CD 137 BINDING FIBRONECTIN TYPE III DOMAINS

FIELD OF THE INVENTION

The present invention relates to fibronectin type III domains that specifically bind to cluster of differentiation 137 (CD137) and methods of making and using the molecules.

BACKGROUND OF THE INVENTION

Advances in understanding of the requirements for tumor antigen recognition and immune effector function indicate that a potential strategy to enhance an anti-tumor immune response is to provide co-stimulation through an auxiliary molecule. The current model for T-cell activation postulates that naive T- cells require two signals for full activation: (i) a signal provided through the binding of processed antigens presented to the T-cell receptor by major histocompatibility complex (MHC) class I molecules; and (ii) an additional signal provided by the interaction of co-stimulatory molecules on the surface of T-cells and their ligands on antigen presenting cells.

CD 137 (4- IBB) is a member of the TNF receptor superfamily and is an activation-induced T-cell costimulatory molecule. The receptor was initially described in mice (B. Kwon et al, P.N.A.S. USA, 86: 1963-7 (1989)), and later identified in humans (M. Alderson et al, Eur. J. Immunol, 24: 2219-27 (1994); Z. Zhou et al, Immunol. Lett., 45:67 (1995)). The interaction of CD137 and the CD 137 ligand (4-1BBL) activates an important costimulatory pathway. Signaling via CD 137 upregulates survival genes, enhances cell division, induces cytokine production, and prevents activation-induced cell death in T cells. The importance of the CD137 pathway has been underscored in a number of diseases, including cancer (see, e.g., U.S. Patent No.: 7,288,638).

Expression of CD 137 has been shown to be predominantly on cells of lymphoid lineage such as activated T-cells, activated Natural Killer (NK) cells, NKT-cells, CD4CD25 regulatory T-cells, and also on activated thymocytes, and intraepithelial lymphocytes. In addition, CD137 has also been shown to be expressed on cells of myeloid origin like dendritic cells, monocytes, neutrophils, and eosinophils. Even though CD137 expression is mainly restricted to immune/inflammatory cells, there have been reports describing its expression on endothelial cells associated with a small number of tissues from inflammatory sites and tumors.

The physiological events observed following CD 137 stimulation on T-cells are mediated by NF-κΒ and PI3K/ERK1/2 signals with separate physiological functions. NF-κΒ signals trigger expression of Bel -XL, an anti-apopotic molecule, thus resulting in increased survival, whereas PI3K and ERK1/2 signals are specifically responsible for CD137-mediated cell cycle progression (H. Lee et al, J. Immunol, 169(9):4882-8 (2002)). The effect of CD137 activation on the inhibition of activation-induced cell death was shown in vitro by Hurtado et al. (J. Hurtado et al, J. Immunol, 158(6):2600-9 (1997)), and in an in vivo system in which anti-CD 137 monoclonal antibodies (mabs) were shown to produce long- term survival of superantigen-activated CD8+ T-cells by preventing clonal deletion (C. Takahashi et al, J. Immunol, 162:5037 (1999)). Later, two reports demonstrated, under different experimental conditions, that the CD 137 signal regulated both clonal expansion and survival of CD8+ T-cells (D. Cooper et al, Eur. J. Immunol, 32(2):521-9 (2002); M. Maus et al, Nat. Biotechnol, 20: 143 (2002)).

Altogether, CD 137 stimulation results in enhanced expansion, survival, and effector functions of newly primed CD8+ T-cells, acting, in part, directly on these cells. Both CD4+ and CD8+ T-cells have been shown to respond to CD137 stimulation, however, it appears that enhancement of T-cell function is greater in CD8+ cells ((W. Shuford et al, J. Exp. Med, 186(l):47-55 (1997); I. Gramaglia et al, Eur. J. Immunol, 30(2):392-402 (2000); C. Takahashi et al., J. Immunol, 162:5037 (1999)). Based on the critical role of CD137 stimulation in CD8+ T-cell function and survival, agonism of the CD137/CD137L system provides a plausible approach for the treatment of tumors and viral pathogens.

