WO2011092233A1

WO2011092233A1 - Yeast mating to produce high-affinity combinations of fibronectin-based binders

Info

Publication number: WO2011092233A1
Application number: PCT/EP2011/051111
Authority: WO
Inventors: Andreas Loew
Original assignee: Novartis Ag
Priority date: 2010-01-29
Filing date: 2011-01-27
Publication date: 2011-08-04

Abstract

The present invention relates to means of using yeast mating for identifying optimal, high-affinity combinations of fibronectin-based binders. The present invention further relates to high-affinity combinations of fibronectin-based binders for use in diagnostic, research and therapeutic applications. The invention further relates to cells comprising such combinations, and polynucleotides encoding such combinations.

Description

YEAST MATING TO PRODUCE HIGH-AFFINITY COMBINATIONS OF

FIBRONECTIN-BASED BINDERS

Related Information

[001] The contents of any patents, patent applications, and references cited throughout this specification are hereby incorporated by reference in their entireties.

Background of the Invention

[002] Molecules capable of specific binding to a desired target epitope are of enormous importance as both therapeutics and medical diagnostic tools. A well-known example of this class of molecules is the monoclonal antibody. Antibodies can be selected that bind specifically and with high affinity to almost any structural epitope. As a result, antibodies are used routinely as research tools and as FDA-approved therapeutics. The worldwide market for therapeutic and diagnostic monoclonal antibodies is currently worth approximately $30 billion.

[003] However, monoclonal antibodies have a number of shortcomings. For example, classical antibodies are large and complex molecules. They have a heterotetrameric structure comprising two light chains and two heavy chains connected together by both inter- and intra-disulphide linkages. This structural complexity precludes easy expression of antibodies in simple prokaryotic systems and requires that antibodies are produced in more elaborate (and expensive) mammalian cell systems. The large size of antibodies also limits their therapeutic effectiveness since they are often unable to efficiently penetrate certain tissue spaces. In addition, therapeutic antibodies, because they possess an Fc region, occasionally trigger undesired effector cell function and/or clotting cascades.

[004] Alternative binding molecules capable of specific binding to a desired target have been explored. Fibronectin is a large protein which plays essential roles in the formation of extracellular matrix and cell-cell interactions; it consists of many repeats of three types (types I,

II, and III) of small domains (Baron et al. 1991 Trends Biochem. Sci. 16: 13-17).

[005] Fn3 (fibronectin type III) itself is the paradigm of a large subfamily which includes portions of cell adhesion molecules, cell surface hormone and cytokine receptors, chaperoning, and carbohydrate-binding domains. For reviews see Bork et al. 1992 Proc. Natl. Acad. Sci. USA.

89: 8990-4; Bork et al. 1994 J. Mol. Biol. 242: 309-20; Campbell et al. 1994 Structure. 2: 333-7; and Harpez et al. 1994 J. Mol. Biol. 238: 528-39.

[006] Due to their structure, fibronectins function in a manner characteristic of antibodies and also possess structural advantages. Specifically, the structure of these molecules has been designed for optimal folding, stability, and solubility, even under conditions that normally lead to the loss of structure and function in antibodies. [007] However, there exists a need for an improved method for obtaining combinations of fibronectin-based binders with increased affinity to a target. The present disclosure provides a method for obtaining such improved high-affinity combinations of fibronectin-based binders. Summary of the Invention

[008] The present invention relates to means of using yeast mating for identifying optimal, high-affinity combinations of fibronectin-based binders. Individual haploid yeast cells of one mating type express a member of a library of fibronectin-based binders. Individual haploid yeast cells of a different mating type also express a member of a library of fibronectin-based binders which bind to the same epitope or target (e.g., different epitopes on the same target). Mating of the cells produce diploid yeast cells which express different combinations of fibronectin-based binders, each combination comprising two different fibronectin-based binders. These cells can be screened to identify high-affinity combinations, wherein the combination of fibronectin-based binders binds to an epitope or target with higher affinity than either of its constituent fibronectin- based binders.

[009] Accordingly, the invention provides a method of identifying a high-affinity combination of fibronectin-based binders, the method comprising the steps of: providing a first population of haploid yeast cells, each cell comprising a first heterologous nucleic acid comprising a first promoter and an operably-linked coding region encoding a member of a first library of fibronectin-based binders; providing a second population of haploid yeast cells, each cell comprising a second heterologous nucleic acid comprising a second promoter and an operably- linked coding region encoding a member of a second library of fibronectin-based binders, wherein the cells of the first and second populations are different mating types; mating cells of the first and second populations to produce a population of diploid yeast cells, wherein each cell displays on its surface a member of the first library and a member of the second library; and screening the population of diploid yeast cells to obtain a diploid yeast cell displaying on its surface a high- affinity combination of two fibronectin-based binders.

[0010] In one embodiment, the first and second promoters are the same.

[001 1 ] In one embodiment, the first and second promoters are different.

[0012] In one embodiment, the first and second libraries are the same or different, but both libraries bind to the same target.

[0013] In one embodiment, the first and second heterologous nucleic acids encode selectable markers.

[0014] In one embodiment, the first and second heterologous nucleic acids encode resistance to two different antibiotics.

[0015] In one embodiment, the antibiotics are selected from the group consisting of zeocin, blasticidin, hygromycin and neomyocin. [0016] In one embodiment, the method further comprises a step of selecting for diploid cells using the antibiotics for which the first and second heterologous nucleic acids encode resistance.

[0017] In one embodiment, the step of screening is performed using FACS.

[0018] In one embodiment, the step of screening is performed using a method selected from the group consisting of ELISA, panning, magnetic particle processing, and flow cytometry.

[0019] In one embodiment, either of the first or second population of haploid yeast cells is EBY100 and the other is AWY102.

[0020] In one embodiment, the method further comprises the step of: isolating the nucleotides encoding the high-affinity combination of two fibronectin-based binders from the yeast cell.

[0021] In one embodiment, the method further comprises the step of: preparing a fusion protein comprising the high-affinity combination of two fibronectin-based binders expressed on the diploid cell.

[0022] In one embodiment, the first and second heterologous nucleic acid are integrated into a chromosome.

[0023] In one embodiment, the first and second library of fibronectin-based binders is enriched.

[0024] In one embodiment, the first and second library of fibronectin-based binders bind to different epitopes of the same target.

[0025] In one embodiment of the invention, the high-affinity combinations of fibronectins or fibronectin-based binders prepared by this method can be isolated. Two or more fibronectin-based binders can be combined as a fusion protein. These fusion proteins can be used for both therapeutic and diagnostic purposes. The fusion proteins can be combined with one or more additional moieties which are not fibronectins or fibronectin-based binders to produce conjugates. The high-affinity combinations of fibronectin-based binders, and fusions and conjugates comprising them, can be used as diagnostics and therapeutics.

Brief Description of Drawings

Figure 1 shows a ribbon diagram of a Fn3 -based binding molecule with loop and framework residues constituting the ligand binding surfaces highlighted.

Figure 2 is a schematic showing yeast mating of haploid yeast cells that result in a diploid yeast cell that expresses both high-affinity combinations of fibronectin molecules.

Figure 3 diagrams plasmid pYS6/CT (also known as pYC6/CT).

Figure 4 diagrams plasmid pYS6/CT Zeo (also known as pYS6/CT Zeo).

Detailed Description of the Invention Definitions

[0026] In order to provide a clear understanding of the specification and claims, the following definitions are conveniently provided below.

[0027] Fibronectin-based binders include fibronectins (e.g., an Fn3 or FnlO domain) that have been altered or selected. As used herein, the term "Fibronectin type III domain" or "Fn3 domain" refers to a wild-type Fn3 domain from any organism, as well as chimeric Fn3 domains constructed from beta strands from two or more different Fn3 domains. As is known in the art, naturally- occurring Fn3 domains have a beta-sandwich structure composed of seven beta-strands, referred to as A, B, C, D, E, F, and G, linked by six loops, referred to as AB, BC, CD, DE, EF, and FG loops (see e.g., Bork and Doolittle, Proc. Natl. Acad. Sci. U.S.A 89: 8990, 1992; Bork et al., Nature Biotech. 15: 553, 1997; Meinke et al., J. Bacterid. 175: 1910, 1993; Watanabe et al., J. Biol. Chem. 265: 15659, 1990; Main et al., 1992; Leahy et al., 1992; Dickinson et al., 1994; U.S. patent 6,673,901 ; Patent Cooperation Treaty publication WO/03104418; and, US patent application 2007/0082365, the entire teachings of which are incorporated herein by reference). Three loops are at the top of the fibronectin domain (the BC, DE and FG loops) and three loops are at the bottom of the domain (the AB, CD and EF loops) (see Figure 1). In a particular embodiment of the invention, the Fn3 domain is from the tenth Fn3 domain of human Fibronectin (¹⁰Fn3 or FnlO) (SEQ. ID. NO: 1): VSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKS TATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRTEI (SEQ ID NO: 1)

[0028] As used herein the terms "fibronectin-based binder", "Fn3-based binding molecule", "fibronectin type III (Fn3)-based binding molecule" and the like refer to fibronectin (e.g., an Fn3 domain) that has been altered or selected. For example, the sequence may be altered to contain one or more non-Fn3 binding sequences. In one embodiment, one or more of the bottom AB, CD and/or EF loops are altered compared to the corresponding wild-type Fn3 domain to contain one or more non-Fn3 -binding sequences.

[0029] In one embodiment, one or more of the top BC, DE and FG loops are altered compared to the corresponding wild-type Fn3 domain to contain one or more non-Fn3 -binding sequences. In yet another embodiment, any one of the AB, BC, CD, DE, EF and/or FG loops are altered compared to the corresponding wild-type Fn3 domain to contain one or more non-Fn3 binding sequence.

[0030] In a further embodiment, two or more Fn3 -based binding molecules are linked together to produce "multi-specific Fn3 -based binding molecules".

[0031] The term "non-Fn3 binding sequence" or "non- fibronectin-based binder" refers to an amino acid sequence which is not present in the naturally- occurring (e.g., wild-type) Fn3 or fibronectin domain, and which optionally binds to a specific target. Such non-Fn3 binding sequences are typically introduced by modifying (e.g., by substitution and/or addition) the wild- type Fn3 domain (e.g., within the bottom loops and/or top loop regions). This can be achieved by, for example, random or predetermined mutation of amino acid residues within the wild-type Fn3 domain.

[0032] The term "high-affinity combination of fibronectin-based binder" refers to a group of Fn3 -based binding molecules, preferably two, which bind to an epitope or target with high affinity. For example, both fibronectin-based binders can bind to the same epitope, or to different regions of the same target e.g., different epitopes within the same target. The combination can bind with a higher affinity than the affinities of the constituent fibronectin-based binders. A high- affinity combination of Fn3 -based binding molecules can bind strongly to a target, even if the individual Fn3 -based binding molecule binds weakly. This essentially increases affinity by increasing avidity. As a non-limiting example, the high-affinity combination can comprise two or more mono-specific fibronectin-based binders.

[0033] The term "mono-specific" as used herein refers to an Fn3 -based binding molecule that binds to one target molecule (or one epitope of a single target).

[0034] The term "target" refers to an antigen recognized by Fn3-based binding molecule of the invention. Targets include, but are not limited to, proteins, peptides, carbohydrates, and/or lipids. As used herein, an "epitope" generally means a portion of a target (e.g., a part of a target protein). Generally, a high-affinity combination of fibronectin-based binders can bind to the same target (e.g., the same protein). The constituent binders can bind to the same epitope on the same target, or, preferably, bind non-competitively to different epitopes on the same target (e.g., different, non- overlapping portions of the same protein).

[0035] Suitable targets for Fn3-based binding molecules include, but are not limited to, half-life extenders (e.g., human serum albumin), lysozyme, a cellular receptor, a cellular receptor ligand, a bacteria, or a virus. In a particular embodiment the target is involved in a human disease, e.g., an autoimmune disease, cancer, or an infectious disease.

