops of the stabilized scaffold.

The protein scaffolds of the invention may be used as monomeric units or linked to form polymeric structures with the same or different binding partner specificity. The Tencon protein scaffold-based molecules may be further modified to enhance one or more in vivo properties related to biodistribution, persistence in the body, or therapeutic efficacy such as the association with molecules which alter cellular, particularly, epithelial cell uptake, for example, the Fc region of an antibody, or molecules designed to bind serum proteins such as an albumin binding domain. In further embodiments, the protein scaffolds of the invention may be bound to a nucleic acid molecule that may encode the protein scaffold.

The present invention also provides at least one method for expressing at least one protein scaffold polypeptide whose sequence is related to a consensus sequence of multiple FN3 domains, in a host cell, comprising culturing a host cell as described herein under conditions wherein at least one protein scaffold is expressed in detectable and/or recoverable amounts.

The present invention also provides at least one composition comprising (a) a protein scaffold based on a consensus sequence of multiple FN3 domains and/or encoding nucleic acid as described herein; and (b) a suitable and/or pharmaceutically acceptable carrier or diluent.

The present invention further comprises a method of generating libraries of a protein scaffold based on a fibronectin type III (FN3) repeat protein, preferably, a consensus sequence of multiple FN3 domains and, more preferably, a consensus sequence of multiple FN3 domains from human Tenascin with enhanced thermal and chemical stability. Libraries can be generated by altering the amino acid composition of a single loop or the simultaneous alteration of multiple loops or additional positions of the scaffold molecule. The loops that are altered can be lengthened or shortened 3 accordingly. Such libraries can be generated to include all possible amino acids at each position, or a designed subset of amino acids. The library members can be used for screening by display, such as in vitro display (DNA, RNA, ribosome display, etc.), yeast, bacterial, and phage display.

The protein scaffolds of the present invention provide enhanced biophysical properties, such as stability under conditions of high osmotic strength and solubility at high concentrations. The domains of the scaffold proteins are not disulfide bonded, making them capable of expression and folding in systems devoid of enzymes required for disulfide linkage formation, including prokaryotic systems, such as E. coli, and in in vitro transcription/translation systems, such as the rabbit reticulocyte lysate system.

In an additional aspect, the present invention provides a method of generating a scaffold molecule that binds to a particular target by panning the scaffold library of the invention with the target and detecting binders. In other related aspects, the invention comprises screening methods that may be used to generate or affinity mature protein scaffolds with the desired activity, e.g., capable of binding to target proteins with a certain affinity. Affinity maturation can be accomplished by iterative rounds of mutagenesis and selection using systems, such as phage display or in vitro display. Mutagenesis during this process may be the result of site directed mutagenesis to specific scaffold residues, random mutagenesis due to error-prone PCR, DNA shuffling, and/or a combination of these techniques. The present invention further provides any invention described herein.

BRIEF DESCRIPTION OF THE DRAWING

Figure 1 Figure 1. SDS-PAGE analysis of purified Tencon performed on a NuPAGE 4-12% Bis-Tris gel (Invitrogen) and stained with coomassie blue. N stands for native conditions and R for reduced conditions.

Figure 2 shows a circular dichroism analysis of Tencon in PBS. 4

Figure 3 shows a circular dichroism analysis of the third FN3 domain from tenascin and Tencon in PBS where the melting temperatures of 54°C and 78°C were obtained respectively.

Figure 4 shows phagemid plasmid design of pTencon-pIX. Expression is driven by a Lac promoter and secretion via the OmpA signal sequence.

Figure 5 shows myc-Tencon can be displayed on M13 phage using ELISA demonstrating the binding of phage to anti-Myc coated, CNT095 coated, and uncoated wells.

Figure 6 is a drawing depicting the loop structure of the third FN3 domain of human Tenascin.

Figure 7 shows the screening by ELISA output of IgG selections whereby individual clones were tested for binding to biotinylated IgG or biotinylated HSA as a control.

Figures 8A-B are graphs showing the GdmCl induced denaturation for single mutants (A) and combinatorial mutants (B) as measured by fluorescence excitation of 280 nm and an emission of 360 nm.

DETAILED DESCRIPTION OF THE INVENTION

Abbreviations ADCC = antibody-dependent cellular cytotoxicity; CDC = complement-dependent cytotoxicity; DSC = differential scanning calorimetry; AG = Gibbs Free Energy; IgG = immunoglobulin G; Tm = temperature of melting;

Definitions & Explanation of Terminology

The term “antibody” or “antibody moiety” is intended to encompass antibodies, digestion fragments, specified portions and variants thereof, including, without limitation, antibody mimetics or comprising portions of antibodies that mimic the structure and/or function of an antibody or specified fragment or portion thereof, including, without limitation, single chain antibodies, single domain antibodies, 5 minibodies, and fragments thereof. Functional fragments include antigen-binding fragments that bind to the target antigen of interest. For example, antibody fragments capable of binding to a target antigen or portions thereof, including, but not limited to, Fab (e.g., by papain digestion), Fab' (e.g., by pepsin digestion and partial reduction) and F(ab’)2 (e.g., by pepsin digestion), facb (e.g., by plasmin digestion), pFc’ (e.g., by pepsin or plasmin digestion), Fd (e.g., by pepsin digestion, partial reduction and reaggregation), Fv or scFv (e.g., by molecular biology techniques) fragments, are encompassed by the term antibody. The antibody or fragment may be derived from any mammal, such as, but not limited to, a human, a mouse, a rabbit, a rat, a rodent, a primate, a camelid, a goat, or any combination thereof and includes isolated human, primate, rodent, mammalian, chimeric, humanized and/or CDR-grafted antibodies, immunoglobulins, cleavage products and other specified portions and variants thereof.

The term "epitope" means a protein determinant capable of specific binding to an antibody or engineered binding domain such as one or more loops of a scaffold-based protein. Epitopes usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three-dimensional structural characteristics, as well as specific charge characteristics. Conformational and nonconformational epitopes are distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents. The conformational epitopes result from conformational folding of the target molecule which arise when amino acids from differing portions of the linear sequence of the target molecule come together in close proximity in 3-dimensional space. Such conformational epitopes are typically distributed on the extracellular side of the plasma membrane.

The terms “Fc,” “Fc-containing protein” or “Fc-containing molecule” as used herein refer to a monomeric, dimeric or heterodimeric protein having at least an immunoglobulin CH2 and CH3 domain. The CH2 and CH3 domains can form at least a part of the dimeric region of the protein/molecule (e.g., antibody).

The term "stability" as used herein refers to the ability of a molecule to maintain a folded state under physiological conditions such that it retains at least one of its normal functional activities, for example, binding to a target molecule like a cytokine or serum 6 protein. Measurement of protein stability and protein liability can be viewed as the same or different aspects of protein integrity. Proteins are sensitive or “labile” to denaturation caused by heat, by ultraviolet or ionizing radiation, changes in the ambient osmolarity and pH if in liquid solution, mechanical shear force imposed by small pore-size filtration, ultraviolet radiation, ionizing radiation, such as by gamma irradiation, chemical or heat dehydration, or any other action or force that may cause protein structure disruption. The stability of the molecule can be determined using standard methods. For example, the stability of a molecule can be determined by measuring the thermal melt ("TM") temperature. The TM is the temperature in ° Celsius (°C) at which A of the molecules become unfolded. Typically, the higher the TM, the more stable the molecule. In addition to heat, the chemical environment also changes the ability of the protein to maintain a particular three dimensional structure.

Chemical denaturation can likewise be measured by a variety of methods. A chemical denaturant is an agent known to disrupt non-covalent interactions and covalent bonds within a protein, including hydrogen bonds, electrostatic bonds, Van der Waals forces, hydrophobic interactions, or disulfide bonds. Chemical denaturants include guanidinium hydrochloride, guanadinium thiocyanate, urea, acetone, organic solvents (DMF, benzene, acetonitrile), salts (ammonium sulfate lithium bromide, lithium chloride, sodium bromide, calcium chloride, sodium chloride); reducing agents (e.g. dithiothreitol, beta-mercaptoethanol, dinitrothiobenzene, and hydrides, such as sodium borohydride) , non-ionic and ionic detergents, acids (e.g. hydrochloric acid (HC1), acetic acid (CH3COOH), halogenated acetic acids), hydrophobic molecules (e.g. phosopholipids), and targeted denaturants (Jain R.K and Hamilton A. D., Angew. Chem. 114(4), 2002). Quantitation of the extent of denatureation can rely on loss of a functional property such as ability to bind a target molecule, or by physiochemical properties such tendency to aggregation, exposure of formerly solvent inaccessible residues, or disruption or formation of disulfide bonds.

In terms of loss of stability, i.e. “denaturing” or “denaturation” of a protein is meant the process where some or all of the three-dimensional conformation imparting the functional properties of the protein has been lost with an attendant loss of activity and/or solubility. Forces disrupted during denaturation include intramolecular bonds, including 7 2015271900 17 Dec 2015 but not limited to electrostatic, hydrophobic, Van der Waals forces, hydrogen bonds, and disulfides. Protein denaturation can be caused by forces applied to the protein or a solution comprising the protein such as mechanical force (for example, compressive or shear-force), thermal, osmotic stress, change in pH, electrical or magnetic fields, ionizing 5 radiation, ultraviolet radiation and dehydration, and by chemical denaturants. A “therapeutically effective" treatment or amount as used herein, refers to an amount of sufficient quantity to cause a detectable lessening or amelioration of the cause of a disorder or its symptoms. "Ameliorate" refers to a lessening of the detrimental effect of the disorder in the patient receiving the therapy. The subject of the invention is 10 preferably a human, however, it can be envisioned that any animal in need of a treatment for a deleterious conditions, disorder, or disease can be treated with a scaffold-based protein designed for that purpose.

Overview

The present invention provides an isolated, recombinant and/or synthetic protein 15 scaffold based on a consensus sequence of a fibronectin type III (FN3) repeat protein, including, without limitation, mammalian-derived scaffold, as well as compositions and encoding nucleic acid molecules comprising at least one polynucleotide encoding a protein scaffold based on the consensus FN3 sequence. The present invention further includes, but is not limited to, methods of making and using such nucleic acids and 20 protein scaffolds, including as a discovery platform, and for diagnostic and therapeutic compositions, methods and devices.