Alternatively, while it has been shown that agonistic antibodies to CD 137 and the ligand to CD137 enhance lymphocyte activation, the CD137 protein has the opposite effect. It inhibits proliferation of activated T lymphocytes and induces programmed cell death. These T cell-inhibitory activities of CD 137 require immobilisation of the protein, arguing for transmission of a signal through the ligand/coreceptor (Schwarz et al, Blood 87, 2839-2845 (1996); Michel et al, Immunology 98, 42-46 (1999)).

The known human CD137 ligand is expressed constitutively by monocytes and its expression is inducible in T lymphocytes (Alderson et al., Eur. J. Immunol. 24, 2219-2227 (1994)). Monocytes are activated by immobilised CD137 protein and their survival is profoundly prolonged by CD137. (Langstein et al, J.

Immunol. 160, 2488-2494 (1998); Langstein et al, J. Leuk. Biol. 65, 829-833 (1999)). CD137 also induces proliferation in peripheral monocytes (Langstein et al, 1999b). Macrophage colony- stimulating factor (M-CSF) is essential for the proliferative and survival-enhancing activities of CD137 (Langstein et al, J. Leuk. Biol. 65, 829-833 (1999); Langstein et al, Blood 94, 3161-3168 (1999)).

Signalling through CD 137 ligand has also been demonstrated in B cells where it enhances proliferation and immunoglobulin synthesis. This occurs at interactions of B cells with CD137-expressing T cells or follicular dendritic cells (Pauly et al, J. Leuk. Biol. 72, 35-42 (2002)). It was postulated that similarly to the CD40 receptor/ligand system, which mediates T cell help to B cells after first antigen encounter, the CD 137 receptor/ligand system may mediate co-stimulation of B cells by FDC during affinity maturation (Pauly et al, J. Leuk. Biol. 72, 35-42 (2002)).

Furthermore, soluble forms of CD 137 are generated by differential splicing and are selectively expressed by activated T cells (Michel et al, Eur. J. Immunol. 28, 290-295 (1998)). Soluble CD137 is antagonistic to membrane-bound or immobilised CD137, and levels of soluble CD137 correlate with activation induced cell death in T cells (DeBenedette et al, J. Exp. Med. 181, 985-992 (1995);

Hurtado et al, J. Immunol. 155, 3360-3367 (1995); Michel et al., Cytokine 12, 742- 746 (2000)).

Thus, considering the complicated picture for CD 137 involvement in divergent mechanisms of action and different cell types, there exists a need for reagents to accurately detect CD 137 in tumor tissues and other samples and for new therapeutics that modulate the interaction between CD137 and the 4-1BBL ligand or that modulate the interaction between CD137 and other cellular targets. SUMMARY OF THE INVENTION

The invention provides an isolated FN3 domain that specifically binds to CD 137 protein.

The invention also provides an isolated FN3 domain that specifically binds to CD137 protein comprising the amino acid sequence of SEQ ID NOs: 45, 46, 47,

48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, or 224.

The invention also provides an isolated polynucleotide encoding the FN3 domain that specifically binds to CD 137 protein.

The invention also provides a vector comprising the polynucleotide.

The invention also provides a host cell comprising the vector.

The invention also provides a method of producing the FN3 domain that specifically binds to CD 137 protein, comprising culturing the isolated host cell under conditions that the FN3 domain that specifically binds to CD 137 protein is expressed, and purifying the FN3 domain that specifically binds to CD137 protein.

The invention also provides a pharmaceutical composition comprising the FN3 domain that specifically binds to CD 137 protein and a pharmaceutically acceptable carrier.

The invention also provides an anti-idiotypic antibody that specifically binds the FN3 domain that specifically binds to CD 137 protein.

The invention also provides a kit comprising the FN3 domain.