[0036] In one embodiment, a target is chosen (e.g., human serum albumin), because it extends the serum half-life of a fibronectin-based binder. This specification describes, inter alia, the identification and production of novel, Fn3-based binding molecules that bind to a target, e.g., albumin, using the bottom loops of the Fn3 molecule. Note that in various embodiments of this invention, human serum albumin can be either a target of a fibronectin-based binder, or albumin can be a conjugate partner of a fibronectin-based binder. Furthermore, it was discovered that many independently randomized loops tended to converge to a consensus sequence that is likely to participate in albumin binding. Therefore, it is expected that fibronectin-based binders having this consensus sequences will be useful as albumin-binding agents even when separated from the protein context in which they were identified. [0037] Generally, a mono-specific fibronectin-based binder will employ only the top region or the bottom region in binding to its target. For example, a bottom mono-specific Fn3 -based binding molecule is one that uses only the bottom loops, such as the AB, CD, and/or EF loops, and/or the C-terminal of the Fn3 domain to bind a target. A top mono-specific Fn3-based binding molecule uses only the top loops of the Fn3 domain, such as BC, DE, and/or FG loops, to bind the target. It is to be understood that binding to a target molecule may not involve all three top loops or all three bottom loops. A mono-specific fibronectin-based binder which binds to a single target using only the top loops (or a subset thereof) or the bottom loops (or a subset thereof) is considered "mono-valent". If individual fibronectin-based binders within a chain or complex bind to different targets, the chain or complex is multi-specific and multi-valent.

[0038] A "mono-specific" fibronectin-based binder can also be "bi-valent." In this case, both the top loops (or a subset thereof) and the bottom loops (or a subset thereof) bind to the same target or epitope.

[0039] The mono-specific Fn3 domains can also be linked together (e.g., in a pearl-like fashion) to form chains or complexes. If all the mono-specific fibronectin-based binders in the complex are identical, the complex will be mono-specific (wherein each binder binds to the same epitope), but multi-valent. Alternately, the complex can comprise a variety of different mono-specific binders, which all still bind to the same epitope, in which case the complex would still be monospecific and multi-valent.

[0040] In one embodiment, the chain or complex comprises two or more fibronectin-based binders which are "paratopic." In this case, all the fibronectin-based binders bind to the same target (e.g., the target protein "A") but at different epitopes. The complex thus forms a chain of fibronectin-based binders which bind to different epitopes of the same target (e.g., Al and A2). As one non-limiting example, a target protein (e.g., "B") can comprise, for example, four epitopes (Bl, B2, B3 and B4). Each of these epitopes can be bound by a different mono-specific fibronectin-based binder. The four binders can be physically linked or joined to form a paratopic complex which binds to all four epitopes (Bl, B2, B3 and B4), leading to very tight binding of the target molecule (B).

[0041] In another embodiment, a complex can be formed between a fibronectin-based binder which is mono-specific for a particular target (e.g., protein "A"), and a fibronectin-based binder which is mono-specific for a completely different target (e.g., protein "B"). This results in a "multi-specific" complex. The term "multi-specific" as used herein refers to a Fn3 -based binding molecule that comprises at least two mono-specific Fn3 -based binding molecules linked together. Linking or joining these binders results in a complex which binds to two different targets. As a non-limiting example, such a complex can be used to bring different targets into proximity. For example, if protein "A" is a toxin and protein "B" is an antigen on a cancer cell, the complex can be used to deliver the toxin to a cancer cell. These various bi-specific and multi-specific complexes can be formed from high-affinity combinations of fibronectin-based binders, as provided herein.

[0042] The term "fusion" refers to a physical combination of two or more moieties, preferably, two or more fibronectin (e.g., Fn3) domains or fibronectin-based binders. The fusion protein can be produced via any method known in the art, including, but not limited to, expressing the fusion protein from a composite gene comprising the coding segment for each Fn3 domain; or by isolating each Fn3 domains and then physically linking them. The linkage is preferably sized and flexible enough to allow the two or more Fn3 domains to simultaneously bind to the same target molecule. The fusion protein can, optionally, further comprise additional components, as described below, to form a conjugate.

[0043] The term "conjugate" refers to one or more Fn3 -based binding molecules chemically or genetically linked to one or more non-Fn3 moieties. A conjugate can include a fusion protein comprising two or more fibronectin-based binders chemically or genetically linked to a non- fibronectin-based binder moiety (e.g., a moiety which is not a fibronectin-based binder).

[0044] The terms "non-Fn3 moiety", "non-fibronectin-based binder" and the like refer to a biological or chemical entity that is not a Fn3 or fibronectin-based binder and that imparts additional functionality to a an Fn3 or fibronectin-based binder to which it is attached. In a particular embodiment, the non-Fn3 moiety is a polypeptide, e.g., human serum albumin (HSA), or a chemical entity, e.g., polyethylene gycol (PEG) which increases the half-life of the Fn3-based binding molecule in vivo.

[0045] A constituent fibronectin-based binder in a high-affinity combination can comprise a non- natural amino acid residue.

[0046] The term "non-natural amino acid residue" refers to an amino acid residue that is not present in the naturally- occurring (wild-type) Fn3 domain. Such non-natural amino acid residues can be introduced by substitution of naturally occurring amino acids, and/or by insertion of non- natural amino acids into the naturally occurring amino acid Fn3 sequence. The non-natural amino acid residue also can be incorporated such that a desired functionality is imparted to the Fn3-based binding molecule, for example, the ability to link a functional moiety (e.g., PEG).

[0047] The term "polyethylene glycol" or "PEG" refers to a polyalkylene glycol compound or a derivative thereof, with or without coupling agents or derivatization with coupling or activating moieties.

[0048] The terms "specific binding'", "specifically binds to" and the like refer to the ability of an Fn3 -based binding molecule to bind to a target with an affinity of at least 1 x 10^"6 M, and/or bind to a target with an affinity that is at least two-fold (preferably at least 10 fold) greater than its affinity for a non-specific antigen at room temperature under standard physiological salt and pH conditions, as measured by surface plasmon resonance. I. Fibronectin-Based Binders and Libraries

[0049] The present invention provides high-affinity combinations of fibronectin-based binders that have increased avidity. Fibronectin-based binders are derivatives, variants and/or selected or mutant forms of fibronectins, which bind specifically to one or more targets. Fibronectins are large proteins which play essential roles in the formation of extracellular matrix and cell-cell interactions; they bind a target specifically and consist of many repeats of three types (types I, II, and III) of small domains. Fibronectin type III (Fn3) domains comprise, in order from N-terminus to C-terminus, a beta or beta-like strand, A; a loop, AB; a beta or beta-like strand, B; a loop, BC; a beta or beta-like strand, C; a loop, CD; a beta or beta-like strand, D; a loop, DE; a beta or beta-like strand, E; a loop, EF; a beta or beta-like strand, F; a loop, FG; and a beta or beta-like strand, G. Any or all of loops AB, BC, CD, DE, EF and FG may participate in target binding. The BC, DE, and FG loops are both structurally and functionally analogous to the complementarity determining regions (CDRs) from immunoglobulins. U.S. Pat. No. 7,1 15,396 describes Fn3 domain proteins wherein alterations to the BC, DE, and FG loops result in high-affinity TNF-alpha binders. U.S. Publication No. 2007/0148126 describes Fn3 domain proteins wherein alterations to the BC, DE, and FG loops result in high-affinity VEGFR2 binders.

[0050] Fibronectin type III (Fn3)-based binding molecules can specifically bind to a target antigen and, thus, can be used in a broad variety of therapeutic and diagnostic applications.

Mono-specific Fn3-based binding molecules can bind to a target(s) using the bottom AB, CD, EF loops and/or C-terminal ("bottom mono-specific Fn3 -based binding molecules"), or the top DE, BC, and/or FG loops ("top mono-specific Fn3-based binding molecules"). These Fn3-based binding molecules can be linked together (e.g., in pearl-like fashion) to form multi-specific Fn3- based binding molecules that simultaneously bind to different regions of the same target.

Fibronectin-based binders (or complexes thereof) can be conjugated to one or more non-Fn3 moieties (e.g., functional moieties), such as Human Serum Albumin (HSA), an antibody Fc region or polyethylene glycol (PEG), for example, to improve half-life and stability of the Fn3 -based binding molecule.

[0051 ] The invention further provides methods of screening libraries of Fn3 -based binding molecules for producing high-affinity combinations of fibronectin-based binders with specific binding to a target, typically a target protein, as well as methods for manufacturing combinations of Fn3-based binding molecules in, for example, prokaryotic or eukaryotic systems. Still further, the invention provides compositions (e.g., therapeutic compositions) comprising combinations of Fn3 -based binding molecules, and uses of such compositions in a variety of therapeutic and diagnostic applications. [0052] Also provided by the invention are compositions comprising high-affinity combinations of the Fn3 -based binding molecules and conjugates of the invention, formulated with a suitable carrier.

[0053] The high-affinity combinations of Fn3-based binding molecules and conjugates of the invention can be used in a variety of therapeutic and diagnostic applications including, but not limited to, applications that antibodies can be used in. Such applications include, for example, treatment and diagnosis of autoimmune diseases, cancers and infectious diseases.

[0054] In another aspect, the invention provides a library of Fn3 -based binding molecules, which can be used to identify high-affinity combinations of Fn3 -based binding molecules which bind to a particular desired target. In one embodiment, the library comprises Fn3 -based binding molecules, each of which contains at least one amino acid alteration in one or more of the bottom AB, CD, EF loop regions or C-terminus compared to a wild-type Fn3 domain, such as the human ¹⁰Fn3 (SEQ ID NO: 1). Particular amino acid residues in the AB, CD or EF loop regions and beta strands which can be altered include, for example, amino acids at position 15, 16, 38, 39, 40, 41, 42, 43, 44, 45, 60, 61, 62, 63, 64, 93, 95, or 96 of SEQ ID NO: 1.

[0055] Fibronectin-based binder libraries are known in the art, e.g., that in Published U.S. Patent Appl. No. 20090176654 to Cappuccilli.

[0056] In a another embodiment, the library comprises Fn3-based binding molecules, each of which contains at least one amino acid alteration in one or more of the top BC, DE or FG loop regions compared to a wild-type Fn3 domain, such as the human ¹⁰Fn3 (SEQ ID NO: 1).

Particular amino acid residues in the BC, DE or FG loop regions and beta strands which can be altered include, for example, amino acids at position 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 51, 52, 53, 54, 55, 56, 76, 77, 78, 79, 80, 80, 81, 82, 83, 84, 85, 86, 87, or 88 of SEQ ID NO: 1.

[0057] Library diversity can be generated by, for example, random mutagenesis, "walk though mutagenesis, or "look through mutagenesis of one or more of the disclosed residues in SEQ ID NO: 1 (U.S. Patent Nos. 7,195,880; 6,951,725; 7,078,197; 7,022,479; 5,922,545; 5,830,721; 5,605,793, 5,830,650; 6,194,550; 6,699,658; 7,063,943; and 5,866,344 and Patent Cooperation Treaty publication WO06023144).

[0058] Nucleic acids encoding the library of Fn3-based binding molecules, or variants thereof, described herein can be constructed using art-recognized methods including, but not limited to, PCR-based or enzyme-mediated genetic engineering, ab initio DNA or RNA synthesis, and/or cassette mutagenesis.

[0059] The library can optionally be enriched for fibronectin-based binders with a higher affinity for binding to a desired target. As described below, the libraries can be used to produce high- affinity combinations of fibronectin-based binders.

II. Nucleic Acids and Vectors Encoding Fibronectin-Based Binders [0060] A haploid yeast cell can be provided comprising a heterologous nucleic acid or vector encoding a member of a library of fibronectin-based binders. The nucleic acid can comprise, in an operably-linked order, one or more promoter, coding segment for a signal peptide, and coding segment for a member of a fibronectin-based binder. The nucleic acid can also comprise a cloning site and a marker. The nucleic acid is designed so that the fibronectin-based binder which it encodes is displayed on the surface of the yeast cell. The haploid yeast cell is mated to another haploid yeast cell, which encompasses a different heterologous nucleic acid encoding a different fibronectin-based binder. The mating produces a diploid cell, and a cell producing a high-affinity combination of binders is selected.

[0061 ] A "heterologous nucleic acid" refers to a nucleic acid which is introduced into a cell by artifice. A heterologous nucleic acid may comprise a copy of a gene (e.g., a member of the library of fibronectin-based binders, as described above). Additional optional elements of a heterologous nucleic acid include, as non- limiting examples, one or more restriction sites, a marker, an element for replication (e.g., a replication origin sequence), a promoter (including inducible and constitutive promoters), and a coding segment for a signal peptide sequence. Heterologous nucleic acids can comprise more than one of a particular type of element (e.g., it can comprise two different markers or two different origins of replication suitable for different organisms).