The protein scaffolds of the present invention offer advantages over larger immunogoblulin based biotherapeutics, owing to their small, compact size. In particular, the size and shape of a biologic molecule can impact its ability to be administered locally, 25 orally, or cross the blood-brain barrier; ability to be expressed in low cost systems such as E. coli; ability to be engineered into bi- or multi-specific molecules binding to multiple targets or multiple epitopes of the same target, suitability for conjugation, i.e. to actives, polymers, and probes; ability to be formulated to high concentrations; and the ability of such molecules to effectively penetrate diseased tissues and tumors. 8

Moreover, the protein scaffolds possess many of the properties of antibodies in relation to their fold that mimics the variable region of an antibody. This orientation enables the FN3 loops to be exposed similar to antibody complementarity determining regions (CDRs). They should be able to bind to cellular targets and the loops can be altered, e.g., affinity matured, to improve certain binding or related properties.

Three of the six loops of the protein scaffold of the invention correspond topologically to the binding domains of an antibody positioned at the loops of the variable domain known to be hypervariable in nature (the hypervariable domains loops (HVL), at positions as defined by Kabat as the residues of the complementarity determining regions (CDRs), i.e., antigen-binding regions, of an antibody, while the remaining three loops are surface exposed in a manner similar to antibody CDRs. These loops span or are positioned at or about residues 13-16, 22-28, 38-43, 51-54, 60-64, and 75-81 of SEQ ID NO:16 as shown in Table 3 below and Figure 6. Preferably, the loop regions at or about residues 22-28, 51-54, and 75-81 are altered for binding specificity and affinity. One or more of these loop regions are randomized with other loop regions and/or other strands maintaining their sequence as backbone portions to populate a library and potent binders can be selected from the library having high affinity for a particular protein target. One or more of the loop regions can interact with a target protein similar to an antibody CDR interaction with the protein.

The scaffolds of the present invention may incorporate other subunits, e.g., via covalent interaction. All or a portion of an antibody constant region may be attached to the scaffold to impart antibody-like properties especially those properties associated with the Fc region, e.g., complement activity (ADCC), half-life, etc. For example, effector function can be provided and/or controlled, e.g., by modifying Clq binding and/or FcyR binding and thereby changing CDC activity and/or ADCC activity. “Effector functions" are responsible for activating or diminishing a biological activity (e.g., in a subject). Examples of effector functions include, but are not limited to: Clq binding; complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g., B cell receptor; BCR), etc. Such effector functions may require the Fc region to be combined 9 with a binding domain (e.g., protein scaffold loops) and can be assessed using various assays (e.g., Fc binding assays, ADCC assays, CDC assays, etc.).

Additional moieties may be appended or associated with the scaffold-based polypeptide or variant such as a toxin conjugate, albumin or albumin binders, polyethylene glycol (PEG) molecules may be attached to the scaffold molecule for desired properties. These moieties may be in-line fusions with the scaffold coding sequence and may be generated by standard techniques, for example, by expression of the fusion protein from a recombinant fusion encoding vector constructed using publically available coding nucleotide sequences. Alternatively, chemical methods may be used to attach the moieties to a recombinantly produced scaffold-based protein.

The scaffolds of the present invention can be used as monospecific in monomeric form or as bi- or multi-specific (for different protein targets or epitopes on the same protein target) in multimer form. The attachments between each scaffold unit may be covalent or non-covalent. For example, a dimeric bispecific scaffold has one subunit with specificity for a first target protein or epitope and a second subunit with specificity for a second target protein or epitope. Scaffold subunits can be joined in a variety of conformations that can increase the valency and thus the avidity of antigen binding.

Generation and Production of Scaffold Protein

At least one scaffold protein of the present invention can be optionally produced by a cell line, a mixed cell line, an immortalized cell or clonal population of immortalized cells, as well known in the art. See, e.g., Ausubel, et al., ed., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NY, NY (1987-2001); Sambrook, et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor, NY (1989); Harlow and Lane, Antibodies, a Laboratory Manual, Cold Spring Harbor, NY (1989); Colligan, et al., eds., Current Protocols in Immunology, John Wiley & Sons, Inc., NY (1994-2001); Colligan et al., Current Protocols in Protein Science, John Wiley & Sons, NY, NY, (1997-2001).

Amino acids from a scaffold protein can be altered, added and/or deleted to reduce immunogenicity or reduce, enhance or modify binding, affinity, on-rate, off-rate, 10 avidity, specificity, half-life, stability, solubility or any other suitable characteristic, as known in the art.

Bioactive scaffold-based proteins can be engineered with retention of high affinity for the antigen and other favorable biological properties. To achieve this goal, the scaffold proteins can be optionally prepared by a process of analysis of the parental sequences and various conceptual engineered products using three-dimensional models of the parental and engineered sequences. Three-dimensional models are commonly available and are familiar to those skilled in the art. Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate sequences and can measure possible immunogenicity (e.g., Immunofilter program of Xencor, Inc. of Monrovia, CA). Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate sequence, i.e., the analysis of residues that influence the ability of the candidate scaffold protein to bind its antigen. In this way, residues can be selected and combined from the parent and reference sequences so that the desired characteristic, such as affinity for the target antigen(s), is achieved. Alternatively, or in addition to, the above procedures, other suitable methods of engineering can be used.

Screening

Screening engineered scaffold-based protein or libraries comprising scaffold-based proteins with variegated residues or domains for specific binding to similar proteins or fragments can be conveniently achieved using nucleotide (DNA or RNA display) or peptide display libraries, for example, in vitro display. This method involves the screening of large collections of peptides for individual members having the desired function or structure. The displayed peptide with or without nucleotide sequences can be from 3 to 5000 or more nucleotides or amino acids in length, frequently from 5-100 amino acids long, and often from about 8 to 25 amino acids long. In addition to direct chemical synthetic methods for generating peptide libraries, several recombinant DNA methods have been described. One type involves the display of a peptide sequence on the surface of a bacteriophage or cell. Each bacteriophage or cell contains the nucleotide sequence encoding the particular displayed peptide sequence. 11

The protein scaffolds of the invention can bind human or other mammalian proteins with a wide range of affinities (KD). In a preferred embodiment, at least one protein scaffold of the present invention can optionally bind to a target protein with high affinity, for example, with a Kd equal to or less than about 10'7 M, such as but not limited to, 0.1-9.9 (or any range or value therein) X 10‘8, 10‘9, ΙΟ'10, 10'11, ΙΟ"12, ΙΟ'13, ΙΟ'14, 10'15 or any range or value therein, as determined by surface plasmon resonance or the Kinexa method, as practiced by those of skill in the art.

The affinity or avidity of a protein scaffold for an antigen can be determined experimentally using any suitable method. (See, for example, Berzofsky, et al., “Antibody-Antigen Interactions,” In Fundamental Immunology, Paul, W. E., Ed., Raven Press: New York, NY (1984); Kuby, Janis Immunology, W. H. Freeman and Company: New York, NY (1992); and methods described herein). The measured affinity of a particular protein scaffold-antigen interaction can vary if measured under different conditions (e.g., osmolarity, pH). Thus, measurements of affinity and other antigenbinding parameters (e.g., Kd, Κ,η, K,ff) are preferably made with standardized solutions of protein scaffold and antigen, and a standardized buffer, such as the buffer described herein.

Competitive assays can be performed with the protein scaffold of the present invention in order to determine what proteins, antibodies, and other antagonists compete for binding to a target protein with the protein scaffold of the present invention and/or share the epitope region. These assays as readily known to those of ordinary skill in the art evaluate competition between antagonists or ligands for a limited number of binding sites on a protein. The protein and/or antibody is immobilized, isolated, or captured before or after the competition and the sample bound to the target protein is separated from the unbound sample, for example, by decanting (where the protein/antibody was preinsolubilized) or by centrifuging (where the protein/antibody was precipitated after the competitive reaction). Also, the competitive binding may be determined by whether function is altered by the binding or lack of binding of the protein scaffold to the target protein, e.g., whether the protein scaffold molecule inhibits or potentiates the enzymatic activity of, for example, a label. ELISA and other functional assays may be used, as well known in the art. 12

Nucleic Acid Molecules

Nucleic acid molecules of the present invention encoding protein scaffolds can be in the form of RNA, such as mRNA, hnRNA, tRNA or any other form, or in the form of DNA, including, but not limited to, cDNA and genomic DNA obtained by cloning or produced synthetically, or any combinations thereof. The DNA can be triple-stranded, double-stranded or single-stranded, or any combination thereof Any portion of at least one strand of the DNA or RNA can be the coding strand, also known as the sense strand, or it can be the non-coding strand, also referred to as the anti-sense strand.

Isolated nucleic acid molecules of the present invention can include nucleic acid molecules comprising an open reading frame (ORF), optionally, with one or more introns, e.g., but not limited to, at least one specified portion of at least one protein scaffold; nucleic acid molecules comprising the coding sequence for a protein scaffold or loop region that binds to the target protein; and nucleic acid molecules which comprise a nucleotide sequence substantially different from those described above but which, due to the degeneracy of the genetic code, still encode the protein scaffold as described herein and/or as known in the art. Of course, the genetic code is well known in the art. Thus, it would be routine for one skilled in the art to generate such degenerate nucleic acid variants that code for specific protein scaffolds of the present invention. See, e.g., Ausubel, et al., supra, and such nucleic acid variants are included in the present invention.

As indicated herein, nucleic acid molecules of the present invention which comprise a nucleic acid encoding a protein scaffold can include, but are not limited to, those encoding the amino acid sequence of a protein scaffold fragment, by itself; the coding sequence for the entire protein scaffold or a portion thereof; the coding sequence for a protein scaffold, fragment or portion, as well as additional sequences, such as the coding sequence of at least one signal leader or fusion peptide, with or without the aforementioned additional coding sequences, such as at least one intron, together with additional, non-coding sequences, including but not limited to, non-coding 5’ and 3’ sequences, such as the transcribed, non-translated sequences that play a role in transcription, mRNA processing, including splicing and polyadenylation signals (for 13 example, ribosome binding and stability of mRNA); an additional coding sequence that codes for additional amino acids, such as those that provide additional functionalities. Thus, the sequence encoding a protein scaffold can be fused to a marker sequence, such as a sequence encoding a peptide that facilitates purification of the fused protein scaffold comprising a protein scaffold fragment or portion.

Nucleic Acid Molecules

The invention also provides for nucleic acids encoding the compositions of the invention as isolated polynucleotides or as portions of expression vectors including vectors compatible with prokaryotic, eukaryotic or filamentous phage expression, secretion and/or display of the compositions or directed mutagens thereof.

The isolated nucleic acids of the present invention can be made using (a) recombinant methods, (b) synthetic techniques, (c) purification techniques, and/or (d) combinations thereof, as well-known in the art.