Exemplary sequences include the CEN6/ARS4 and fl origin. The elements can be selected so that the heterologous nucleic acid can function in other hosts in addition to yeast (e.g., phage, bacteria, mammalian cells, insect cells, etc.). The elements of the heterologous nucleic acid can be arranged (e.g., operably-linked) such that the fibronectin gene can be expressed, and the fibronectin protein produced and displayed on the surface of the yeast cell. For example, the coding segment for the fibronectin-based binder should be operably linked to a promoter for expression.

[0062] A number of eukaryotic promoters can be used in a heterologous nucleic acid in a yeast cell. Exemplary eukaryotic promoters include yeast promoters, such as galactose inducible promoters, pGALl , pGALl -10, pGal4, and pGal 10; phosphoglycerate kinase promoter, pPGK, cytochrome c promoter, pCYCl ; and alcohol dehydrogenase I promoter, pADHl . The T7 promoter can also be used in both prokaryotes and eukaryotes if the T7 RNA polymerase is expressed in the cell. The CUP1 and TEF1 promoters can also be used.

[0063] A coding segment for a signal peptide can optionally intervene between the promoter and the coding segment for the fibronectin-based binder. The signal sequence (or leader peptide) operates to direct transport (secretion) of a nascent fibronectin-based binder into or across a cellular membrane. A fibronectin-based binder or fusion thereof can be expressed in a eukaryotic cell from a vector and can be transported to the endoplasmic reticulum (ER) for assembly and transport to the cell surface for extracellular display. An effective signal sequence is functional in a eukaryotic system. Optionally (e.g., in the case of a dual display vector) the signal sequence is effective in a prokaryotic system as well. Polynucleotides encoding a member of a fusion of two or more fibronectin-based binders are typically linked in- frame to a signal sequence. The polynucleotides encoding the fibronectin-based binder and the signal peptide can be immediately adjacent to each other, or a space sequence can intervene. In both cases, a conjugate of a signal peptide and a fibronectin-based binder is produced.

[0064] The signal sequence may be native to the host or heterologous, so long as it is operable to effect extracellular transport of the fibronectin-based binder to which it is conjugated. Several signal sequences are known to persons skilled in the art (e.g., Mf-alpha-1 prepro, Mf-alpha-1 pre, acid phosphatase Pho5, Invertase SUC2 signal sequences operable in yeast; pill, PelB, OmpA, PhoA signal sequences operable in E. coli; gp64 leader operable in insect cells; IgK leader, honeybee melittin secretion signal sequences operable in mammalian cells). The signal sequences can be derived from native secretory proteins of the host cell. Other exemplary eukaryotic signal sequences include those of alpha-mating factor of yeast (e.g., of S. cerevisiae), alpha-agglutinin of yeast (e.g., of S. cerevisiae), invertase of yeast (e.g., of S. cerevisiae), inulinase of Kluyveromyces, and the signal peptide of the Aga2p subunit of a-agglutinin (e.g., of S. cerevisiae) (especially in embodiments where the anchoring polypeptide to be used is the Aga2p polypeptide). Additional suitable hybrid leader sequences are listed in Rakestraw et al. Biotechnol. Bioeng. 2009 Aug 15; 103 (6): 1 192-201.

[0065] The heterologous nucleic acid or vector can further comprise a marker. The marker can be a selectable marker, a dominant marker, or a counter-selectable marker (e.g., URA3). Typically, the marker is a marker that enables cells that have lost the marker to be selectively identified, e.g., by growth or other phenotype. The marker can be functional in a yeast cell; e.g., the marker can be a yeast gene. In a preferred embodiment, the marker is a gene that encodes resistance to an antibiotic, e.g., zeocin, blasticidin, hygromycin or neomyocin. Kill curves can be experimentally performed to determine appropriate levels of antibiotics, for example. Other suitable markers include TRP1 , LEU and HIS.

[0066] These various components are arranged and the heterologous nucleic acid is designed such that the fibronectin-based binder it encodes is expressed on the surface of the yeast cell ("surface display"). The signal peptide facilitates delivering the fibronectin-based binder to the cell surface. There the fibronectin-based binder can, optionally, be immobilized. Immobilization ("tethering" or "display") can be achieved by linkage to an immobilization moiety or anchor embedded in the cell surface. The linkage can be covalent or non-covalent. In one embodiment, the linkage is covalent (e.g., via a peptide, disulfide, amide, or other bond). For example, a fibronectin-based binder may be linked to an anchor via the high-affinity interaction of the Jun and Fos leucine zippers c-jun/fos linkage) (e.g., see Crameri and Suter, 1993 Gene 137: 69-75; Crameri and Blaser 1996 Exp. Med. Bio. 409: 103-1 10). Generally, each fibronectin-based binder has only one point of attachment to the host cell surface. However, more than one fibronectin-based binder may be linked to a single anchor. If a particular fibronectin-based binder is a member of a joined complex or conjugate of binders, only one binder needs to be attached to the cell. In one embodiment, a coding segment for a protein anchor can be inserted adjacent to the coding segment for the fibronectin-based binder, and they can be co-expressed along with the

(optional) signal peptide. Multiple methods of anchoring a fibronectin-based binder to a yeast cell surface are known in the art. Many anchors are known in the art. The anchor can be, for example, a surface-expressed protein native to the host cell, e.g., either a transmembrane protein or a protein linked to the cell surface via a glycan bridge. Several operable anchor proteins can be used, depending on the host cell (e.g., .about. l 1 1 , pVI, pVIIl , LamB, PhoE, Lpp-OmpA, Flagellin (FliC), or at least the transmembrane portions thereof, operable in prokaryotes/phage; platelet- derived growth factor receptor (PDGFR) transmembrane domain, glycosylphosphatidylinositol (GPI) anchors, operable in mammalian cells; gp64 anchor in insect cells, and the like). Where yeast is the host, the anchor protein can be .alpha.-agglutinin, a-agglutinin (having subcomponents Agalp and Aga2p), or FLO, which naturally form a linkage to the yeast cell surface. The CWP2 and FS anchor can also be used, as can the adapter system using different pairs of proteins for cell surface display, as described in Wang et al. J Immunol Methods. 2010 Jan 21.

[0067] In addition to the promoter, coding segments, replication origin, marker and other components, the heterologous nucleic acid can also, optionally, encode a tag. In an exemplary fibronectin-based binder conjugate, one or more fibronectin-based binders is linked to a molecular tag. A coding segment for a tag can be operably linked to the coding segment of the fibronectin- based binder. Alternatively, the tag can be separately expressed from the binder and the two can be physically linked. Exemplary tags include epitope tags (e.g., tags generated by conjugating a peptide sequence that is recognized as an antigenic determinant to the polypeptide of interest) and tags that chelate a metal (e.g., polyHis tags). Examples of epitope tags include the HA tag and myc 12CA5 tag, flag-tag, s-tag,v5-tag etc . Such tags can be useful in purifying the fibronectin- based binder.

[0068] In addition to the above-identified components, a heterologous nucleic acid or vector can comprise one or more cloning sites. These sites facilitate insertion, deletion and transfer of the polynucleotide sequences (e.g., the aforementioned promoters, coding segments, markers, etc.). Such cloning sites can include at least one restriction endonuclease recognition site. A restriction site which only occurs once in a vector (a "unique" site) is particularly useful. The sites can be positioned to facilitate excision and insertion, in reading frame, of coding segments. Any of the restriction sites known in the art may be utilized in the vector construct; most commercially- available vectors already contain multiple cloning sites or polylinkers. In addition, genetic engineering techniques useful to incorporate new and unique restriction sites into a vector are known and routinely practiced by persons of ordinary skill in the art. [0069] The various components described above can be incorporated into a single vector suited for use in a yeast cell. One exemplary vector is disclosed in U.S. S. No. 60/326,320 and WO 03/029456. Various vectors suitable for expressing a fibronectin-based binder are known in the art. The pYS6/CT (or pYC6/CT plasmid, as diagrammed in Fig. 3) can be used. Additional components of vectors typically used by one of ordinary skill in the art can also be used (e.g., EM7, URA3, pUC origin, CYC1, 6s His, v5 epitope, T7 promoter, etc.)

[0070] In one preferred embodiment, the heterologous nucleic acid is a vector suitable for use in yeast, comprising a promoter and a coding segment for a fibronectin-based binder, and optionally, one or more of the following: a coding segment for an anchor, a coding segment for a tag, a marker, a replication origin, and a cloning site.

III. Yeast Mating and

Screening for High- Affinity Combinations of Fibronectin-Based Binders

[0071] The heterologous nucleic acid as described above is introduced into a haploid yeast cell. A separate heterologous nucleic acid is separately introduced into a different haploid yeast cell of a different mating type. Each nucleic acid expresses a member of a library of fibronectin-based binders which bind to the same target or epitope. The haploid yeast cells are mated, producing diploid yeast cells which express both fibronectin-based binders. The diploid cells are screened for high-affinity combinations of fibronectin-based binders.

[0072] By "yeast" is meant a eukaryotic micro-organism classified in the kingdom Fungi, including about 1,500 known species. For example, the term "yeast" includes various strains of S. cerevisiae and S. pombe. Yeasts suitable for the present invention include those which are amenable to molecular biology techniques, and capable of maintaining vectors, expressing the proteins encoded therein, displaying such proteins on their surfaces, and capable of mating with similarly capable yeasts.

[0073] Each yeast cell expresses a member of a library of fibronectin-based binders. The haploid cells are then mated. The first cell comprises vector expressing a first fibronectin-based binder and has a first mating type, e.g., MAT-a; the second cell expressed a second fibronectin-based binder and has a second mating type different from the first, e.g., MAT-alpha. The two cells are contacted with one another, and yeast mating produces a single diploid cell (e.g., MAT-a/alpha.) with a nucleus formed by the fusion of the respective genomes of both the first and second cells. The fusion event brings the fibronectin-based binder-encoding nucleic acid of the first cell and that of the second cell into the same nucleus and provides them with an opportunity for simultaneous expression. Simultaneous expression allows both fibronectin-based binders to be expressed and displayed on a single yeast cell. This is diagrammed schematically in Figure 2, in which fibronectin-based binder is abbreviated as "Fn".

[0074] The population of diploid yeast cells, each expressing two fibronectin-based binders, is screened for a cell expressing a high-affinity combination of fibronectin-based binder.

[0075] Screening involves the use of the appropriate target molecule. The selection of a proper target depends on the binding characteristics of the original fibronectin-based binders. In one embodiment of the invention, a target protein ("A") comprises two non- overlapping epitopes, "Al" and "A2." One set of haploid cells can express a fibronectin which binds to epitope Al , while the other set of haploid cells can express a fibronectin which binds to epitope A2. These can be mated to produce diploid cells. The diploid cells can be screened using a target protein A (which comprises both Al and A2). This results in the selection of a high-affinity combination of fibronectin-based binders, wherein one binder binds to Al and one binds to A2.

[0076] In another embodiment, the method of the invention can be used to create chains or complexes of fibronectin-based binders which all bind to the same target. For example, a target protein ("B") can comprise, for example, four epitopes, "B l", "B2", "B3," and "B4." A linked chain or complex or fibronectin-based binders can be created, wherein each member of the chain binds to a different epitope (Bl or B2 or B3 or B4). For example, a population of haploid cells expressing binders which bind to B 1 can be mated to haploid cells expressing binders which bind to B2. This mating produces diploid cells, which can be screened using a target comprising Bl and B2. This produces a high-affinity combination of fibronectin-based binders binding to both Bl and B2. Separately, a population of haploid cells expressing binders which bind to B3 can be mated a population of haploid cells expressing binders which bind to B4. This mating produces a population of diploid cells, which can be screened with a target comprising B3 and B4. This produces a high-affinity combination of fibronectin-based binders which bind to both B3 and B4. The two high-affinity combinations can be combined, producing a set of fibronectin-based binders which bind to multiple epitopes (Bl , B2, B3 and B4) within the same target. These various binders can be co-expressed or conjugated or otherwise linked into a chain or complex. Because this chain comprises multiple binding sites, the chain can bind to the target very tightly.

[0077] In a different embodiment, a population of haploid cells can be prepared, each cell expressing a member of a library of fibronectin-based binders. A separate population of haploid cells can also be prepared, each cell expressing a different member of a library of fibronectin- based binders. The haploid cells are mated to produce a population of diploid cells, each expressing two fibronectin-based binders. In this embodiment, rather than screening for a single cell expressing a high-affinity combination of binders, a group of diploid cells is selected. This group of cells represents a "high-affinity combination library." The binders or combinations or binders in this population can be re-sorted using an additional step of mating. This will yield a cell or group of cells expressing an even higher affinity combination of fibronectin-based binders (a "super-high-affinity combination of fibronectin-based binders"). This process of identifying high-affinity combinations and repeating the mating step can be repeated as many times as necessary to produce a cell(s) expressing a combination with the desired high level of affinity.

[0078] In one embodiment, an extra screening step is added to the method prior to mating the haploid cells. For example, a population of haploid cells expressing fibronectin-based binders which bind to a particular target can be created. This library can be screened with the target molecule to isolate a smaller library, sub-populations of cells or individual cells which tightly bind the target. These libraries, sub-populations or individual haploid cells can then be used in mating with other haploid cells to produce diploid cells, as described above, to screen for high- affinity combinations of fibronectin-based binders.

[0079] After diploid cells expressing high-affinity combinations of fibronectin-based binders are identified, plasmids encoding the fibronectin-based binders are isolated from the cells. The plasmids are digested with restriction enzymes that cleave uniquely on either side of a region that includes the coding segment for the fibronectin-based binder.

[0080] Various methods known in the art are suitable for screening the diploid cells for high- affinity combinations of fibronectin-based binders. Generally, high-affinity combinations of fibronectin-based binders can be identified from a cell-display library by one or more cycles of selection. Some exemplary selection processes are as follows. These methods include, but are not limited to, FACS, panning, magnetic particle processing, using a capillary device, ELISA, flow cytometry, and any other method known in the art.

[0081] FACS. It is also possible to use fluorescent cell sorting to sort cells from a cell-display library. The cells can be contacted with a target that is fluorescently labeled. Cells that interact with the target can be detected by the FACS sorter and deflected into a container. Further, the cells can be contacted with an unlabeled target to form cell-target complexes. The cell-target complexes can then be labeled, e.g., using a fluorescent reagent that is specific for the target.

[0082] Panning. The target molecule is immobilized to a solid support such as a surface of a microtitre well, matrix, particle, or bead. The cell-display library is contacted to the support. Library members that have affinity for the target are allowed to bind. Non-specifically or weakly- bound members are washed from the support. Then the bound library members are recovered (e.g., by elution) from the support. Recovered library members are collected for further analysis (e.g., sequencing, purification, additional screening, etc.) or pooled for an additional round of selection.

[0083] Magnetic Particle Processor. One example of an automated selection uses magnetic particles and a magnetic particle processor. In this case, the target is immobilized on the magnetic particles, e.g., as described below. The KingFisher™ system, a magnetic particle processor from Thermo LabSystems (Helsinki, Finland), is used to select cell-display library members against the target. The cell-display library is contacted to the magnetic particles in a tube. The beads and library are mixed. Then a magnetic pin, covered by a disposable sheath, retrieves the magnetic particles and transfers them to another tube that includes a wash solution. The particles are mixed with the wash solution. In this manner, the magnetic particle processor can be used to serially transfer the magnetic particles to multiple tubes to wash non-specifically or weakly-bound library members from the particles. After washing, the particles are transferred to a tube that includes an elution buffer to release specifically and/or strongly bound library members from the particles. These eluted library members can be individually isolated for analysis (e.g., sequencing, purification, additional screening, etc.) or pooled for an additional round of selection.

[0084] Capillary Device for Washing Magnetic Beads. U.S. S. No. 60/337,755 describes an apparatus and methods that can, in one implementation, be used to wash magnetic particles in a capillary tube. On exemplary apparatus features a capillary that houses magnetic particles. The chamber is located between a first magnet and a second magnet. The magnets and are attached to a frame that can be actuated from a first position to a second position. When the frame is actuated, the magnetic particles in the capillary are agitated. To use the Capillary Devise for cell-display library screening, cell-display library members are contacted to magnetic particles that have an attached target. The particles are disposed in the capillary (before, during, or after the contacting). Then, the particles are washed in the capillary with cycles of agitation and liquid flow to remove non-specifically or weakly-bound library members. After washing, bound library members can be eluted or dissociated from the particles and recovered. Cells can also be grown in the device, e.g., during a selection, to amplify bound cells.

[0085] A screen for binding high-affinity combinations of fibronectin-based binders can include measures to identify fibronectin-based binders that bind to a particular epitope of a target or that have a desired specificity. This can be done, for example, by using competing non-target molecules that lack the desired epitope or are mutated within the epitope, e.g., with alanine. Such non-target molecules can be used in a negative selection procedure as described below, as competing molecules when binding a cell-display library to the target, or as a pre-elution agent, e.g., to capture in a wash solution dissociating cell-display library members that are not specific to the target.

[0086] Iterative Selection. In one embodiment, cell-display library technology is used in an iterative mode. A first cell-display library is used to identify a high-affinity combination of fibronectin-based binders for a target. These identified fibronectin-based binders are then varied by recombination to form a second cell-display library. Higher affinity fibronectin-based binders are then selected from the second library, e.g., by using higher stringency or more competitive binding and washing conditions.

In one example of iterative selection, the methods described herein are used to first identify a fibronectin-based binder from a cell-display library that binds a target with at least a minimal binding specificity for a target or a minimal activity, e.g., an equilibrium dissociation constant for binding of greater than 1 nM, 10 nM, or 100 nM. The nucleic acid sequence encoding the initial identified fibronectin-based binder are used as a template nucleic acid for the introduction of variations, e.g., to identify a second fibronectin-based binder that has enhanced properties (e.g., binding affinity, kinetics, or stability) relative to the initial fibronectin-based binder.

[0087] Off-Rate Selection. Since a slow dissociation rate can be predictive of high-affinity, particularly with respect to interactions between fibronectin-based binders and their targets, the methods described herein can be used to isolate combinations of fibronectin-based binders with a desired kinetic dissociation rate (i.e. reduced) for a binding interaction to a target.

[0088] To select for slow-dissociating combinations of fibronectin-based binders from a cell- display library, the library is contacted to an immobilized target. The immobilized target is then washed with a first solution that removes non-specifically or weakly-bound biomolecules. Then the immobilized target is eluted with a second solution that includes a saturation amount of free target, i.e., replicates of the target that are not attached to the particle. The free target binds to biomolecules that dissociate from the target. Rebinding is effectively prevented by the saturating amount of free target relative to the much lower concentration of immobilized target.

[0089] The second solution can have solution conditions that are substantially physiological or that are stringent. Typically, the solution conditions of the second solution are identical to the solution conditions of the first solution. Fractions of the second solution are collected in temporal order to distinguish early from late fractions. Later fractions include fibronectin-based binders that dissociate at a slower rate from the target than fibronectin-based binders in the early fractions.

[0090] Further, it is also possible to recover combinations of cell-display library members that remain bound to the target even after extended incubation. These can dissociated, e.g., by cleaving the target (along with the cell) from the support. Alternatively, the target and the associated cell can be incubated under conditions that encourage cell division. Eventually the cells will outnumber the available targets; cells not bound to a target, but expressing the desired fibronectin- based binder will arise.

[0091] Selecting or Screening for Specificity. The cell-display library screening methods described herein can include a selection or screening process that discards cell-display library members that bind to a non-target molecule. In one implementation, a so-called "negative selection" step is used to discriminate between the target and a related, but distinct, non-target molecule. The cell-display library or a pool thereof is contacted to the non-target molecule.

Members of the sample that do not bind the non-target are collected and used in subsequent selections for binding to the target molecule or even for subsequent negative selections. The negative selection step can be prior to or after selecting library members that bind to the target molecule. [0092] Screening Individual Library Members. After selecting candidate combinations of cell- display library members that bind to a target, each candidate combination can be further analyzed, e.g., to further characterize its binding properties for the target. Each candidate combination can be subjected to one or more secondary screening assays. The assay can be for a binding property, a catalytic property, a physiological property (e.g., cytotoxicity, renal clearance, immunogenicity), a structural property (e.g., stability, conformation, oligomerization state) or another functional property. The same assay can be used repeatedly, but with varying conditions, e.g., to determine pH, ionic, or thermal sensitivities.

[0093] As appropriate, the assays can use the combinations of cell-display library member directly, a recombinant fibronectin-based binder produced from the nucleic acid encoding a displayed fibronectin-based binder, or a synthetic fibronectin-based binder synthesized based on the sequence of a displayed fibronectin-based binder. Exemplary assays for binding properties include the following.

[0094] ELISA. Combinations of fibronectin-based binders encoded by a cell-display library can also be screened for binding properties using an ELISA assay. For example, each diploid cell expressing a combination of fibronectin-based binders is contacted to a microtitre plate whose bottom surface has been coated with the target, e.g., a limiting amount of the target. The plate is washed with buffer to remove non-specifically bound cells. Then the amount of the combination of fibronectin-based binders bound to the plate is determined by probing the plate with an antibody that recognizes the fibronectin-based binder. The antibody is linked to an enzyme such as alkaline phosphatase, which produces a calorimetric product when appropriate substrates are provided. The combination of fibronectin-based binders can be purified from the cells or assayed in a cell-display library format, e.g., as a cell surface protein.

[0095] In another version of the ELISA assay, each combination of fibronectin-based binders is used to coat a different well of a microtitre plate. The ELISA then proceeds using a constant target molecule to query each well.

[0096] Homogeneous Binding Assays. The binding interaction of a candidate combination of fibronectin-based binders with a target can be analyzed using a homogenous assay; i.e., after all components of the assay are added, additional fluid manipulations are not required. For example, fluorescence resonance energy transfer (FRET) can be used as a homogenous assay (see, for example, Lakowicz et ah, U.S. Pat. No. 5,631 ,169; Stavrianopoulos et ah , U.S. Pat. No.

4,868,103). A fluorophore label on the fibronectin-based binder is selected such that its emitted fluorescent energy can be absorbed by a fluorescent label on a second molecule (e.g., the target) if the second molecule is in proximity to the first molecule. The fluorescent label on the second molecule fluoresces when it absorbs the transferred energy. Since the efficiency of energy transfer between the labels is related to the distance separating the molecules, the spatial relationship between the molecules can be assessed. In a situation in which binding occurs between the molecules, the fluorescent emission of the ^"acceptor^" molecule label in the assay should be maximal. A binding event that is configured for monitoring by FRET can be conveniently measured through standard fluorometric detection means well known in the art (e.g., using a fluorimeter). By titrating the amount of the first or second binding molecule, a binding curve can be generated to estimate the equilibrium binding constant.

[0097] Another example of a homogenous assay is Alpha Screen (Packard Bioscience, Meriden Conn.). Alpha Screen uses two labeled beads. One bead generates singlet oxygen when excited by a laser. The other bead generates a light signal when singlet oxygen diffuses from the first bead and collides with it. The signal is only generated when the two beads are in proximity. One bead can be attached to the cell-display library member, the other to the target. Signals are measured to determine the extent of binding.

[0098] The homogenous assays can be performed while the candidate fibronectin-based binder is attached to the cell surface of the displaying cell.

[0099] Cell Arrays. In still another embodiment, a cell-display library generated by

recombination is formatted as a cellular array. Individual cells of the library or small pools are manipulated onto a grid and cultivated. A labeled target can be contacted to the grid to identify library members that bind the target. The cellular array can likewise be screened for any appropriate detectable activity.

[00100] U.S. Ser. No. 10/309,391 describes, among other things, methods of automation which can be used in conjunction with a method described herein.

[00101 ] The various methods discussed herein, and others known in the art, can be used alone or in combination to screen cell-display libraries to generate high-affinity combinations of fibronectin-based binders. These combinations can be used to generate bi-specific and multi- specific fibronectin-based binders, and also variants, fusions and conjugates of fibronectin-based binders.

IV. Multi- and bi-specific high-affinity combinations

of fibronectin-based binder molecules [00102] The methods of the present invention can be used to create high-affinity combinations of fibronectin-based binders which are bi-specific or multi-specific. For example, a combination can be produced wherein each constituent fibronectin-based binder in a combination binds to two targets (e.g., proteins "C" and "D"). In another non-limiting example, a combination is multi- specific. For example, a combination can comprise one binder which binds to two targets (e.g., proteins "E" and "F"), and another binder which binds to a different set of two targets (e.g., proteins F and "G"). Thus the combination binds to three total proteins, E, F and G. These are described in more detail below. [00103] In one embodiment, bi-specific high-affinity combinations of fibronectin-based binders can be produced. For example, an individual fibronectin-based binder comprises a set of top loops and a set of bottom loops. The top loops (or a subset thereof) can bind to one target (e.g., protein C), while the bottom loops bind to a separate target (e.g., protein D), producing a bi- specific binder. A library of bi-specific binders can be produced, wherein each binder binds strongly to protein C (e.g., each member of the library has the same set of top loops which bind to protein C). However, each member of the library has a different set of bottom loops which bind to protein D. Members of such libraries can be expressed in populations of haploid cells of two different mating types. These cells can be mated, and screened for binding to protein D. This results in a high-affinity combination of bi-specific fibronectin-based binders which bind with high-affinity to both targets, proteins C and D. Variants of the method of this invention can be performed to produce other high-affinity combinations of bi-specific fibronectin-based binders.