The polynucleotides useful in the practice of the present invention will encode a functional portion of the protein scaffold described herein. The polynucleotides of this invention embrace nucleic acid sequences that can be employed for selective hybridization to a polynucleotide encoding a protein scaffold of the present invention. The present invention provides isolated nucleic acids that hybridize under selective hybridization conditions to a polynucleotide disclosed herein. Thus, the polynucleotides of this embodiment can be used for isolating, detecting, and/or quantifying nucleic acids comprising such polynucleotides. For example, polynucleotides of the present invention can be used to identify, isolate, or amplify partial or full-length clones in a deposited library. In some embodiments, the polynucleotides are genomic or cDNA sequences isolated, or otherwise complementary to, a cDNA from a human or mammalian nucleic acid library.

The nucleic acids can conveniently comprise sequences in addition to a polynucleotide of the present invention. For example, a multi-cloning site comprising one or more endonuclease restriction sites can be inserted into the nucleic acid to aid in isolation of the polynucleotide. Also, translatable sequences can be inserted to aid in the isolation of the translated polynucleotide of the present invention. For example, a hexa-histidine marker 14 sequence provides a convenient means to purify the proteins of the present invention. The nucleic acid of the present invention, excluding the coding sequence, is optionally a vector, adapter, or linker for cloning and/or expression of a polynucleotide of the present invention.

Additional sequences can be added to such cloning and/or expression sequences to optimize their function in cloning and/or expression, to aid in isolation of the polynucleotide, or to improve the introduction of the polynucleotide into a cell. Use of cloning vectors, expression vectors, adapters, and linkers is well known in the art.

As indicated herein, nucleic acid molecules of the present invention which comprise a nucleic acid encoding a protein scaffold can include, but are not limited to, those encoding the amino acid sequence of a protein scaffold fragment, by itself; the coding sequence for the entire protein scaffold or a portion thereof; the coding sequence for a protein scaffold, fragment or portion, as well as additional sequences, such as the coding sequence of at least one signal leader or fusion peptide, with or without the aforementioned additional coding sequences, such as at least one intron, together with additional, non-coding sequences, including but not limited to, non-coding 5’ and 3’ sequences, such as the transcribed, non-translated sequences that play a role in transcription, mRNA processing, including splicing and polyadenylation signals (for example, ribosome binding and stability of mRNA); an additional coding sequence that codes for additional amino acids, such as those that provide additional functionalities. Thus, the sequence encoding a protein scaffold can be fused to a marker sequence, such as a sequence encoding a peptide that facilitates purification of the fused protein scaffold comprising a protein scaffold fragment or portion.

For bacterial expression including phage infected bacteria, a preferred secretion signal is a pelB or ompA secretion signal but other secretion signal polypeptide domains may be used as described in U.S. Pat. No. 5,658,727. In phage display, a downstream translatable DNA sequence encodes a filamentous phage coat protein, e.g. pill or pIX protein. Preferred phage proteins are obtainable from filamentous phage Ml3, fl, fd, and the like equivalent filamentous phage. Thus, a downstream translatable DNA sequence encodes an amino acid residue sequence that corresponds, and preferably is identical, to 15 the filamentous phage gene III or gene IX coat polypeptide. The sequences of such coat proteins are known and accessible in public databases such as the NCBI. A cDNA or genomic library can be screened using a probe based upon the sequence of a polynucleotide of the present invention, such as those disclosed herein. Probes can be used to hybridize with genomic DNA or cDNA sequences to isolate homologous genes in the same or different organisms. Those of skill in the art will appreciate that various degrees of stringency of hybridization can be employed in the assay; and either the hybridization or the wash medium can be stringent. As the conditions for hybridization become more stringent, there must be a greater degree of complementarity between the probe and the target for duplex formation to occur. The degree of stringency can be controlled by one or more of temperature, ionic strength, pH and the presence of a partially denaturing solvent, such as formamide. For example, the stringency of hybridization is conveniently varied by changing the polarity of the reactant solution through, for example, manipulation of the concentration of formamide within the range of 0% to 50%. The degree of complementarity (sequence identity) required for detectable binding will vary in accordance with the stringency of the hybridization medium and/or wash medium. The degree of complementarity will optimally be 100%, or 70-100%, or any range or value therein. However, it should be understood that minor sequence variations in the probes and primers can be compensated for by reducing the stringency of the hybridization and/or wash medium.

In one aspect of the invention, the polynucleotides are constructed using techniques for incorporation of randomized codons in order to variegate the resulting polypeptide at one or more specific residues or to add residues at specific locations within the sequence.

Various strategies may be used to create libraries of altered polypeptide sequences including random, semi-rational and rational methods. Rational and semi-rational methods have the advantage over the random strategies in that one has more control over the consequences of changes introduced into the coding sequence. In addition, by focusing the variation in certain regions of the gene, the universe of all possible amino acid variants can be explored in chosen positions. 16 A library built on the common NNK or NNS diversification scheme introduce a possible 32 different codons in every position and all 20 amino acids. Such a library theoretically grows by 32n for every n number of residues. In practical terms, however, phage display is limited to sampling libraries of 109 to 1010 variants implying that only 6-7 residues can be targeted for variegation if full sequence coverage is to be achieved in the library. Thus, semi-rational or “focused” methods to generate libraries of scaffold variants by identifying key positions to be variegated and choosing the diversification regime according can be applied. A "codon set" refers to a set of different nucleotide triplet sequences used to encode desired variant amino acids. A standard form of codon designation is that of the IUB code, which is known in the art and described herein. A "non-random codon set" refers to a codon set that encodes select amino acids. Synthesis of oligonucleotides with selected nucleotide "degeneracy" at certain positions is well known in that art, for example the TRIM approach (Knappek et al.; J. Mol. Biol. (1999), 296:57-86); Garrard & Henner, Gene (1993), 128:103). Such sets of nucleotides having certain codon sets can be synthesized using commercially available nucleotide or nucleoside reagents and apparatus. A codon set is a set of different nucleotide triplet sequences used to encode desired variant amino acids. Codon sets can be represented using symbols to designate particular nucleotides or equimolar mixtures of nucleotides as shown in below according to the IUB code. IUB Codes G Guanine A Adenine T Thymine C Cytosine R(AorG) Y(CorT) M (A or C) 17 K(GorT) S (C or G) W (A or T) H (A or C or T) B (C orGorT) V (A or C or G) D (A or G or T) N (A or C or G or T)

For example, in the codon set DVK, D can be nucleotides A or G or T; V can be A or G or C; and K can be G or T. This codon set can present 18 different codons and can encode amino acids Ala, Trp, Tyr, Lys, Thr, Asn, Lys, Ser, Arg, Asp, Glu, Gly, and Cys.

Focused (e.g., non-random) libraries can be generated using NNK codons and focusing the varigation at selected residues or, alternatively, variants with non-random substitutions can be generated using for example DVK codons, which encodes 11 amino acids (ACDEGKNRSYW) and one stop codon. Alternatively, Kunkel mutagenesis can be used to variegate the desired residues or regions of the polypeptide (Kunkel et al., Methods Enzymol. 154:367-382,1987).

Standard cloning techniques are used to clone the libraries into a vector for expression. The library may be expressed using known system, for example expressing the library as fusion proteins. The fusion proteins can be displayed on the surface of any suitable phage. Methods for displaying fusion polypeptides comprising antibody fragments on the surface of a bacteriophage are well known (US 6,969,108 to Griffith; US 6,172,197 to McCafferty; US 5,223,409 to Ladner; US 6,582,915 to Griffiths; US6472147 to Janda). Libraries for de novo polypeptide isolation can be displayed on pIX (W02009085462A1). The libraries can also be translated in vitro, for example using ribosome display (Hanes and Pluckthun, Proc. Natl. Acad. Scie. USA, 94:4937, 1997), mRNA display (Roberts and Szostak, Proc. Natl. Acad. Sci. USA, 94:12297,1997), CIS-display (Odegrip et. al., Proc. 18

Natl. Acad. Sci. USA, 101:2806, 2004) or other cell-free systems (US 5,643,768 to Kawasaki).

Libraries with diversified regions can be generated using vectors comprising the polynucleotide encoding the Tencon sequence (SEQ ID NO: 16) or a predetermined mutant thereof. The template construct may have a promoter and signal sequences for the polypeptide chain. To make scaffold libraries, mutagenesis reactions using oligonucleotides that coded for loop regions (A:B, B:C, C:D, D:E, E:F, and F:G) of the scaffold are used. To ensure the incorporation of all chosen positions into the randomization scheme, a stop codon (such as TAA) can be incorporated in each region desired to be intended to be diversified. Only clones where the stop codons have been replaced will occur.

Modified Scaffold Polypeptides

Modified protein scaffolds and fragments of the invention can comprise one or more moieties that are covalently bonded, directly or indirectly, to another protein.

In the case of the addition of peptide residues, or the creation of an in-line fusion protein, the addition of such residues may be through recombinant techniques from a polynucleotide sequence as described herein. In the case of an appended, attached or conjugated peptide, protein, organic chemical, inorganic chemical or atom, or any combination thereof, the additional moiety that is bonded to a protein scaffold or fragment of the invention is typically via other than a peptide bond. The modified protein scaffolds of the invention can be produced by reacting a protein scaffold or fragment with a modifying agent. For example, the organic moieties can be bonded to the protein scaffold in a non-site specific manner by employing an amine-reactive modifying agent, for example, an NHS ester of PEG. Modified protein scaffolds and fragments comprising an organic moiety that is bonded to specific sites of a protein scaffold of the present invention can be prepared using suitable methods, such as reverse proteolysis (Fisch et al., Bioconjugate Chem., 3:147-153 (1992); Werlen et al., Bioconjugate Chem., 5:411-417 (1994); Kumaran et al., Protein Sci. 6(10):2233-2241 (1997); Itoh et al., Bioorg. Chem., 24(1): 59-68 (1996); Capellas et al., Biotechnol. Bioeng., 56(4):456-463 (1997)), and the methods described in Hermanson, G. T., Bioconjugate Techniques, Academic Press: San Diego, CA (1996). 19

Where a polymer or chain is attached to the scaffold protein, the polymer or chain can independently be a hydrophilic polymeric group, a fatty acid group or a fatty acid ester group. As used herein, the term “fatty acid” encompasses mono-carboxylic acids and di-carboxylic acids. A “hydrophilic polymeric group,” as the term is used herein, refers to an organic polymer that is more soluble in water than in octane. For example, polylysine is more soluble in water than in octane. Thus, a protein scaffold modified by the covalent attachment of polylysine is encompassed by the invention. Hydrophilic polymers suitable for modifying protein scaffolds of the invention can be linear or branched and include, for example, polyalkane glycols (e.g., PEG, monomethoxy-polyethylene glycol (mPEG), PPG and the like), carbohydrates (e.g., dextran, cellulose, oligosaccharides, polysaccharides and the like), polymers of hydrophilic amino acids (e.g., polylysine, polyarginine, polyaspartate and the like), polyalkane oxides (e.g., polyethylene oxide, polypropylene oxide and the like) and polyvinyl pyrolidone. Preferably, the hydrophilic polymer that modifies the protein scaffold of the invention has a molecular weight of about 800 to about 150,000 Daltons as a separate molecular entity. For example, PEG5000 and PEG2o,ooo, wherein the subscript is the average molecular weight of the polymer in Daltons, can be used. The hydrophilic polymeric group can be substituted with one to about six alkyl, fatty acid or fatty acid ester groups. Hydrophilic polymers that are substituted with a fatty acid or fatty acid ester group can be prepared by employing suitable methods. For example, a polymer comprising an amine group can be coupled to a carboxylate of the fatty acid or fatty acid ester, and an activated carboxylate (e.g., activated with N, N-carbonyl diimidazole) on a fatty acid or fatty acid ester can be coupled to a hydroxyl group on a polymer.