[00104] In another embodiment of this invention, high affinity, multi-specific combinations of fibronectin-based binders can be created. For example, a library of bi-specific fibronectin-based binders can be created which bind to, for example, proteins E and F. A separate library of bi- specific binders can be created which bind to proteins G and F. These libraries can be expressed in haploid cells of different mating types. The diploid cells can be screened (with proteins E, F and G) to produce multi-specific combinations of binders which bind to three separate targets (proteins E, F and G).

[00105] Bi-specific and multi-specific fibronectin-based binders comprise either a single fibronectin-based binder which binds to multiple targets (e.g., the top and bottom binders bind to different targets), or to combinations of (or fusions or conjugates comprising) two or more fibronectin-based binders which bind to more than one target.

[00106] Multi-specific Fn3-based (or fibronectin-based) binding molecules can be prepared by chemically joining individual Fn3 -based binding molecules using methods known in the art. A variety of coupling or cross-linking agents can be used for covalent conjugation. Examples of cross-linking agents include, e.g., protein A, carbodiimide, N-succinimidyl-S-acetyl-thioacetate (SATA), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), o-phenylenedimaleimide (oPDM), N- succinimidyl-3-(2-pyridyldithio)propionate (SPDP), and sulfosuccinimidyl 4-(N- maleimidomethyl) cyclohaxane-l-carboxylate (sulfo-SMCC) (see e.g., Karpovsky et al. (1984) J. Exp. Med. 160: 1686; Liu, MA et al. (1985) Proc. Natl. Acad. Sci. U.S.A 82: 8648). Other methods include those described in Paulus (1985) Behring Ins. Mitt. No. 78, 118-132; Brennan et al. (1985) Science 229: 81-83), and Glennie et al. (1987) J. Immunol. 139: 2367-2375). Preferred conjugating agents are SATA and sulfo-SMCC, both available from Pierce Chemical Co.

(Rockford, IL). Cysteine residues can be introduced into the Fn3 -binding molecules at specific positions and then crosslink with reagents to sulfhydryl such as DPDPB or DTME (available from Pierce) to link two molecules together. [00107] The Examples describe the identification and production of novel, bi-specific fibronectin- based binders (e.g., Fn3 molecules) that bind to two different targets, human serum albumin and VEGFR2, using two separate mono-specific Fn3 molecules that are linked together with a linker sequence. In this particular Example, one monomelic Fn3 -based binding molecule uses the bottom loops to bind to albumin while the other mono-specific Fn3 -based binding molecule uses the top loops to bind VEGFR2. Hundreds of clones have been generated using the methods of the invention and a representative few have characterized, in particular clone 89.

[00108] The skilled artisan will appreciate that any number of bi-specific and multi-specific Fn3- based binding molecules can be generated using the methods of the invention.

[00109] The high-affinity combinations of fibronectin-based binders produced by the present invention can be physically or genetically linked to produce fusions.

IV. Fusions of high-affinity combinations of fibronectin-based binders [00110] In one embodiment, the constituent fibronectin-based binders in a high-affinity combination can be fused together. The fusion can be produced by genetic or chemical linkage of two or more fibronectin-based binders. For example, the genes encoding the fibronectin-based binders can be operably linked and positioned in- frame to each other, so they are expressed together into a single fusion protein. This fusion protein can also optionally include other elements such as linking polypeptides and signal polypeptides. Alternatively, the fibronectins can be expressed and purified separately and then the proteins physically linked using any chemical linkage known in the art. Such chemical linkages are described in more detail below in the discussion of conjugates.

[00111] Fusions of fibronectin-based binders can be mono-specific (if all the components of the fusion bind to the same target or same epitope). Alternatively, the fusions of fibronectin-based binders can be bi-specific (wherein the fusion comprises two fibronectin-based binders which bind to different epitopes or targets), or multi-specific (wherein the fusion comprises two or more fibronectin-based binders which bind to two or more epitopes or targets).

[00112] In one embodiment, the method of the invention can be used to create chains of fibronectin-based binders which all bind to the same target. For example, as described in more detail above, a target protein ("B") can comprise, for example, four epitopes, "Bl", "B2", "B3," and "B4." A linked chain or complex or fibronectin-based binders can be created, wherein each member of the chain binds to a different epitope (Bl or B2 or B3 or B4). These various binders can be co-expressed or conjugated or otherwise linked into a chain or complex. Because this chain comprises multiple binding sites, the chain can bind to the target very tightly.

[00113] The constituent binders in a fusion can be chemically linked using a variety of coupling or cross-linking agents. Examples of cross-linking agents include protein A, carbodiimide, N- succinimidyl-S-acetyl-thioacetate (SATA), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), o- phenylenedimaleimide (oPDM), N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), and sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohaxane-l-carboxylate (sulfo-SMCC) (see e.g., Karpovsky et al. (1984) J. Exp. Med. 160: 1686; Liu, MA et al. (1985) Proc. Natl. Acad. Sci. U.S.A 82: 8648). Other methods include those described in Paulus (1985) Behring Ins. Mitt. No. 78, 118-132; Brennan et al. (1985) Science 229: 81-83), and Glennie et al. (1987) J. Immunol. 139: 2367-2375). Preferred conjugating agents are SATA and sulfo-SMCC, both available from Pierce Chemical Co. (Rockford, IL).

[00114] Alternatively, the constituent fibronectin-based binders can be encoded in the same vector and expressed as a single protein in a host cell. Methods for producing protein fusions are described, for example, in U.S. Patent Number 5,260,203; U.S. Patent Number 5,455,030; U.S.

Patent Number 4,881,175; U.S. Patent Number 5,132,405; U.S. Patent Number 5,091,513; U.S.

Patent Number 5,476,786; U.S. Patent Number 5,013,653; U.S. Patent Number 5,258,498; and

U.S. Patent Number 5,482,858. Other methods of producing fusions are known in the art.

[00115] The fusions comprising two or more fibronectin-based binders can be combined with non- fibronectin-based binder molecules to form conjugates.

V. Conjugates of High- Affinity Combinations of Fibronectin-Based Binders [00116] Another aspect the invention provides conjugates comprising a high-affinity combination of Fn3 -based binding molecules linked to one or more non-Fn3 moieties (moieties which are not Fn3 or fibronectin-based binders). Exemplary Fn3-based binding molecule conjugates of the present invention include an Fn3 -based binding molecule linked to a non-Fn3 -based binding molecule, e.g., another peptide or protein {e.g., an antibody or ligand for a receptor), to generate a molecule that binds to at least two different binding sites or target molecules.

[00117] Such non-Fn3 moieties can, for example, impart additional functional or physiochemical properties to the Fn3 -based binding molecule. In one embodiment, the Fn3 -based binding molecule is linked or conjugated to an antibody Fc domain (or a portion thereof). Similar methods for conjugating molecules to Fc domains are known in the art (see, e.g., U.S. 5,428,130).

[00118] In another embodiment, one or more Fn3-based binding molecules is conjugated to one or more human serum albumin (HSA) polypeptides (or a portion thereof). Human serum albumin, a protein of 585 amino acids in its mature form, is responsible for a significant proportion of the osmotic pressure of serum and also functions as a carrier of endogenous and exogenous ligands. The role of albumin as a carrier molecule and its inert nature are desirable properties for use as a carrier and transporter of polypeptides in vivo. Thus, an exemplary conjugate can be constructed comprising a fibronectin-based binder specific for a particular target (for example, a cancer cell, for example, a liver cancer cell), conjugated to a human serum albumin. The albumin can be loaded with a cancer drug, and the conjugate can deliver the drug to the liver cell. Alternatively, the albumin moiety in a conjugate can serve to increase stability and half-life in the blood, rather aid in drug delivery. In some embodiments of the present invention, the albumin moiety in a conjugate both aids in drug binding and delivery and increases serum half-life.

[00119] The use of albumin as a carrier in a protein conjugate with various proteins has been suggested in WO 93/15199, WO 93/15200, and EP 413 622. The use of N-terminal fragments of albumin for conjugation to polypeptides has also been proposed (EP 399 666). Accordingly, by genetically or chemically conjugating the molecules of the present invention to albumin, or a fragment (portion) or variant of albumin or a molecule capable of binding human serum albumin (an "anti-human serum albumin binder") that is sufficient to stabilize the protein and/or its activity, the molecule is stabilized to extend the shelf-life, and/or to retain the molecule's activity for extended periods of time in solution, in vitro and/or in vivo.

[00120] Conjugation of albumin to another protein may be achieved by genetic manipulation, such that the DNA coding for albumin, or a fragment thereof, is joined to the DNA coding for the other protein. A suitable host is then transformed or transfected with the conjugated nucleotide sequences, so arranged on a suitable plasmid to express a polypeptide conjugate. The expression may be effected in vitro from, for example, prokaryotic or eukaryotic cells, or in vivo e.g. from a transgenic organism. Additional methods pertaining to albumin conjugates can be found, for example, in WO 2001077137 and WO 200306007, incorporated herein by reference. In a specific embodiment, the expression of the protein conjugate is performed in mammalian cell lines, for example, CHO cell lines.

[00121] The Fn3 -based binding molecule conjugates of the present invention can be prepared by linking the constituent molecules using methods known in the art. For example, the constituent molecules can be chemically linked using a variety of coupling or cross-linking agents. Examples of cross-linking agents include protein A, carbodiimide, N-succinimidyl-S-acetyl-thioacetate (SATA), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), o-phenylenedimaleimide (oPDM), N- succinimidyl-3-(2-pyridyldithio)propionate (SPDP), and sulfosuccinimidyl 4-(N- maleimidomethyl) cyclohaxane-l-carboxylate (sulfo-SMCC) (see e.g., Karpovsky et al. (1984) J. Exp. Med. 160: 1686; Liu, MA et al. (1985) Proc. Natl. Acad. Sci. U.S.A 82: 8648). Other methods include those described in Paulus (1985) Behring Ins. Mitt. No. 78, 118-132; Brennan et al. (1985) Science 229: 81-83), and Glennie et al. (1987) J. Immunol. 139: 2367-2375). Preferred conjugating agents are SATA and sulfo-SMCC, both available from Pierce Chemical Co.

(Rockford, IL).

[00122] Alternatively, the constituent molecules can be encoded in the same vector and expressed as a single protein in a host cell. Methods for producing such protein conjugates are described, for example, in U.S. Patent Number 5,260,203; U.S. Patent Number 5,455,030; U.S. Patent Number 4,881,175; U.S. Patent Number 5,132,405; U.S. Patent Number 5,091,513; U.S. Patent Number 5,476,786; U.S. Patent Number 5,013,653; U.S. Patent Number 5,258,498; and U.S. Patent Number 5,482,858.

[00123] In yet another embodiment, the invention provides Fn3 -based binding molecules that are conjugated to polyethylene glycol (PEG), for example, to increase the biological (e.g., serum) half-life of the molecule. Methods for PEGylating proteins are well known in the art. For example, the Fn3-based binding molecule can be reacted with a PEG moiety, such as a reactive ester or aldehyde derivative of PEG, under conditions in which one or more PEG groups become attached to the molecule.

[00124] The terms "PEGylation moiety", "polyethylene glycol moiety", "PEG moiety" and the like include a polyalkylene glycol compound or a derivative thereof, with or without coupling agents or derivatization or activating moieties (e.g., thiol, triflate, tresylate, azirdine, oxirane, or preferably with a maleimide moiety, e.g., PEG-maleimide). Other appropriate polyalkylene glycol compounds include, but are not limited to, maleimido monomethoxy PEG, activated PEG polypropylene glycol, but also charged or neutral polymers of the following types: dextran, colominic acids, or other carbohydrate based polymers, polymers of amino acids, and biotin derivatives.

[00125] The choice of the suitable functional group for the PEG derivative is based on the type of available reactive group on the fibronectin-based binder. Typical reactive amino acids include lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine, tyrosine. The N-terminal amino group and the C-terminal carboxylic acid can also be used.