Fatty acids and fatty acid esters suitable for modifying protein scaffolds of the invention can be saturated or can contain one or more units of unsaturation. Fatty acids that are suitable for modifying protein scaffolds of the invention include, for example, n-dodecanoate (C12, laurate), n-tetradecanoate (C14, myristate), n-octadecanoate (Cig, stearate), n-eicosanoate (C20, arachidate), n-docosanoate (C22, behenate), n-triacontanoate (C30), n-tetracontanoate (C40), c/.v-A9-octadccanoate (Cig, oleate), all cis-A5,8,l 1,14-eicosatetraenoate (C20, arachidonate), octanedioic acid, tetradecanedioic acid, octadecanedioic acid, docosanedioic acid, and the like. Suitable fatty acid esters include 20 mono-esters of dicarboxylic acids that comprise a linear or branched lower alkyl group. The lower alkyl group can comprise from one to about twelve, preferably, one to about six, carbon atoms.

Fc-containing proteins can be compared for functionality by several well-known in vitro assays. In particular, affinity for members of the FcyRI, FcyRII, and FcyRIII family of Fey receptors is of interest. These measurements could be made using recombinant soluble forms of the receptors or cell-associated forms of the receptors. In addition, affinity for FcRn, the receptor responsible for the prolonged circulating half-life of IgGs, can be measured, for example, by BIAcore using recombinant soluble FcRn. Cell-based functional assays, such as ADCC assays and CDC assays, provide insights into the likely functional consequences of particular variant structures. In one embodiment, the ADCC assay is configured to have NK cells be the primary effector cell, thereby reflecting the functional effects on the FcyRIIIA receptor. Phagocytosis assays may also be used to compare immune effector functions of different variants, as can assays that measure cellular responses, such as superoxide or inflammatory mediator release. In vivo models can be used as well, as, for example, in the case of using variants of anti-CD3 antibodies to measure T cell activation in mice, an activity that is dependent on Fc domains engaging specific ligands, such as Fey receptors.

Host Cell Selection or Host Cell Engineering

As described herein, the host cell chosen for expression of the scaffold-based protein is an important contributor to the final composition, including, without limitation, the variation in composition of the oligosaccharide moieties decorating the protein, if desirable, for example in the immunoglobulin CH2 domain when present. Thus, one aspect of the invention involves the selection of appropriate host cells for use and/or development of a production cell expressing the desired therapeutic protein.

Further, the host cell may be of mammalian origin or may be selected from COS-1, COS-7, HEK293, BHK21, CHO, BSC-1, Hep G2, 653, SP2/0, 293, HeLa, myeloma, lymphoma, yeast, insect or plant cells, or any derivative, immortalized or transformed cell thereof. 21

Alternatively, the host cell may be selected from a species or organism incapable of glycosylating polypeptides, e.g. a prokaryotic cell or organism, such as and of the natural or engineered E. coli spp, Klebsiella spp., or Pseudomonas spp.

Selecting Binding Domains

The polypeptides or fusion proteins or components and domains thereof may also be obtained from selecting from libraries of such domains or components, e.g., a phage library. A phage library can be created by inserting a library of random oligonucleotides or a library of polynucleotides containing sequences of interest, such as antibody domains from the B-cells of an immunized animal or human (Smith, G.P. 1985. Science 228: 1315-1317). Antibody phage libraries contain heavy (H) and light (L) chain variable region pairs in one phage allowing the expression of single-chain Fv fragments or Fab fragments (Hoogenboom, et al. 2000, Immunol. Today 21(8) 371-8). The diversity of a phagemid library can be manipulated to increase and/or alter the specificities of the polypeptides of the library to produce and subsequently identify additional, desirable, molecular properties and the polynucleotides encoding them.

Other libraries of target binding components which may include other than antibody variable regions are ribosome display, CIS-display, yeast display, bacterial displays and mammalian cell display. Ribosome display is a method of translating mRNAs into their cognate proteins while keeping the protein attached to the RNA. The nucleic acid coding sequence is recovered by RT-PCR (Mattheakis, L.C. et al. 1994.

Proc. Natl. Acad. Sci. USA 91, 9022). CIS-display is an alternative in vitro display method in which the library is constructed as a fusion protein with RepA. During in vitro translation, RepA binds in cis to the DNA which it was made from, providing a direct linkage between genotype and phenotype (Odegrip et. al., Proc. Natl. Acad. Sci. USA, 101:2806,2004). Yeast display is based on the construction of fusion proteins of the membrane-associated alpha-agglutinin yeast adhesion receptor, agal and aga2, a part of the mating type system (Broder, et al. 1997. Nature Biotechnology, 15:553-7). Bacterial display is based on fusion of the target to exported bacterial proteins that associate with the cell membrane or cell wall (Chen and Georgiou 2002. Biotechnol Bioeng, 79:496-503). Similarly, mammalian display systems are based on the creation of a fusion protein 22 between the polypeptide containing randomized sequences and a secreted, membrane anchor protein.

Uses of Scaffold-based Molecules

The compositions of the scaffold-based molecules described herein and generated by any of the above described methods may be used to diagnose, monitor, modulate, treat, alleviate, help prevent the incidence of, or reduce the symptoms of human disease or specific pathologies in cells, tissues, organs, fluid, or, generally, a host. A scaffold-based molecule engineered for a specific purpose may be used to treat an immune-mediated or immune-deficiency disease, a metabolic disease, a cardiovascular disorder or disease; a malignant disease; neurologic disorder or disease; an infection such as a bacterial, viral or parasitic infection; or other known or specified related condition including swelling, pain, and tissue necrosis or fibrosis.

Such a method can comprise administering an effective amount of a composition or a pharmaceutical composition comprising at least one scaffold protein to a cell, tissue, organ, animal or patient in need of such modulation, treatment, alleviation, prevention, or reduction in symptoms, effects or mechanisms. The effective amount can comprise an amount of about 0.001 to 500 mg/kg per single (e.g., bolus), multiple or continuous administration, or to achieve a serum concentration of 0.01-5000 ug/ml serum concentration per single, multiple, or continuous administration, or any effective range or value therein, as done and determined using known methods, as described herein or known in the relevant arts.

Compositions Comprising Scaffold-based Proteins

The target binding scaffold proteins which are modified or unmodified, monovalent, bi- or multivalent, and mono-, bi- or multi-targeting, can be isolated using separation procedures well known in the art for capture, immobilization, partitioning, or sedimentation and purified to the extent necessary for commercial applicability.

For therapeutic use, the scaffold-base proteins may be formulated of an appropriate mode of administration including but not limited to parenteral, subcutaneous, intramuscular, intravenous, intrarticular, intrabronchial, intraabdominal, intracapsular, 23 intracartilaginous, intracavitary, intracelial, intracerebellar, intracerebroventricular, intracolic, intracervical, intragastric, intrahepatic, intramyocardial, intraosteal, intrapelvic, intrapericardiac, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical, intralesional, bolus, vaginal, rectal, buccal, sublingual, intranasal, or transdermal means. At least one protein scaffold composition can be prepared for use in the form of tablets or capsules; powders, nasal drops or aerosols; a gel, ointment, lotion, suspension or incorporated into a therapeutic bandage or “patch” delivery system as known in the art. The invention provides for stable formulations of an scaffold-base proteins, which is preferably an aqueous phosphate buffered saline or mixed salt solution, as well as preserved solutions and formulations as well as multi-use preserved formulations suitable for pharmaceutical or veterinary use, comprising at least one scaffold-base protein in a pharmaceutically acceptable formulation. Suitable vehicles and their formulation, inclusive of other human proteins, e.g., human serum albumin, are described, for example, in e.g. Remington: The Science and Practice of Pharmacy, 21st Edition, Troy, D.B. ed., Lipincott Williams and Wilkins, Philadelphia, PA 2006, Part 5, Pharmaceutical Manufacturing pp 691-1092, See especially pp. 958-989.

The compositions may be used with, or incorporate within a single formulation, other actives known to be beneficial for treatment of the indicated disorder, condition, or disease or may be a tested by preparing combinations of scaffold-based proteins with novel compositions and actives.

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. 24

EXAMPLE 1. CONSTRUCTION OF Fc GLYCOSYLATION VARIANTS

Tencon Design

The third FN3 domain from human Tenascin (SEQ ID NO: 3) can be used as an alternative scaffold capable of being engineered to bind to specific target molecules via surface exposed loops structurally analogous to antibody complementarity determining regions (CDR). The melting temperature of this domain is 54°C in PBS in its native form. In order to produce a scaffold molecule with a similar structure and improved physical properties, such as an improved thermal stability, a consensus sequence was designed based on an alignment of 15 FN3 domains from human Tenascin (SEQ ID NOS: 1-15).

Analysis of the multiple sequence alignment in Table 1 shows that these 15 domains have sequence identities to each other ranging from 13 to 80%, with an average sequence identity among pairs of 29%. A consensus sequence (SEQ ID NO: 16) was designed by incorporating the most conserved (frequent) amino acid at each position from the alignment shown in Table 1. In pairwise alignments, the consensus sequence of the present invention (SEQ ID NO: 16), designated as Tencon, is identical to the FN3 domains from Tenascin at 34 - 59% of positions with an average sequence identity of 43%.