[00126] Preferably, the pegylation is carried out via an acylation reaction or an alkylation reaction with a reactive PEG molecule (or an analogous reactive water-soluble polymer). As used herein, the term "polyethylene glycol" is intended to encompass any of the forms of PEG that have been used to derivatize other proteins, such as mono (CI -CIO) alkoxy- or aryloxy-poly ethylene glycol or polyethylene glycol-maleimide. Methods for pegylating proteins are known in the art and can be applied to the present invention. See for example, WO 2005056764, U.S. Patents 7,045,337, U.S.7,083,970, and U.S.6,927,042, and EP 0 154 316 by Nishimura et al._L and EP 0 401 384 by Ishikawa et al. Fn3 -based binding molecules can be engineered to include at least one cysteine amino acid or at least one non-natural amino acid to facilitate pegylation.

[00127] Binding of the Fn3 -based binding molecule conjugates to their specific targets can be confirmed by various assays. For example, the conjugate can be radioactively labeled and used in a radioimmunoassay (RIA) (see, for example, Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques, The Endocrine Society, March, 1986, which is incorporated by reference herein). The radioactive isotope can be detected by such means as the use of a γ-counter or a scintillation counter or by autoradiography.

[00128] Other Fn3-based binding molecule or fibronectin-based binder conjugates of the present invention include an Fn3-based binding molecule linked to a tag (e.g., biotin) or a chemical (e.g., an immunotoxin or chemotherapeutic agent). Such chemicals include a cytotoxic agent which is any agent that is detrimental to (e.g., kills or prevents the growth of) cells. Examples include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1 -dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, and analogs or homologs thereof. Therapeutic agents also include, for example, anti-metabolites (e.g., methotrexate, 6- mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis- dichlorodiamine platinum (II) (DDP) cisp latin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g., vincristine and vinblastine). Other examples of therapeutic cytotoxins that can be conjugated to Fn3-based binding molecule of the invention include duocarmycins, calicheamicins, maytansines and auristatins, and derivatives thereof.

[00129] Cytotoxins can be conjugated to the Fn3 -based binding molecules of the invention using any linker technology available in the art. Linkers can be sized and flexible enough to allow two linked fibronectin-based binders to bind to the same target. Preferably, the linker is designed so that the adjacent fibronectin-based binder and non- fibronectin-based binder moieties can each still retain their normal functions. Examples of linker types that have been used to conjugate a cytotoxin include, but are not limited to, hydrazones, thioethers, esters, disulfides and peptide- containing linkers. A linker can be chosen that is, for example, susceptible to cleavage by low pH within the lysosomal compartment or susceptible to cleavage by proteases, such as proteases preferentially expressed in tumor tissue such as cathepsins (e.g., cathepsins B, C, D). For further discussion of types of cytotoxins, linkers and methods for conjugating therapeutic agents, see also Saito, G. et al. (2003) Adv. Drug Deliv. Rev. 55: 199-215; Trail, P.A. et al. (2003) Cancer Immunol. Immunother. 52: 328-337; Payne, G. (2003) Cancer Cell 3: 207-212; Allen, T.M. (2002) Nat. Rev. Cancer 2: 750-763; Pastan, I. and Kreitman, R. J. (2002) Curr. Opin. Investig. Drugs 3 : 1089- 1091 ; and Senter, P.D. and Springer, C.J. (2001 ) Adv. Drug Deliv. Rev. 53 : 247- 264.

[00130] Fn3 -based binding molecules of the present invention also can be conjugated to a radioactive isotope to generate cytotoxic radiopharmaceuticals, also referred to as

radioconjugates. Examples of radioactive isotopes that can be conjugated to Fn3-based binding molecules for use diagnostically or therapeutically include, but are not limited to, iodine¹³¹, indium¹¹¹, yttrium⁹⁰ and lutetium¹⁷⁷. Methods for preparing radioimmunoconjugates are established in the art. Examples of radioimmunoconjugates are commercially available, including ibritumomab, tiuxetan, and tositumomab, and similar methods can be used to prepare

radioconjugates using the fibronectin-based binders of the invention.

[00131] The Fn3 -based binding molecule conjugates of the invention can be used to modify a given biological response, and the drug moiety is not to be construed as limited to classical chemical therapeutic agents. For example, the drug moiety may be a protein or polypeptide possessing a desired biological activity. Such proteins may include, for example, an

enzymatically active toxin, or active fragment thereof, such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor or interferon-γ; or, biological response modifiers such as, for example, lymphokines, interleukin-1 ("IL-1 "), interleukin-2 ("IL- 2"), interleukin-6 ("IL-6"), granulocyte macrophage colony stimulating factor ("GM-CSF"), granulocyte colony stimulating factor ("G-CSF"), or other growth factors.

[00132] Techniques for conjugating such therapeutic moiety are well known and can be applied to the molecules of the present invention, see, e.g., Arnon et al, "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al, "Antibodies For Drug Delivery", in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review", in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); "Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy", in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al, "The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates", Immunol. Rev., 62: 119- 58 (1982).

[00133] Fn3 -based binding molecules of the present invention also can be modified by hesylation, which utilizes hydroxyethyl starch ("HES") derivatives linked to drug substances in order to modify the drug characteristics. HES is a modified natural polymer derived from waxy maize starch which is metabolized by the body's enzymes. This modification enables the prolongation of the circulation half-life by increasing the stability of the molecule, as well as by reducing renal clearance, resulting in an increased biological activity. Furthermore, HESylation potentially alters the immunogenicity or allergenicity. By varying different parameters, such as the molecular weight of HES, a wide range of HES drug conjugates can be customized.

[00134] DE 196 28 705 and DE 101 29 369 describe possible methods for carrying out the coupling of hydroxyethyl starch in anhydrous dimethyl sulfoxide (DMSO) via the corresponding aldonolactone of hydroxyethyl starch with free amino groups of hemoglobin and amphotericin B, respectively. Since it is often not possible to use anhydrous, aprotic solvents specifically in the case of proteins, either for solubility reasons or else on the grounds of denaturation of the proteins, coupling methods with HES in an aqueous medium are also available. For example, coupling of hydroxy ethyl starch which has been selectively oxidized at the reducing end of the chain to the aldonic acid is possible through the mediation of water-soluble carbodiimide EDC (1- ethyl-3-(3-dimethyl-aminopropyl)carbodiimide) (PCT/EP 02/02928). Additional hesylation methods which can be applied to the present invention are described, for example, in published U.S. Patent Applications 20070134197, U.S. 20060258607, U.S. 20060217293, U.S.

20060100176, and U.S.20060052342.

[00135] Fn3-based binding molecules of the invention also can be modified via sugar residues. Methods for modifying sugar residues of proteins or glycosylating proteins are known in the art (see, for example, Borman (2006) Chem. & Eng. News 84(36): 13-22, and Borman (2007) Chem. & Eng. News 85: 19-20) and can be applied to the molecules of the present invention.

[00136] Additionally or alternatively, Fn3 -based binding molecules of the invention can be made that have an altered type of glycosylation, such as a hypofucosylated pattern having reduced amounts of fucosyl residues, or increased bisecting GlcNac structures. Such carbohydrate modifications can be accomplished by, for example, expressing the Fn3 -based binding molecule in a host cell with altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used to produce Fn3 -based binding molecules with altered glycosylation. For example, EP 1,176,195 by Hang et al describes a cell line with a functionally disrupted FUT8 gene, which encodes a fucosyl transferase, such that antibodies expressed in such a cell line exhibit hypofucosylation. PCT Publication WO 03/035835 by Presta describes a variant CHO cell line, Lecl3 cells, with reduced ability to attach fucose to Asn(297)- linked carbohydrates, also resulting in hypofucosylation of antibodies expressed in that host cell (see also Shields, R.L. et al, 2002 J. Biol. Chem. 277: 26733-26740). Methods to produce polypeptides with human- like glycosylation patterns have also been described by EP1297172B1 and other patent families originating from Glycofi.

[00137] In various embodiments, complex molecules can be constructed comprising one or more fibronectin-based binder (including a fusion), conjugated to one or more non-fibronectin based binder (e.g., one or more PEG, albumin, radiolabel, antibody, biological response modifier, hydroxyethyl starch, and/or any other component known in the art). Any and all of the techniques and materials described herein can be used, alone or in combination, or substituted by or in combination with any method or material known in the art, to produce variants, fusions and conjugates of fibronectin-based binders.

VI. Compositions of High- Affinity Combinations of Fibronectin-Based Binders [00138] The high-affinity combinations of Fn3-based binders of the present invention have in vivo therapeutic utilities. Accordingly, the present invention also provides compositions, e.g., a pharmaceutical composition, containing a combination of Fn3 -based binding molecules (or variants, fusions, and conjugates thereof), formulated together with a pharmaceutically acceptable carrier. Pharmaceutical compositions of the invention also can be administered in combination therapy, i.e. , combined with other agents. For example, the combination therapy can include a composition of the present invention with at least one or more additional therapeutic agents, such as anti-inflammatory agents, anti-cancer agents, and chemotherapeutic agents.

[00139] The pharmaceutical compositions of the invention can also be administered in conjunction with radiation therapy. Co-administration with other Fn3 -based molecules are also encompassed by the invention.

[00140] As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, that are physiologically compatible. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration {e.g. , by injection or infusion). Depending on the route of administration, the active compound, i.e., antibody, bi-specific and multi-specific molecule, may be coated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.

[00141 ] A "pharmaceutically acceptable salt" refers to a salt that retains the desired biological activity of the parent compound and does not impart any undesired toxicological effects (see e.g. , Berge, S.M., et al. (1977) J. Pharm. Sci. 66: 1 -19). Examples of such salts include acid addition salts and base addition salts. Acid addition salts include those derived from nontoxic inorganic acids, such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic, phosphorous and the like, as well as from nontoxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids and the like. Base addition salts include those derived from alkaline earth metals, such as sodium, potassium, magnesium, calcium and the like, as well as from nontoxic organic amines, such as Ν,Ν'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine and the like.

[00142] A high-affinity combination of fibronectin-based binders of the present invention can be administered by a variety of methods known in the art. As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending upon the desired results. The active compounds can be prepared with carriers that will protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are patented or generally known to those skilled in the art. See, e.g., Sustained and Controlled Release Drug Delivery Systems, J.R. Robinson, ed., Marcel Dekker, Inc., New York, 1978. [00143] To administer a high-affinity combination of fibronectin-based binders by certain routes of administration, it may be necessary to coat the compound with, or co-administer the compound with, a material to prevent its inactivation. For example, the compound may be administered to a subject in an appropriate carrier, for example, liposomes, or a diluent. Pharmaceutically acceptable diluents include saline and aqueous buffer solutions. Liposomes include water-in-oil- in- water CGF emulsions as well as conventional liposomes (Strejan et al. (1984) J.

Neuroimmunol. 7: 27).

[00144] Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is known in the art.

Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the pharmaceutical compositions of the invention is contemplated. Supplementary active compounds can also be incorporated into the compositions.

[00145] Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.

[00146] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterilization microfiltration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying (lyophilization) that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[00147] Dosage regimens are adjusted to provide the optimum desired response {e.g. , a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. For example, the high-affinity combination of fibronectin-based binders may be administered once or twice weekly by subcutaneous injection or once or twice monthly by subcutaneous injection.

[00148] It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals.

[00149] Examples of pharmaceutically-acceptable antioxidants include: (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium

metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha- tocopherol, and the like; and (3) metal-chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

[00150] For the therapeutic compositions, formulations of the present invention include those suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal and/or parenteral administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any methods known in the art of pharmacy. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the subject being treated, and the particular mode of administration. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the composition which produces a therapeutic effect. Generally, out of one hundred per cent, this amount will range from about 0.001 per cent to about ninety percent of active ingredient, preferably from about 0.005 per cent to about 70 per cent, most preferably from about 0.01 per cent to about 30 per cent.

[00151] Formulations of the present invention which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such carriers as are known in the art to be appropriate. Dosage forms for the topical or transdermal administration of compositions of this invention include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The active compound may be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants which may be required.

[00152] The phrases "parenteral administration" and "administered parenterally" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion.

[00153] Examples of suitable aqueous and nonaqueous carriers which may be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[00154] These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of presence of microorganisms may be ensured both by sterilization procedures, supra, and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.

[00155] When the high-affinity combinations of fibronectin-based binders are administered as pharmaceuticals, to humans and animals, they can be given alone or as a pharmaceutical composition containing, for example, 0.001 to 90% (more preferably, 0.005 to 70%, such as 0.01 to 30%)) of active ingredient in combination with a pharmaceutically acceptable carrier.

[00156] Regardless of the route of administration selected, the high-affinity combinations of fibronectin-based binders, which may be used in a suitable hydrated form, and/or the

pharmaceutical compositions of the present invention, are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those of skill in the art.

[00157] Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present invention employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts. A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the compounds of the invention employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. In general, a suitable daily dose of a composition of the invention will be that amount of the compound which is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above. It is preferred that administration be intravenous, intramuscular, intraperitoneal, or subcutaneous, preferably administered proximal to the site of the target. If desired, the effective daily dose of therapeutic compositions may be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms. While it is possible for a compound of the present invention to be administered alone, it is preferable to administer the compound as a

pharmaceutical formulation (composition).

[00158] Therapeutic compositions comprising high-affinity combinations of fibronectin-based binders can be administered with medical devices known in the art. For example, in a preferred embodiment, a therapeutic composition of the invention can be administered with a needleless hypodermic injection device, such as the devices disclosed in U.S. Patent Nos. 5,399,163, 5,383,851, 5,312,335, 5,064,413, 4,941,880, 4,790,824, or 4,596,556. Examples of well-known implants and modules useful in the present invention include: U.S. Patent No. 4,487,603, which discloses an implantable micro-infusion pump for dispensing medication at a controlled rate; U.S. Patent No. 4.,486,194, which discloses a therapeutic device for administering medicants through the skin; U.S. Patent No. 4,447,233, which discloses a medication infusion pump for delivering medication at a precise infusion rate; U.S. Patent No. 4,447,224, which discloses a variable flow implantable infusion apparatus for continuous drug delivery; U.S. Patent

No. 4,439,196, which discloses an osmotic drug delivery system having multi-chamber compartments; and U.S. Patent No. 4,475,196, which discloses an osmotic drug delivery system. Many other such implants, delivery systems, and modules are known to those skilled in the art.

[00159] In certain embodiments, the molecules of the invention can be formulated to ensure proper distribution in vivo. For example, the blood-brain barrier (BBB) excludes many highly hydrophilic compounds. To ensure that the therapeutic compounds of the invention cross the BBB (if desired), they can be formulated, for example, in liposomes. For methods of

manufacturing liposomes, see, e.g., U.S. Patents 4,522,811 ; 5,374,548; and 5,399,331. The liposomes may comprise one or more moieties which are selectively transported into specific cells or organs, thus enhance targeted drug delivery {see, e.g., V.V. Ranade (1989) J. Clin. Pharmacol. 29: 685). Exemplary targeting moieties include folate or biotin (see, e.g., U.S. Patent 5,416,016 to Low et al); mannosides (Umezawa et al, (1988) Biochem. Biophys. Res. Commun. 153: 1038); antibodies (P.G. Bloeman et al. (1995) FEBS Lett. 357: 140; M. Owais et al. (1995) Antimicrob. Agents Chemother. 39: 180); surfactant protein A receptor (Briscoe et al. (1995) Am. J. Physiol. 1233: 134), different species of which may comprise the formulations of the inventions, as well as components of the invented molecules; and pl20 (Schreier et al. (1994) J. Biol. Chem. 269:

9090); see also K. Keinanen; M.L. Laukkanen (1994) FEBS Lett. 346: 123; J.J. Killion; I.J. Fidler (1994) Immunomethods 4: 273. In one embodiment of the invention, the therapeutic compounds of the invention are formulated in liposomes; in a more preferred embodiment, the liposomes include a targeting moiety. In a most preferred embodiment, the therapeutic compounds in the liposomes are delivered by bolus injection to a site proximal to the tumor or infection. The composition must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.

[00160] In a further embodiment, the combinations of the molecules of the invention can be formulated to prevent or reduce the transport across the placenta. This can be done by methods known in the art, e.g., by PEGylation of the Fn3-based binding molecule. Further references can be made to "Cunningham-Rundles C, Zhuo Z, Griffith B, Keenan J. (1992) Biological activities of polyethylene-glycol immunoglobulin conjugates. Resistance to enzymatic degradation. J Immunol Methods. 152: 177-190; and to "Landor M. (1995) Maternal-fetal transfer of immunoglobulins, Ann Allergy Asthma Immunol 74: 279-283. This is particularly relevant when the Fn3-based binding molecules are used for treating or preventing recurrent spontaneous abortion.

[00161] The ability of a compound to inhibit cancer and the approximate suitable dosage can be evaluated in an animal model system predictive of efficacy in human tumors. Alternatively, this property of a composition can be evaluated by examining the ability of the compound to inhibit, such inhibition in vitro by assays known to the skilled practitioner. A therapeutically effective amount of a therapeutic compound can decrease tumor size, or otherwise ameliorate symptoms in a subject. One of ordinary skill in the art would be able to determine such amounts based on such factors as the subject's size, the severity of the subject's symptoms, and the particular composition or route of administration selected.

[00162] The composition must be sterile and fluid to the extent that the composition is deliverable by syringe. In addition to water, the carrier can be an isotonic buffered saline solution, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by use of coating such as lecithin, by maintenance of required particle size in the case of dispersion and by use of surfactants. In many cases, it is preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol or sorbitol, and sodium chloride in the composition. Long-term absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate or gelatin. [00163] When the active compound is suitably protected, as described above, the compound may be orally administered, for example, with an inert diluent or an assimilable edible carrier.

VIII. Therapeutic and Diagnostic Applications of

High- Affinity Combinations of Fibronectin-Based Binders

[00164] The combinations of Fn3 -based binding molecules described herein may be constructed to bind any antigen or target of interest. Such targets include, but are not limited to, cluster domains, cell receptors, cell receptor ligands, growth factors, interleukins, protein allergens, bacteria, and/or viruses (see, Figure 1). The Fn3-based binding molecules described herein may also be modified to have increased stability and half-life, as well as additional functional moieties. Accordingly, these molecules may be employed in place of antibodies in all areas in which antibodies are used, including in the research, therapeutic, and diagnostic fields. In addition, because these molecules possess solubility and stability properties superior to antibodies, the antibody mimics described herein may also be used under conditions which would destroy or inactivate antibody molecules.

[00165] For example, these molecules can be administered to cells in culture, e.g. in vitro or ex vivo, or in a subject, e.g., in vivo, to treat, prevent or diagnose a variety of disorders. The term "subject" as used herein in intended to includes human and non-human animals. Non-human animals include all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cats, cows, horses, chickens, amphibians, and reptiles. When the Fn3 molecules are administered together with another agent, the two can be administered in either order or simultaneously.

[00166] In one embodiment, the combinations of Fn3 -based binding molecules (and variants, fusions, and conjugates thereof) of the invention can be used to detect levels of the target bound by the molecule and/or the targets bound by a bi-specific/multi-specific Fn3 -based binding molecule. This can be achieved, for example, by contacting a sample (such as an in vitro sample) and a control sample with the molecule under conditions that allow for the formation of a complex between the fibronectin-based binder and the target(s). Any complexes formed between the fibronectin-based binder and the target(s) are detected and compared in the sample and the control. For example, standard detection methods, well-known in the art, such as ELISA, FACS, and flow cytometric assays, can be performed using the compositions of the invention.

[00167] Also within the scope of the invention are kits comprising the compositions {e.g. , combinations of Fn3 -based binding molecules, variants, fusions, and conjugates thereof) of the invention and instructions for use. The kit can further contain a least one additional reagent, or one or more additional Fn3-based binding molecules of the invention {e.g. , an antibody having a complementary activity which binds to an epitope on the target antigen distinct from the first molecule). Kits typically include a label indicating the intended use of the contents of the kit. The term label includes any writing, or recorded material supplied on or with the kit, or which otherwise accompanies the kit.

[00168] As described above, the combinations of molecules of the present invention may be employed in all areas of the research, therapeutic, and diagnostic fields. Exemplary

diseases/disorders which can be treated using the Fn3 -based binding molecules of the present invention (and variants, fusions, and conjugates thereof) include autoimmune disorders, cancers, infections, and other pathogenic indications.

[00169] Specific examples of autoimmune conditions in which the Fn3 -based binding molecules of the invention can be used include, but are not limited to, the following: multiple sclerosis and other demyelinating diseases; rheumatoid arthritis; inflammatory bowel disease; systemic lupus erythematosus; Type I diabetes; inflammatory skin disorders; Sjogren's Syndrome; and transplant rejection.

[00170] Specific examples of cancers in which the Fn3 -based binding molecules of the invention can be used include, but are not limited to, the following: lung; breast; prostate; bladder;

melanoma; non-Hodgkin lymphoma; colon and rectal; pancreatic; endometrial; kidney; skin (non- melanoma); leukemia; and thyroid.

[00171 ] Specific examples of diseases associated with VEGF include for example, a number of conditions associated with inappropriate angiogenesis, including but not limited to autoimmune disorders (e.g., rheumatoid arthritis, inflammatory bowel disease or psoriasis); cardiac disorders (e. g., atherosclerosis or blood vessel restenosis); retinopathies (e.g., proliferative retinopathies generally, diabetic retinopathy, age-related macular degeneration or neovascular glaucoma), renal disease (e.g., diabetic nephropathy, malignant nephrosclerosis, thrombotic microangiopathy syndromes; transplant rejection; inflammatory renal disease; glomerulonephritis;

mesangioproliferative glomerulonephritis; haemolytic-uraemic syndrome; and hypertensive nephrosclerosis); hemangioblastoma; hemangiomas; thyroid hyperplasias; tissue transplantations; chronic inflammation; Meigs's syndrome; pericardial effusion; pleural effusion; autoimmune diseases; diabetes; endometriosis; chronic asthma; undesirable fibrosis (particularly hepatic fibrosis) and cancer, as well as complications arising from cancer, such as pleural effusion and ascites. The Fn3-based binding molecules can be used for the treatment of prevention of hyperproliferative diseases or cancer and the metastatic spread of cancers. Non-limiting examples of cancers include bladder, blood, bone, brain, breast, cartilage, colon kidney, liver, lung, lymph node, nervous tissue, ovary, pancreatic, prostate, skeletal muscle, skin, spinal cord, spleen, stomach, testes, thymus, thyroid, trachea, urogenital tract, ureter, urethra, uterus, or vaginal cancer. Additional treatable conditions can be found in U.S.P.N. 6,524, 583, herein incorporated by reference. Other references describing uses for VEGFR-2 binding polypeptides include:

McLeod DS et al , Invest Ophthalmol Vis Sci. 2002 Feb; 43 (2): 474-82; Watanabe et al Exp Dermatol. 2004 Nov; 13 (11): 671-81 ; Yoshiji H et al, Gut. 2003 Sep; 52 (9): 1347-54; Verheul et al, Oncologist. 2000; 5 Suppl 1 : 45-50; Boldicke et al, Stem Cells. 2001 ; 19 (1) : 24-36. As described herein, angiogenesis-associated diseases include, but are not limited to, angiogenesis- dependent cancer, including, for example, solid tumors, blood born tumors such as leukemias, and tumor metastases; benign tumors, for example hemangiomas, acoustic neuromas, neurofibromas, trachomas, and pyogenic granulomas; inflammatory disorders such as immune and non- immune inflammation; chronic articular rheumatism and psoriasis; ocular angiogenic diseases, for example, diabetic retinopathy, retinopathy of prematurity, macular degeneration, corneal graft rejection, neovascular glaucoma, retrolental fibroplasia, rubeosis; Osier- Webber Syndrome; myocardial angiogenesis; plaque neovascularization; telangiectasia; hemophiliac joints;

angiofibroma; and wound granulation and wound healing; telangiectasia psoriasis scleroderma, pyogenic granuloma, cororany collaterals, ischemic limb angiogenesis, corneal diseases, rubeosis, arthritis, diabetic neovascularization, fractures, vasculogenesis, hematopoiesis (see e.g.,

WO2005056764).

[00172] Specific examples of infections in which the Fn3 -based binding molecules of the invention can be used include, but are not limited to, the following: cellular, fungal, bacterial, and viral.

[00173] The present invention is further illustrated by the following examples which should not be construed as further limiting. The contents of all figures and all references, patents and published patent applications cited throughout this application are expressly incorporated herein by reference.