Protein expression and purification

The amino acid sequence of Tencon (SEQ ID NO: 16) was back translated, resulting in the DNA sequence shown in SEQ ID NO: 17. This sequence was assembled by overlapping PCR, subcloned into a modified pET15 vector, transformed into BL21Star(DE3) E. coli (Invitrogen) and plated onto LB agar plates containing 75 pg/mL carbenicillin. A single colony was picked and grown overnight at 37°C in 50 ml of TB media containing 2% glucose and 100 pg/mL carbenicillin. This culture was used to seed 500 mL of autoinduction media (Overnight Express Instant TB media, Novagen) in a 2.5L Ultra Yield flask (Thomson Instrument Company). The growth and expression was done using a dual program (3 hours at 37°C, 300 rpm, followed by 16 hours at 30°C, 250 rpm) in an ATR Multitron shaking incubator. 25

The culture was harvested and centrifuged at 7000 rpm for 15 minutes in a JL8.1 rotor to pellet the cells. The cells were resuspended in 30 ml buffer containing 20 mM sodium phosphate, pH 7.5, 500 mM NaCl, 10% glycerol, 20 mM imidazole, 0.37 mg/mL lysozyme, IX Complete Protease inhibitor (EDTA-free; Roche) and Benzonase (Sigma-Aldrich, 0.25 μΐ/ml final) and lysed with a Misonix XL2020 sonicator for 5 minutes on ice in pulse mode (5 seconds on, 30 seconds off). The insoluble material was removed by centrifugation at 17,000 rpm for 30 minutes in a JA-17 rotor.

The Tencon protein was purified from the soluble lysate in a 2-step chromatographic process. First, the protein was captured by immobilized metal affinity chromatography, adding 2 mL Ni-NTA agarose beads (Qiagen) to the lysate and placing it on a rocking platform for 1 hour at 4°C. The resin was then packed into a Poly-Prep column (Bio-Rad) and washed with 20 mM sodium phosphate, pH 7.5, 500 mM NaCl, 10% glycerol and 20 mM imidazole to remove the unbound material. The proteins were eluted from the resin with 20 mM sodium phosphate, pH 7.5, 500 mM NaCl, 10% glycerol and 500 mM imidazole. The fractions were analyzed by SDS-PAGE, both by Coomassie stain and by Western blot using an HRP-conjugated anti-His antibody (Immunology Consultants Laboratory). The desired fractions were pooled and dialyzed into PBS pH 7.4. As a second purification step the protein was loaded onto a Superdex-75 HiLoad 16/60 column (GE Healthcare) equilibrated in PBS. The fractions were analyzed by SDS-PAGE, and the fractions containing Tencon were pooled and concentrated using a Centriprep UltraCel YM-3 concentrator (Amicon).

Protein concentration was determined using a BioTek plate reader to measure the absorbance of the sample at 280nm. The final preparation was analyzed by Coomassie stain (Figure 1), Western blot with anti-His antibody, and by HPLC-SEC using a G3000SW-XL column (TOSOH Biosciences) equilibrated in PBS. SDS-PAGE analysis shows that Tencon migrates between 6 and 14 kDa, in agreement with the expected mass of 10.7 kDa for the monomeric protein. A yield of >50 mg of pure Tencon protein per liter of culture was obtained. 26

Biophysical Characterization

The structure and stability of Tencon was characterized by circular dichroism spectroscopy and differential scanning calorimetry respectively. CD measurements were made on an AVIV spectrometer at 20°C in PBS and a concentration of 0.2 mg/mL. The spectrum in Figure 8 shows a minimum at 218 nm, suggestive of beta-sheet structure as expected for a protein belonging to the FN3 family as designed. DSC data was obtained by heating 0.5 mg/mL solutions of the 3rd FN3 domain from Tenascin or Tencon in PBS from 35°C to 95°C at a rate of l°C/minute in an N-DSCII calorimeter (Applied Thermodynam i cs). First, the curve for the buffer blank was subtracted to produce the profiles shown in Figure 3. From this data, melting temperatures of 54°C and 78°C were calculated for the 3rd FN3 domain and Tencon, respectively, using CpCalc (Applied Thermodynamics) software. The folding and unfolding of both domains is reversible at these temperatures.

Immunogenicity Analysis A computer program that models for immunogenicity to human of amino acid sequences was used to compare the predicted immunogenicity of amino acid sequences representing the 3rd FN3 domain of human Tenascin, Tencon, and several therapeutic antibodies (shown in Table 2). Chimeric mAbs and a human mAb (adalimumab) analyzed with the program were followed by application of a tolerance threshold (removes 9-mer peptides with 100% identity to human germline encoded sequence). The tolerance threshold was not applied to Tenascin or Tencon. The tolerance threshold assumes broad T cell tolerance to germline encoded mAb sequences and focuses analyses on novel sequence primarily in CDRs and flanking domains.

These analyses predict a low immunogenic risk for both Tenascin and Tencon based on the likelihood that a 9-mer peptide, derived from the analyzed sequence will bind one or more HLA molecules. The score is weighted with respect to the prevalence of each HLA allele. The scores for the models were summed for each sequence to provide a single number describing the overall PIR of each sequence (score sum). The results from this analysis are summarized in Table 2. Tenascin was shown to have the lowest overall Score (11.9). Tencon, like Tenascin, scored primarily non-binders and low 27 predicted immunogenic risk agretopes (Score = 13.2). The Tenascin and Tencon sequences scored favorably as compared to the therapeutic antibodies.

Display of Tencon on M13 phage by pIX fusion

The gene encoding the Tencon amino acid sequence was subcloned into the phagemid expression vector pPep9 by PCR and restriction digest cloning, resulting in the vector pTencon-pIX. This system expresses N-terminally Myc-tagged Tencon as a C-terminal fusion to the N-terminus of the M13 pIX protein (Figure 4). The Lac promoter allows for lower levels of expression without IPTG and increased expression after the addition of IPTG. The OmpA signal sequence was appended to the N-terminus of Tencon to promote efficient translocation to the periplasm. A short TSGGGGS linker (SEQ ID NO: 141) was constructed between Tencon and pIX to prevent steric interactions between these proteins.

For confirmation of display on the surface of the M13 phage particle, pTencon-pIX was transformed into XL 1-Blue E. coli and a single colony was used to innocualte a 5 mL LB culture supplemented with ampicillin. This culture was grown at 37°C until reaching mid-log phase at which point 610 pfu of VCSM13 helper phage was added and the culture incubated at 37°C for 10 minutes without shaking followed by 50 minutes with shaking. The helper phage rescued culture was then diluted into 50 mL of 2YT media supplemented with ampicillin and kanamycin and grown at 37°C with shaking until O.D •6oo reached 0.7, at which point IPTG ivas added to a final concentration of 1 mM and the temperature reduced to 30°C. After 16 hours, the culture was centrifuged at 4000 X g for 20 minutes and the supernatant collected and stored at 4°C for analysis.

Binding of the phage particles to an anti-Myc antibody (Invitrogen) was used to confirm the display of the Myc-Tencon construct on the Ml 3 phage surface. A Maxisorp plate was coated overnight at a concentration of 2.5 ug/mL with anti-Myc or an anti-av antibody (negative control) and blocked with SuperBlock T20 (Pierce). Two-fold serial dilutions of the phagemid culture supernatant described above were made in PBS and added to the wells of the coated plate. After 1 hour, the plate was washed with TBST and a anti-M13 HRP antibody was added to each well and washed with TBST following a 1-hour incubation. The Roche BD ELISA POD substrate was added and luminescence 28 2015271900 17 Dec 2015 detected on a plate reader (Tecan). Figure 5 shows that the Myc-Tencon phage particles bind to the anti-myc, but not the anti-αν antibody coated wells or the uncoated control wells of the plate in a concentration dependent manner, confirming the specific display of Myc-Tencon on the M13 phage particle. 5 An additional phagemid vector can be constructed to display Tencon and library members (see Example 2) on Ml3 phage as fusions to coat protein pill. For this system, the gene for pIX is replaced with a gene encoding a truncated version of pill (Bass et al. 1990). Additional changes as compared to the system shown in Figure 4 include the replacement of the OmpA signal sequence with the signal sequence for DsbA, as 10 secretion using this sequence has been shown to be beneficial for the display of stable alternative scaffold molecules (Steiner et al. 2006). EXAMPLE 2: Generation of Tencon Libraries

Tencon variant libraries can be made by many different methods, depending on the desired complexity and the relative location of mutations in the molecule. DNA synthesis 15 methods are preferred to create mutations scattered throughout the Tencon gene. Restriction enzyme cloning can also be used to recombine DNA fragments containing mutations in different regions of the gene. Saturating mutagenesis in a small-defined region, such as a single Tencon loop, can be introduced by using a degenerate oligo-nucleotide and oligonucleotide directed mutagenesis (Kunkel et al. 1987). 20 A Tencon library, library FG7, designed to replace the FG loop with 7 random amino acids using oligonucleotide directed mutagenesis was constructed. An oligonucleotide (TconFG7-For-5’pho) was synthesized to have a 21 base pair (bp) degenerate sequence of NNS at the positions encoding the FG loop and two flanking 20-27 bp nucleotide sequences of complementarity to the Tencon coding sequence. In this 25 design, all twenty amino acids are capable of being represented in the FG loop. The calculated diversity at nucleotide level is 1.3xl09. 29

TconFG7-For5’pho: (SEQ ID NO: 18)

GAATACACCGTTTCTATCTACGGTGTTNNSNNSNNSNNSNNSNNSNNSCCGCT

GTCTGCGGAATTCAC

The template for oligonucleotide directed mutagenesis, pDsbA-Tencon-Asc-loop-Myc-pIII, was constructed by replacing the Tencon F:G loop encoding sequence with a stem loop sequence containing an AscI restriction site. This system allows the elimination of background template DNA after mutagenesis by digesting the resulting DNA with AscI prior to transformation. To purify a single-stranded DNA template for mutagenesis, a single colony of E. coli CJ236 harboring pDsbA-Tencon-Asc-loop-Myc-pIII, was picked into 5 mL of 2YT growth medium with carbenicillin (50ug/ml final concentration) and Chloramphenicol (lOug/ml). After 6 hours, VCSM13 helper phage was added to a final concentration of 1010 pfu/ml and incubated without shaking for 10 minutes before being transferred to 150 mL of 2YT with carbenicillin (lOug/ml) and uridine (0.25 ug/ml) and incubated at 37°C with shaking at 200 rpm overnight. The cells were pelleted by centrifugation and the supernatant collected and the phage pelleted with PEG NaCl. Single strand DNA was purified from this pellet using a QIAprep Spin M13 kit (Qiagen) according to the manufacturer instructions.

To anneal the degenerate oligonucleotide to the template, 5 ug of template DNA was combined with oligo TconFG7-For-5-pho at a molar ratio of 10:1 in Tris-HCl (50 mM, pH7.5) and MgC12 (10 mM) and incubated at 90°C for 2 minutes, 60°C for 3 minutes, and 20°C for 5 minutes. After the annealing reaction, ATP (lOmM), dNTPs (25mM each), DTT (100 mM), T4 ligase (7 units), and T7 DNA polymerase (10 units) were added to the reaction mixture and incubated at 14°C for 6 hours followed by 20°C for 12 hours. The resulting DNA was purified using a PCR purification kit (Qiagen) and recovered in 100 uL of water. The library DNA was digested with 10 units of AscI for 4 hours and then purified again with Qiagen PCR purification kit. The final library DNA was recovered in 50 uL of water. The resulting double stranded DNA product was then transformed into into E. coli MC1061F’ by electroporation.