Exemplification Example 1

Production of Libraries of Fibronectin-Based Binding Molecules

[00174] In general, the practice of the present invention employs, unless otherwise indicated, conventional techniques of chemistry, molecular biology, recombinant DNA technology, immunology (especially, e.g., antibody technology), and standard techniques in polypeptide preparation. See, e.g., Sambrook, Fritsch and Maniatis, Molecular Cloning: Cold Spring Harbor Laboratory Press (1989); Antibody Engineering Protocols (Methods in Molecular Biology) , 510, Paul, S., Humana Pr (1996); Antibody Engineering: A Practical Approach (Practical Approach Series, 169), McCafferty, Ed., Irl Pr (1996); Antibodies: A Laboratory Manual, Harlow et al, C.S.H.L. Press, Pub. (1999); and Current Protocols in Molecular Biology, eds. Ausubel et al, John Wiley & Sons (1992). Other methods, techniques, and sequences suitable for use in carrying out the present invention are found in U.S. Pat. Nos. 7,153,661 ; 7,119,171 ; 7,078,490; 6,703,199; 6,673,901 ; and 6,462,189. [00175] The 10th human fibronectin protein (Fn3 ) is used to design, isolate, and engineer monospecific binders. Initial binders are first independently isolated from two libraries using standard selection methods. This enriched population is then mutagenized, and successive rounds of random mutagenesis and enrichment is conducted to attain desired mono-specific binders.

[00176] Library Construction

Wildtype Fn3¹⁰ sequence as shown in SEQ ID NO: 1 is used as the basis to

generate libraries of binders that utilize the top or bottom loops.

VSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKS TATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRTEI (SEQ ID NO: 1)

[00177] Using computational modeling, two sets of variable regions of the wildtype

Fn3¹⁰ are chosen to be randomized. The first set comprising the solvent exposed top_

loops BC, DE and FG is designated library A and is randomized in the region

shown in boldface in SEQ ID NO: 2.

[00178] Library A (beta-sandwich with solvent exposed top_ loops BC, DE and FG).

VSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATIS GLKPGVDYTITVYAVTGRGDSPASSKPISINYRTEI (SEQ ID NO: 2)

[00179] The second set comprising the solvent exposed bottom loops AB, CD, EF and C-terminus is designated library B and is randomized in the underlined regions shown in italics. [00180] Library B (beta-sandwich with solvent exposed bottom loops AB, CD, EF and C- terminus).

VSDVPRDLEWAATfT SLLISWDAPAVTVRYYRITYG^rGGNSf FOEFTVPGSKSTATISG LKPG EOYTITVYAVTGRGD SPASSKPISINYRT£7 (SEQ ID NO: 3)

[00181 ] The DNA sequences corresponding to the two libraries are optimized for expression in s. coli at Geneart AG, Germany. Regions in boldface or underlined italics from SEQ ID NOs: 2 and

3, respectively, are synthesized as degenerated positions. The libraries are assembled from synthetic degenerated oligonucleotides and genes corresponding to full length fragments gel purified. Amplification is performed with terminal primers and subsequent ligation of the amplified library into a cloning vector, e.g., pCR-Script, yielding the starting libraries. Libraries

A and B are then independently screened to identify mono-specific binders. [00182] In a separate example of library construction, the display vectors are constructed from the low-copy-number centromeric vector pYC6/CT (Invitrogen) (also designated pYS6/CT, and diagrammed in Fig. 3), which carries a blasticidin (Bis) resistance selectable marker. The fibronectins are anchored to the yeast surface by the 69-amino-acid Aga2p subunit via the Agalp cell surface anchor protein. A second version of this vector is created (designated pYC6/CT Zeo or pYS6/CT Zeo and diagrammed in Fig. 4) by replacing the blasticidin marker with the zeocin (Zeo) marker. After initial enrichment of the library for a given target the plasmid DNA from the pYC6/CT vector is recovered according to standard protocols and the Aga2-Fn conjugates subcloned into pYC6/CT Zeo vector to create an identical copy of the enriched pool. The first pool cloned into pYC6/CT is subsequently transformed into yeast strain EBY100 (MAT-a AGA1 : : GAL1 -AGA1 : : URA3 ura3-52 trpl leu2deltal his3delta200 pep4: : HIS3 prbldeltal.6R canl GAL) (Boder and Wittrup, 1997 Nature Biotech. 15: 553-557) to give a haploid Fibronectin libray with a selectable Blasticidin marker. The second pool cloned into pYC6/CT Zeo is transformed into AWY102 (MAT-alpha AGA1 : : GAL1-AGA1 : : URA3 ura3-52 trpl leu2deltal his3delta200 pep4: : HIS3 prbldeltal .6R canl GAL) (Wentz and Shusta, 2007 Biotech. Prog. 24: 748-756 Nature Biotech. 15: 553-557) to give a haploid fibronectin library with a selectable Zeocin marker. Example 2

Yeast mating

[00183] In one example of yeast mating, yeast strain EBY100 (MAT-a) is transformed with a cloning vector (e.g., pCR-Script), into which a fibronectin library is cloned. Another yeast strain, BJ5457 (MAT-alpha) is transformed with a cloning vector (e.g., pCR-Script) into which the same or a different fibronectin library is cloned. One vector can encode an antibiotic marker (e.g., zeocin), while the other vector can encode a different marker (e.g., blasticidin). The cells are grown overnight at 30 °C in a selective medium. For repertoires, a minimum inoculation of five times the initial library size is made into 500 ml of selective medium. The following day, the OD₆oo mn is determined and equal amounts of each haploid culture are centrifuged and resuspended gently in 10-20 ml of YPD (10 g/1 yeast extract, 20 g/1 peptone, and 20 g/1 glucose) followed by mixing prior to plating onto solid YPD media. Gentle spreading is performed to minimize the mechanical disruption of the mating process. An optimum plating density on solid medium is between 10⁷ and 10^s cells/cm². Zygotes are allowed to form at 30 °C for 5 h on YPD agar plates after which cells are collected by gentle scraping in the presence of double-selective culture medium (containing both zeocin and blasticidin). The mating cell population is finally transferred onto double-selective plates (containing both zeocin and blasticidin) and grown for 3 days at 30 °C. Both haploid cultures and diploid cultures are titrated on selective plates to calculate the mating efficiency. Mating efficiency is calculated as the titration on double-selective plates of the diploid repertoire divided by the titration of the haploid parent culture on single-selective plates given as a percentage.

[00184] In another example of yeast mating, yeast strains EBY100 (MAT-a) transformed with pYC6/CT and AWY102 (MAT-alpha) transformed with pYC6/CT Zeo are grown overnight at 30 °C in selective medium of YPD + 200 ug/ml Blasticidin (EBY100) or YPD + 150ug/ml Zeocin (AWY102) . For repertoires, a minimum inoculation of five times the initial library size is made into 500 ml of selective medium. The following day, the OD600 nm is determined and equal amounts of each haploid culture are centrifuged and re-suspended gently in 10-20 ml of YPD (10 g/1 yeast extract, 20 g/1 peptone, and 20 g/1 glucose) followed by mixing prior to plating onto solid YPD media. Gentle spreading is performed to minimize the mechanical disruption of the mating process. An optimum plating density on solid medium is between 10⁷ and 10^s cells/cm². Zygotes are allowed to form at 30 °C for 5 h on YPD agar plates, after which cells are collected by gentle scraping in the presence of double-selective culture medium YPD + Zeo + Bis. The mating cell population is finally transferred onto double-selective plates (YPD Agar supplemented with Blasticidin and Zeocin) and grown for 3 days at 30 °C. Both haploid cultures and diploid cultures are titrated on selective plates to calculate the mating efficiency. Mating efficiency is calculated as the titration on double-selective plates of the diploid repertoire divided by the titration of the haploid parent culture on single-selective plates given as a percentage. For repeated cycles of fibronectin shuffling, pools of selected combinations of two fibronectins from the first cycle of shuffling are rescued by lysing of yeast cultures and recovering of the plasmid DNA. The purified DNA pools are directly transformed into fresh cultures of EBY100 and AWY102 and grown on selective media. AWY102 transformants are grown on YPD media supplemented with Zeocin, allowing the preferential growth of cells containing the pYC6/CT Zeo expression plasmid.

EBY100 transformants are grown on YPD media supplemented with Blasticidin only, allowing the preferential growth of cells containing the pYC6/CT expression plasmid.

[00185] Diploid yeast cells are grown to logarithmic phase overnight at 30 °C with shaking in YPD + Zeo +Bls. For the growth of haploid EBY100 cells containing the pYC6/CT expression vector, the medium is YPD + Bis, and for haploid AWY102 cells containing pYC6/CT Zeo expression vector, the medium is YPD + Zeo. The following day, yeast cells are pelleted and resuspended in selective medium supplemented with 2% (wt/vol) galactose and grown for 24 h at 30 °C . Flow cytometric analysis of yeast cells is performed on an FACS ARIA flow cytometer (Beckman Coulter) as described previously.

Example 3

Identification of high-affinity combinations of fibronectin-based binders [00186] In one example of identifying high-affinity combinations of two fibronectin-based binders, one can use the yeast mating display technology as published by DYAX (Blaise et ah, Gene 2004, Nov 24, 342(2): 211).

[00187] In analogy to this approach the libraries of fibronectin-based binders are screened using the standard protocol for several rounds to enrich a pool that specifically binds to a given target. At this point the pooled library is isolated and cloned into two different vectors such as pYES6 with one set under the blasticidin marker and the second set under the zeocin marker. The pooled libraries are then transformed into compatible mating strains such as EBYIOO (Invitrogen, Boder, E. T. and Wittrup, K. D. 1997 Nat. Biotechnol. 15, 553-557) or AWY102 (Wentz and Shusta, 2007 App. Env. Microb. 73: 1189-1198). The cells are then mated using the protocol published in Blaise et al.

[00188] After mating the newly generated diploid strain is then plated out on double selective media (such as Blasticidin/Zeocin) to select for colonies carrying two fibronectin-based binders. The newly generated library carries now two different fibronectin molecules on the surface of each yeast cell and can be used for further identification of highly avid binders. The best combination of two binders should be easily identified by FACS sorting. It is expected that the combination of two different binders will yield a significant increase in affinity over the individual binders and should be easily detected by FACS sorting.

[00189] In these examples, the cycles of library preparation, mating, and selection can be repeated as necessary until a satisfactory high-affinity combination of fibronectin-based binders is achieved.

Equivalents

[00190] Those skilled in the art will recognize or be able to ascertain, using no more than routine experimentation, many equivalents of the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

Claims:

1. A method of identifying a high-affinity combination of fibronectin-based binders, the method comprising the steps of:

providing a first population of haploid yeast cells, each cell comprising a first heterologous nucleic acid comprising a first promoter and an operably-linked coding region encoding a member of a first library of fibronectin-based binders;

providing a second population of haploid yeast cells, each cell comprising a second heterologous nucleic acid comprising a second promoter and an operably-linked coding region encoding a member of a second library of fibronectin-based binders,

wherein the cells of the first and second populations are different mating types;

mating cells of the first and second populations to produce a population of diploid yeast cells, wherein each cell displays on its surface a member of the first library and a member of the second library;

screening the population of diploid yeast cells to obtain a diploid yeast cell displaying on its surface a high-affinity combination of two fibronectin-based binders.

2. The method of claim 1, wherein the first and second promoters are the same.

3. The method of claim 1, wherein the first and second promoters are different.

4. The method of claim 1, wherein the first and second libraries are the same or different, but both libraries bind to the same target.

5. The method of claim 1, wherein the first and second heterologous nucleic acids encode selectable markers.

6. The method of claim 5, wherein the first and second heterologous nucleic acids encode resistance to two different antibiotics.

7. The method of claim 6, wherein the antibiotics are selected from the group consisting of zeocin, blasticidin, hygromycin and neomyocin.

8. The method of claim 1, further comprising a step of selecting for diploid cells using the antibiotics for which the first and second heterologous nucleic acids encode resistance.

9. The method of claim 1, wherein the step of screeening is performed using FACS.

10. The method of claim 1, wherein the step of screening is performed using a method selected from the group consisting of ELISA, panning, magnetic particle processing, and flow cytometry.

11. The method of claim 1 , wherein either of the first or second population of haploid yeast cells is DSY-5 and the other is DSY-6.

12. The method of claim 1, further comprising the step of:

isolating the nucleotides encoding the high-affinity combination of two fibronectin-based binders from the yeast cell.

13. The method of claim 1, further comprising the step of:

preparing a fusion protein comprising the high-affinity combination of two fibronectin-based binders expressed on the diploid cell.

14. The method of claim 1, wherein the first and second heterologous nucleic acid are integrated into a chromosome.

15. The method of claim 1, wherein the first and second library of fibronectin-based binders is enriched.

16. The method of claim 1, wherein the first and second library of fibronectin-based binders bind to different epitopes of the same target.