The transformants were collected in 20 mL SOC medium and allowed to recover for 1 hour at 37°C. At the end of the recovery, an aliquot of the transformation was serial 30 diluted and plated on Carbenicillin (lOOug/ml) plates containing 1% glucose to assess the total transformant number. The remaining SOC culture was then used to inoculate 1 L of 2xYT medium with Carbinicillin and 1% glucose and grown until OD6oo reached 0.6. 100 mL of this culture was inoculated with M13 helper phage to 1010/mL and incubated at 37°C before centrifugation. The resulting cell pellet was resuspended in 500 mL fresh 2xYT medium containing Carbenicillin (lOOug/mL) and Kanamycin (35ug/mL) and grown at 30°C overnight before centrifugation. Phage particles were precipitated by the addition of PEG/NaCl and stored at -80°C. A second library, BC6/FG7, was designed to introduce diversity within the B:C and F:G loops of Tencon simultaneously. In order to do so, two oligonucleotides, Tc-BC6-For-5’phos and POP 149 were synthesized. The forward oligo was phosphorylated and contained 18 bases of NNS codon at each position encoding the B:C loop, while the reverse oligo was biotinylated at the 5’ end and contained 21 bases of NNS codon at each position encoding the F:G loop. Both oligonucleotides are flanked by two 18 bp nucleotide sequences identical to the region preceding and following the region to be mutagenized (see below for primer detail).

Tc-BC6-For-5’nhos: (SEP ID NO: 191

gactctctgcgtctgtcttggNN SNN SNN SNN SNN SNN STTCGACT CTTT CCTG AT CC AGT A cc POP 2149: iSEOIDNO:20!

GT G AATT CCGC AG AC AGCGGSNN SNN SNN SNN SNN SNN SNN AAC ACCGT AG AT A GAAACGGTG

To construct the library, sixteen 100 uL PCR reactions were performed using t oligos Tc-CB6-For5’phos and POP2149 to amplify the Tencon DNA template, introducing NNS codons into the B:C and F:G loops simultaneously in the process. The double-stranded PCR product was mixed with magnetic streptavidin beads (Dynal) in B&W buffer (lOmM Tris-HCl, pH7.5, ImM EDTA, 2M NaCl, 0.1% Tween-20) and incubated for 20 minutes, pulled down with a magnet and washed with B&W buffer twice. The forward 31 strand was eluted from the beads with 300 uL of 150 mM NaOH. This “megaprimer,” a mixture of long primers with more than 8x1016 in theoretical diversity, was used to anneal to a single strand library template. Library construction was carried out as described above for the FG7 library. EXAMPLE 3: Selection of IgG binders

In order to perform selections of Tencon library members that bind to IgG, recombinant IgG (human IgGl subtype) was biotinylated using sulfo-NHS-LC-Biotin (Pierce) before dialyzing into PBS. For selections, 200 uL of phage displaying libraries FG7 or BC6/FG7 were blocked with 200 uL of chemiblocker before the addition of biotinylated IgG at concentrations of 500 nM (round 1) or 100 nM (rounds 2 and 3). Bound phages were recovered by Neutravidin magnetic beads (Seradyne) in round 1 or streptavidin magnetic beads (Promega) in rounds 2 and 3. Unbound phages were washed from the beads using 5-10 washes with 1 mL of Tris buffered saline with tween (TBST) followed by 2 1 mL washes with Tris buffered saline (TBS). Bound phages were eluted from the beads by the addition of mid-log phase E. coli MCI 06 IF’. Infected cells were plated on LB agar plates supplemented with carbenicillin and glucose. The next day, cells were scraped from the plate and grown to mid-log phase before rescue with VCSM13 helper phage and grown overnight. Phage particles isolated by PEG/NaCl precipitation and used for the next round of selections.

After 3 rounds of panning against IgG, the output was subcloned into a pET27 vector modified to include a ligase independent cloning site by amplifying the Tencon gene by PCR. This PCR product was annealed to the vector and transformed into BL21-GOLD(DE3) cells (Stratagene). Individual colonies were picked into 1 mL cultures in 96 deep well plates (Coming) and grown to saturation overnight at 37°C. The next day, 50 microL of the overnight culture was used to inoculate a fresh 1 mL culture. Cultures were grown at 37°C for 2 hours before adding IPTG to 1 mM and reducing the temperature to 30°C. Cells were harvested by centrifugation 16 hours after induction and lysed with 100 microL of BugBuster (Novagen). The resulting lysates were clarified by centriguation and used to test for binding to IgG by ELISA. 32 2015271900 17 Dec 2015

Maxisorp plates (Nunc) were coated with 0.1 pg of anti-HIS antibody (Qiagen) overnight, washed with TBST, and blocked with Starting Block T20 (Thermo Scientific). Clarified lysates diluted 1:4 in Starting Block were added to the plates and allowed to bind for 1 hour before washing with TBST. Biotinylated IgG or biotinylated HSA was added at a 5 concentration of 1 ug/ml and washed with TBST after a 1-hour incubation. Detection of bound IgG or HSA was accomplished by adding streptavidin-HRP (Jackson Immunoresearch) and detecting with POD chemiluminescence substrate. Results of the ELISA are shown in Figure 7. Constructs that bound biotinylated IgG more than 10-fold over biotinylated HSA as judged by ELISA signal were sequenced. After completion of 10 several selection experiments, 60 unique binding sequences from library FG7 and 10 unique sequences from library BC6FG7 were obtained; Table 4 shows representative sequences of IgGbinders in which the B:C and/or F:G loops are shown to the extent they are different than those of SEQ ID NO: 16. Also shown in Table 4 are numerous mutations in other regions of the scaffold. 15 The Tencon protein designed, expressed, and purified here has a thermal stability improved by 26°C with respect to that of the 3rd FN3 domain from human Tenascin, which has been used as an alternative scaffold molecule. Based on this stability increase, this scaffold molecule is likely to be more amenable to amino acid substitution and easier to manufacture. Mutations that decrease protein stability are likely to be better tolerated in the 20 context of a more stable scaffold and thus a scaffold with enhanced stability is likely to yield more functional, well folded binders from a library of scaffold variants. 33 2015271900 17 Dec 2015

Table 1 (1) 1 ,10 ,20 ,30 40 JQ ,60 JO JO ,90 100 1 (1) SPPKDLVVTEVTEETVNLAWDN-EMRVTE YLWYTPTH—EGGLEMQFRVPGDQTSTIIQELEPGVE YFIRVFAILENKKSIPVSARVAT------- 2 (1) TYLPAPEGLKFKSIKETSVEVEWDPLDIAFETWEIIFRNMN-KEDEGEITKSLRRPETSYRQTGLAPGQEYEISLHIVKNNTRGPGLKRVTTTRLD---- 3 (1) DAPSQIEVKDVTDTTALITWFKPLAEIDGIELTYGIKD—VPGDRTTIDLTEDENQYSIGNLKPDTEYEVSLISRRGDMSSNPAKETFTT-------

4 (1) TGLDAPRNLRRVSQTDNSITLEWRNGKAAIDSYRIKYAPISGGDHAEVDVPKSQQATTKTTLTGLRPGTEYGIGVSAVKEDKESNPATINAATELDTPKD 5 (1)---DTPKDLQVSETAETSLTLLWKTPLAKFDRYRLNYSLPT----GQWVGVQLPRNTTSYVLRGLEPGQEYNVLLTAEKGRHKSKPAKSKPARVK----- 6 (1) -QAPELENLTVTEVGWDGLRLNWTAADQAYEHFIIQVQEAN—KVEAARNLTVPGSLRAVDIPGLKAATPYTVSIYGVIQGYRTPVLSAEASTGE----- 7 (1) -ETPNLGEWVAEVGWDALKLNWTAPEGAYEYFFIQVQEAD—TVEAAQNLTVPGGLRSTDLPGLKAATHYTITIRGVTQDFSTTPLSVEVLTE------ 8 (1) -EVPDMGNLTVTEVSWDALRLNWTTPDGTYDQFTIQVQEAD—QVEEAHNLTVPGSLRSMEIPGLRAGTPYTVTLHGEVRGHSTRPLAVEWTE------ 9 (1)-DLPQLGDLAVSEVGWDGLRLNWTAADNAYEHEVIQVQEVN—KVEAAQNLTLPGSLRAVDIPGLEAATPYRVSIYGVIRGYRTPVLSAEASTAKEPE— 10 (1)-KEPEIGNLNVSDITPESFNLSWMATDGIFETFTIEIIDSN—RLLETVEYNISGAERTAHISGLPPSTDFIVYLSGLAPSIRTKTISATATTE------ 11 (1) -ALPLLENLTISDINPYGFTVSWMASENAFDSFLVTWDSG—KLLDPQEFTLSGTQRKLELRGLITGIGYEVMVSGFTQGHQTKPLRAEIVTE------ 12 (1)-AEPEVDNLLVSDATPDGFRLSWTADEGVFDNEVLKIRDTK—KQSEPLEITLLAPERTRDLTGLREATEYEIELYGISKGRRSQTVSAIATTAM----- 13 (1)---GSPKEVIFSDITENSATVSWRAPTAQVESFRITYVPITG---GTPSMVTVDGTKTQTRLVKLIPGVEYLVSIIAMKGFEESEPVSGSFTTAL----- 14 (1) DGPSGLVTANITDSEALARWQPAIATVDS YVIS YTGEK----VPEITRTVSGNTVEYALTDLEPATE YTLRIFAEKGPQKSSTITAKFTTDL----- 15 (1)---DSPRDLTATEVQSETALLTWRPPRASVTGYLLVYESVD----GTVKEVIVGPDTTSY SLADLSPSTHYTAKIQALNGPLRSNMIQTIFTTIGL----

Table 2.

Sequence Description 1st Score sum 2nd Score sum Score sum (chain) Score sum (molecule) Tenascin Alt. Scaff. 6.01 5.85 11.86 11.86 Tencon Alt. Scaff. 5.83 7.37 13.20 13.20 adalimumab Vh humanized mAb 9.45 8.06 17.50 45.42 VI 15.29 12.63 27.92 cetuximab Vh Chimeric mAb 17.63 16.89 34.52 64.44 VI 14.45 15.47 29.92 Rituximab Vh Chimeric mAb 16.57 14.38 30.96 61.65 VI 16.63 14.06 30.69 basiliximab Vh Chimeric mAb 16.48 13.40 29.89 58.98 VI 16.05 13.05 29.09 34 2015271900 17 Dec 2015

Table 3. Loops Loop Residues ofSEQ ID NO: 16 Amino Acid Sequence A-B 13-16 TEDS B-C 22-28 TAPDAAF C-D 38-43 SEKVGE D-E 51-54 GSER E-F 60-64 GLKPG F-G 75-81 KGGHRSN

Table 4. Scaffolds binding to IgG Clone No. B:C Loop Residues 22-28 (SEQ ID NO) F:G Loop Residues 75-81 (SEQ ID NO) Scaffold Mutations 1 SYGFNN (21) QIGPIIP (46) 2 TYEGES (22) QIGPIIP (46) 3 TYESES (23) QIGPIIP (46) 4 TNWMDS (24) SIRTIDS (47) 5 KSVFIM (25) PKFHSPL (48) 6 YSSYAT (26) WKTTIWF (49) 7 RFHPFP (27) RKNWKTR (50) 8 MMCMPL (28) RLFRIYQ (51) 9 YCRVRD (29) WLSRSYD (52) 10 SYGFNN (21) WLSRSYD (52) 11 MDCFMG (30) WLSRSCD (53) 12 TYRFNS (31) WMGPYCD (54) 13 ASRRSL (32) RRRRYSF (55) 35 2015271900 17 Dec 2015

Clone No. B:C Loop Residues 22-28 (SEQ ID NO) F:G Loop Residues 75-81 (SEQ ID NO) Scaffold Mutations 14 TIESES (33) HIVPMVP (56) 15 TL*MQS (34) QIEPIIR (57) 16 IYDSES (35) PSAANNP (58) 17 VRLRYVQ (59) 18 QVGPLIP (60) 19 RIGPILP (61) 20 QIGPLLP (62) 21 RIGPLLP (63) 22 QVGPLLP (64) 23 RIGPMLP (65) 24 QIGPVLP (66) 25 RIGPVLP (67) 26 QIGPMMP (68) 27 QVGPLVP (69) 28 QIGPMLP (70) R18P 29 QVGPILP (71) 30 QVGPLLP (64) 31 QVGPMLP (72) 32 QIGPIVP (73) 133V 33 MIGPLLP (74) 34 QIGPLFP (75) 35 QIGPVLP (66) T59A 36 QIGPMVP (76) 37 QIGPIVP (77) 36 2015271900 17 Dec 2015

Clone No. B:C Loop Residues 22-28 (SEQ ID NO) F:G Loop Residues 75-81 (SEQ ID NO) Scaffold Mutations 38 RIEPILP (78) V7 4G 39 VAGSVWP (79) 40 REGATLY (80) 41 KQIPPIL (81) S38G 42 LSLSSVL (82) 43 HMLLPLP (83) V7 4 A 44 MIGPLIP (84) 45 TIGPHIP (85) 46 EIGPCLP (86) 47 EIGPVLP (87) 48 KIGPCLP (88) Y35H 49 MIGPVLP (89) 50 QIGPILP (90) S52P 51 QIGPILP (90) Q36R 52 QIGPILP (90) 53 EVGPILP (91) 54 QVGPLLP (92) A2 3T 55 QIGPVMP (93) 56 QIGPCVP (94) 57 QIGPLVP (95) 58 RGLVMPM (96) V7 4 A 59 MIGPILP (97) 60 QIGPILP (90) E37G 61 QIGPILP (90) T68A 37 2015271900 17 Dec 2015

Clone No. B:C Loop Residues 22-28 (SEQ ID NO) F:G Loop Residues 75-81 (SEQ ID NO) Scaffold Mutations 62 QIGPILP (90) T221 63 QIGPILP (90) S52F 64 QIGPILP (90) Y56H 65 QIGPILP (90) A4 4V 66 QIGPILP (90) P24S 67 RIGPILP (61) 68 CIGPMVP (98) 69 FIGPVLP (99) 70 HIGPILP (100) 71 HIGPIMP (101) 72 HIGPYLP (102) 73 HVGPILP (103) 74 IIGPLLP (104) 75 LIGPLLP (105) 76 MVGPLLP (106) 77 NIGPYLP (107) 78 NIGPYLP (108) 79 QIGPHLP (109) 80 QIGPIIP (46) 82 QIGPILG (110) 83 QIGPILS (111) 83 QIGPILT (112) 84 QIGPIMP (113) 85 QIGPIPI (114) 38 2015271900 17 Dec 2015

Clone No. B:C Loop Residues 22-28 (SEQ ID NO) F:G Loop Residues 75-81 (SEQ ID NO) Scaffold Mutations 86 QIGPLLN (115) 87 QIGPLLP (62) 88 QIGPVFP (116) 89 QIGPVLS (117) 90 QIGPWLP (118) 92 QVGPILP (71) 93 QVGPILR (118) 94 QVGPIMN (119) 95 QVGPIMP (120) 96 QVGPIVP (121) 97 QVGPLLS (122) 98 QVGPVLP (123) 99 QVGPVLT (124) 100 RIGPIMP (125) 101 RIGPIVP (126) 102 RIGPMFP (127) 103 RIGPMIP (128) 104 RIGPMVP (129) 105 RIGPVIP (130) 106 RVGPILP (131) 107 RVGPLLP (132) 108 TVGPHIP (133) 109 DRKRFI (36) PSWRSNW (134) 110 EFWRGS (37) QIGPLLP (62) 111 GLLDPL (38) ALRATLE (135) 39 2015271900 17 Dec 2015

Clone No. B:C Loop Residues 22-28 (SEQ ID NO) F:G Loop Residues 75-81 (SEQ ID NO) Scaffold Mutations 112 GLVLPE (39) KYGYLTP (136) 113 MASDGL (40) RIGPMLP (137) 114 NKTETN (41) NPFCSRF (138) 115 QAERKV (42) QIGPLLP (62) 116 QAERKV (42) RIGPLLP (63) 117 SQVCTL (43) YYLHQWC (139) 118 YFDKDS (44) QIGPLLP (62) 119 YFECEP (45) HIVPLLR (140)

Sequences: SEQ ID No. 1: sppkdlvvtevteetvnlawdnemrvteylvvytpthegglemqfrvpgdqtstiiqelepgveyfirvf ailenkksipvsarvat SEQ ID No. 2: tylpapeglkfksiketsvevewdpldiafetweiifrnmnkedegeitkslrrpetsyrqtglapgqey eislhivknntrgpglkrvtttrld SEQ ID No. 3: 10 dapsqievkdvtdttalitwfkplaeidgieltygikdvpgdrttidltedenqysignlkpdteyevsl isrrgdmssnpaketftt SEQ ID No. 4 tgldaprnlrrvsqtdnsitlewrngkaaidsyrikyapisggdhaevdvpksqqattkttltglrpgte ygigvsavkedkesnpatinaateldtpkd 15 SEQ ID No. 5 dtpkdlqvsetaetsltllwktplakfdryrlnyslptgqwvgvqlprnttsyvlrglepgqeynvllta ekgrhkskpakskparvk SEQ ID No. 6 40 qapelenltvtevgwdglrlnwtaadqayehfiiqvqeankveaarnltvpgslravdipglkaatpytv siygviqgyrtpvlsaeastge 2015271900 17 Dec 2015 SEQ ID No. 7 etpnlgevvvaevgwdalklnwtapegayeyffiqvqeadtveaaqnltvpgglrstdlpglkaathyti tirgvtqdfsttplsvevlte SEQ ID No. 8 evpdmgnltvtevswdalrlnwttpdgtydqftiqvqeadqveeahnltvpgslrsmeipglragtpytv tlhgevrghstrplavevvte SEQ ID No. 9 dlpqlgdlavsevgwdglrlnwtaadnayehfviqvqevnkveaaqnltlpgslravdipgleaatpyrv siygvirgyrtpvlsaeastakepe SEQ ID No. 10 kepeignlnvsditpesfnlswmatdgifetftieiidsnrlletveynisgaertahisglppstdfiv ylsglapsirtktisatatte SEQ ID No. 11 alpllenltisdinpygftvswmasenafdsflvtvvdsgklldpqeftlsgtqrklelrglitgigyev mvsgftqghqtkplraeivte SEQ ID No. 12 aepevdnllvsdatpdgfrlswtadegvfdnfvlkirdtkkqsepleitllapertrdltglreateyei elygiskgrrsqtvsaiattam SEQ ID No. 13 gspkevifsditensatvswraptaqvesfrityvpitggtpsmvtvdgtktqtrlvklipgveylvsii amkgfeesepvsgsfttal SEQ ID No. 14 25 dgpsglvtanitdsealarwqpaiatvdsyvisytgekvpeitrtvsgntveyaltdlepateytlrifa ekgpqksstitakfttdl SEQ ID No. 15 dsprdltatevqsetalltwrpprasvtgyllvyesvdgtvkevivgpdttsysladlspsthytakiqa lngplrsnmiqtifttigl 30 SEQ ID No. 16

LPAPKNLVVSEVTEDSLRLSWTAPDAAFDSFLIQYQESEKVGEAINLTVPGSERSYDLTGLKPGTEYTVS

IYGVKGGHRSNPLSAEFTT 41 SEQ ID No. 17 2015271900 17 Dec 2015 ctgccggcgccgaaaaacctggttgtttctgaagttaccgaagactctctgcgtctgtcttggaccgcgc cggacgcggcgttcgactctttcctgatccagtaccaggaatctgaaaaagttggtgaagcgatcaacct gaccgttccgggttctgaacgttcttacgacctgaccggtctgaaaccgggtaccgaatacaccgtttct atctacggtgttaaaggtggtcaccgttctaacccgctgtctgcggaattcaccacc

Tencon Sequence showing loops (SEQ ID NO: 16)

A-B B-C C-D 1-LPAPKNLVVSEVTEDSLRLSWTAPDAAFDS FLIQYQESEKVGEA-4 4

D-E E-F F-G 45-INLTVPGSERSYDLTGLKPGTEYTVSIYGVKGGHRSNPLSAEFTT-89 EXAMPLE 4: Stablizing Mutations of Tencon

Mutants were designed to improve the folding stability of Tencon scaffold described herein above (SEQ ID NO: 16). Several point mutations were made to produce substitution of individual residues of SEQ ID NO: 16, such as N46V (Tenconl7 - SEQ ID NO: 142), E14P (Tenconl8 - SEQ ID NO:143), El IN (Tenconl9 - SEQ ID NO: 144), E37P (Tencon20 - SEQ ID NO:145), and G73Y (Tencon21 - SEQ ID NO: 146) which were predicted to improve stability by the program PoPMuSiC v2.0 (Dehouck, Grosfils et al. 2009). The mutant E86I (Tencon22 - SEQ ID NO: 147) had been previously found to stabilize a homologous protein, the 3rd FN3 domain from human Tenascin (W02009/086116A2). Finally, the L17A mutation was found to significantly stabilize Tencon during alanine scanning experiments in which all loop residues of Tencon were replaced with alanine independently (data not shown). Following an initial round of stability assays (see 25 below), the combinatorial mutants N46V/E86I (Tencon 23 - SEQ ID NO: 148), E14P/N46V/E86I (Tencon24 - SEQ ID NO: 149), and L17A/N46V/E86I (Tencon25 - SEQ ID NO:150) were produced to further increase stability. 30 42

Expression and Purification 2015271900 17 Dec 2015

Mutations in the Tencon coding sequence were made using a QuikChange mutagenesis kit (Stratagene). The resulting plasmids were transformed into BL21-GOLD (DE3) E. coli (Stratagene) for expression. A single colony was picked and grown overnight at 37°C in 2 mL of TB media containing 100 qg/ml ampicillin. This culture was used to seed 100 mL of autoinduction media (Overnight Express Instant TB media, Novagen) in a 500 mL baffled flask and grown at 37°C for 16 hours.

The culture was harvested by centrifugation at 4000xg for 20 min and the pelleted cells resuspended 5 mL of BugBuster HT (Novagen) per gram of wet cell pellet. After 30 minutes of incubation at room temperature, lysates were clarified by centrifugation at 30,000xg for 20 minutes and loaded onto a 3 mL Ni-NTA superflow column (Novagen) by gravity. After loading, each column was washed with 15 mL of a buffer containing 50 mM sodium phosphate pH 7.4, 500 mM NaCl, and 10 mM imidazole. Bound protein was then eluted from the column using 10 mL of a buffer containing 50 mM sodium phosphate pH 7.4, 500 mM NaCl, and 250 mM imidazole. Protein purity was assessed by SDS-PAGE. Prior to biophysical analysis, each mutant was dialyzed thoroughly into PBS pH 7.4. 28-33 mg of purified protein was obtained for each mutant from 100 mL of culture.

Characterization of Thermal Stability

The thermal stabilities of the parent Tencon and each mutant were measured by capillary differential scanning calorimetry (DSC). Each sample was dialyzed extensively against PBS pH 7.4 and diluted to a concentration of 2-3 mg/mL. Melting temperatures were measured for these samples using a VP-DSC instrument equipped with an autosampler (MicroCal, LLC). Samples were heated from 10°C to 95°C or 100°C at a rate of 1°C per minute. A buffer only scan was completed between each sample scan in order to calculate a baseline for integration. Data were 25 fit to a two state unfolding model following subtraction of the buffer only signal. Reversibility of thermal denaturation was determined by repeating the scan for each sample without removing it from the cell. Reversibility was calculated by comparing the area under the curve from the 1st scan with the 2nd scan. Results of the DSC experiments are presented in Table 5 as the values derived from complete melting curves. Single mutants Tencon 17, Tenconl8, Tencon 19, and 30 Tencon22 improved the thermal stability compared to the parent tencon sequence. Only 43

Tencon21 was significantly destabilizing. Combinatorial mutant samples Tencon23, Tencon24, and Tencon25 all had a significantly larger enhancement of the stability, indicating that the designed mutations are additive with respect to improving thermal stability. 2015271900 17 Dec 2015

Denaturation by Guandine Hydrochloride

The abilities of Tencon and each mutant to remain folded upon treatment with increasing concentrations of guanidine hydrochloride (GdmCl) as measured by tryptophan fluorescence were used to assess stability. Tencon contains only one tryptophan residue. The tryptophan residue is buried within the hydrophobic core and thus fluorescence emission at 360 nm is a sensitive measure of the folded state of this protein. 200 uL of a solution containing 50 mM sodium phosphate pH 7.0, 150 mM NaCl, and variable concentrations of GdmCl from 0.48 to 6.63 M were pipetted into black, non-binding, 96-well plates (Greiner) in order to produce a 17 point titration. 10 uL of a solution containing the tencon mutants were added to each well across the plate to make a final protein concentration of 23 uM and mixed by pipetting up and down gently. After incubation at room temperature for 24 hours, fluorescence was read using a Spectramax M5 plate reader (Molecular Devices) with excitation at 280 nm and emission at 360 nm. The data generated from such curves is shown in Figure 8. Fluorescence signal was converted to fraction unfolded using the equation (Pace 1986 Methods Enzymol 131: 266-80):

Where yF is the fluorescence signal of the folded sample and yu of the unfolded sample.

The mid-points of the unfolding transition and slope of the transition were determined by fitting to the equation below (Clarke, Hamill et al. 1997): F- % +#*·!$]} + fcHEtapMH - GEMOT)

Where F is the fluorescence at the given denaturant concentration, and are the y-intercepts of the native and denatured state, and {%are the slopes of the baselines for the native and 25 denatured state, [D] is the concentration of GdmCl, [iffgg^the GdmCl concentration at which point 50% of the sample is denatured, m the slope of the transition, R the gas constant, and T the 44 temperature. The free energy of folding for each sample was estimated using the equation (Pace 1986 supra; Clarke, Hamill et al. 1997 J Mol Biol 270(5): 771-8): M? = ?&[!?] 2015271900 17 Dec 2015

It is often difficult to accurately measure the slope of the transition, m, for such curves. Additionally, the mutations described here are not expected to alter the folding mechanism of tencon. Thus, the m value for each mutant was measured and the values averaged (Pace 1986 supra) to produce an m = 3544 cal/mol/M used for all free energy calculations. The results of these calculations are presented in Table 5. The results for GdmCl unfolding experiments demonstrate that the same mutants that stabilize Tencon with respect to thermal stability also stabilize the protein against GdmCl induced denaturation.

Size Exclusion Chromatography

Size exclusion chromatography (SEC) was used to assess the aggregation state of WT tencon and each mutant. 5 uL of each sample were injected onto a Superdex 75 5/150 column (GE Healthcare) at a flow rate of 0.3 mL/min with a PBS mobile phase. Elution from the column was monitored by absorbance at 280 nm. In order to assess the aggregation state, the column was previously calibrated with globular molecular weight standards (Sigma). All of the samples tested, with the exception of Tencon21, eluted in one peak at an elution volume consistent with that of a monomeric sample. Tencon21 eluted with 2 peaks, indicating the presence of aggregates. 45 2015271900 17 Dec 2015

Table 5. Construct Mutations Tm (Kcal) [D]so% (M) AG(H20) (kcal/mol) Tencon 16 (SEQ ID NO: 16) 78.04 3.4 12.0 Tenconl7 (SEQ ID NO: 142) N46V 81.88 3.6 12.8 Tenconl8 (SEQ ID NO: 143) E14P 82.77 3.5 12.4 Tencon 19 (SEQ ID NO: 144) El IN 79.00 3.4 12.0 Tencon20 (SEQ ID NO: 145) E37P 77.40 3.4 12.0 Tencon21 (SEQ ID NO: 146) G73Y 67.56 2.4 8.5 Tencon22 (SEQ ID NO: 147) E86I 82.78 3.7 13.1 Tencon23 (SEQ ID NO: 148) N46V/E86I 86.65 4.1 14.5 Tencon24 (SEQ ID NO: 149) E14P/N46V/E86I 87.47 4.0 14.2 Tencon25 (SEQ ID NO: 150) L17A/N46V/E86I 92.73 5.1 18.1 Tencon26 (SEQ ID NO:151) L17A 84.9 4.6 16.2

It will be clear that the invention can be practiced otherwise than as particularly described in the foregoing description and examples. Numerous modifications and variations of the 46 2015271900 17 Dec 2015 present invention are possible in light of the above teachings and, therefore, are within the scope of the appended claims. 47

Claims (10)

1. A protein scaffold comprising loop domains topologically similar to loop domains of a fibronectin type III (FN3) domain, the protein scaffold having an amino acid sequence based on the consensus amino acid sequence of SEQ ID NO: 16, wherein one or more specific residues of SEQ ID NO: 16 are replaced to enhance melting temperature (Tm) and chemical stability of the protein scaffold as compared to the Tm and chemical stability of a protein scaffold having the amino acid sequence of SEQ ID NO: 16, and the replacements of SEQ ID NO: 16 are selected from the group consisting of N46V, E14P, L17A, E86I, a combination of N46V and E86I, a combination of N46V and E86I, and a combination of L17A, N46V and E86I.

2. The protein scaffold of claim 1, wherein the Tm is measured by differential scanning calorimetry, the chemical stability is measured as [D] by resistance to guanidinium chloride denaturation, and the Tm is increased as compared to the protein scaffold having the amino acid sequence of SEQ ID NO: 16 from about 1 Kcal to about 12 Kcal.

3. The protein scaffold of claim 1, comprising loop regions for contacting a target protein, wherein the loop regions have been modified to encompass alternate residues to positions in the amino acid sequence of SEQ ID NOS: 16, 142, 143 and 147-151 at one or more positions selected from the group consisting of residues 13, 15, 16, 22-28, 38-43, 51-54, 60-64, and 75-81 of an amino acid sequence selected from the group consisting of SEQ ID NOS: 16, 142, 143 and 147-151.

4. The protein scaffold of claim 3, wherein said protein scaffold binds a target protein with at least one affinity selected from a KD of at least 10"9M, at least 10"10M, at least 10"nM, at least 10"12M, at least 10~13M, at least 10~14M, and at least 10"15M, as determined by surface plasmon resonance or the Kinexa method.

5. A protein scaffold comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 142-144 and 147-151.

6. A method of generating a protein scaffold binding to a specific target with a predefined binding affinity, comprising the steps of: (a) contacting a library of a scaffold-based protein derived from a stability enhanced consensus sequence of a fibronectin type III domain incorporating randomized codons wherein a polynucleotide sequence encoding a polypeptide comprising a stability enhanced consensus sequence of a fibronectin type III domain selected from the group consisting of SEQ ID NOs: 142-151 is provided, randomizing codons are introduced into the polynucleotide sequence at selected positions and copies of the polynucleotide sequence are propagated to form a library of polynucleotides encoding variant scaffold proteins, with the specific target; and (b) isolating a protein binding to the specific target with the predefined affinity.

7. The method of claim 6, wherein the isolating step comprises isolating scaffold molecules binding to the specific target and testing the isolated scaffold molecules for binding affinity to the specific target.

8. The method of claim 7, wherein the isolating step comprises panning the library with the specific target, capturing scaffold molecules bound to the specific target, and isolating the binding scaffold molecules.

9. The method of claim 7, wherein the predefined binding affinity as measured by Kd is _~j less than or equal to about 10" M.

10. A protein scaffold when generated by the method of any one of claims 6 to 9.