The invention also provides a method of detecting CD137-expressing cancer cells in a tumor tissue, comprising obtaining a sample of the tumor tissue from a subject; and

detecting whether CD 137 protein is expressed in the tumor tissue by contacting the sample of the tumor tissue with the FN3 domain that specifically binds CD137 protein comprising the amino acid sequence of one of SEQ ID NOs: 45-224 and detecting the binding between CD137 protein and the FN3 domain.

The invention also provides a method of isolating CD 137 expressing cells, comprising obtaining a sample from a subject;

contacting the sample with the FN3 domain that specifically binds to CD137 protein comprising the amino acid sequence of one of SEQ ID NOs: 45-224, and

isolating the cells bound to the FN3 domains.

The invention also provides a method of detecting CD137-expressing cancer cells in a tumor tissue, comprising

conjugating the FN3 domain that specifically binds to CD 137 protein comprising the amino acid sequence of one of SEQ ID NOs: 45-224 to a detectable label to form a conjugate;

administering the conjugate to a subject; and

visualizing the CD137 expressing cancer cells to which the conjugate is bound.

DETAILED DESCRIPTION OF THE INVENTION

As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a cell" includes a combination of two or more cells, and the like.

"Fibronectin type III (FN3) domain" (FN3 domain) refers to a domain occurring frequently in proteins including fibronectins, tenascin, intracellular cytoskeletal proteins, cytokine receptors and prokaryotic enzymes (Bork and Doolittle, Proc Nat Acad Sci USA 89:8990-8994, 1992; Meinke et al, J Bacteriol 175: 1910-1918, 1993; Watanabe et al, J Biol Chem 265: 15659-15665, 1990). Exemplary FN3 domains are the 15 different FN3 domains present in human tenascin C, the 15 different FN3 domains present in human fibronectin (FN), and non-natural synthetic FN3 domains as described for example in U.S. Pat. No. 8,278,419. Individual FN3 domains are referred to by domain number and protein name, e.g., the 3 ^rd FN3 domain of tenascin (TN3), or the 10 ^th FN3 domain of fibronectin (FN10).

"Centyrin" refers to a FN3 domain that is based on the consensus sequence of the 15 different FN3 domains present in human tenascin C.

The term "capture agent" refers to substances that bind to a particular type of cells and enable the isolation of that cell from other cells. Exemplary capture agents are magnetic beads, ferrofluids, encapsulating reagents, molecules that bind the particular cell type and the like.

"Sample" refers to a collection of similar fluids, cells, or tissues isolated from a subject, as well as fluids, cells, or tissues present within a subject.

Exemplary samples are tissue biopsies, fine needle aspirations, surgically resected tissue, organ cultures, cell cultures and biological fluids such as blood, serum and serosal fluids, plasma, lymph, urine, saliva, cystic fluid, tear drops, feces, sputum, mucosal secretions of the secretory tissues and organs, vaginal secretions, ascites fluids, fluids of the pleural, pericardial, peritoneal, abdominal and other body cavities, fluids collected by bronchial lavage, synovial fluid, liquid solutions contacted with a subject or biological source, for example, cell and organ culture medium including cell or organ conditioned medium and lavage fluids and the like.

"Substituting" or "substituted" or 'mutating" or "mutated" refers to altering, deleting of inserting one or more amino acids or nucleotides in a polypeptide or polynucleotide sequence to generate a variant of that sequence.

"Variant" refers to a polypeptide or a polynucleotide that differs from a reference polypeptide or a reference polynucleotide by one or more modifications for example, substitutions, insertions or deletions.

"Specifically binds" or "specific binding" refers to the ability of the FN3 domain of the invention to bind CD 137 with a dissociation constant (KD) of about lxl O ^"6 M or less, for example about lxlO ^"7 M or less, about lxl O ^"8 M or less, about lxl O ^"9 M or less, about lxlO ^"10 M or less, about lxl O ^"11 M or less, about lxlO ^"12 M or less, or about lxl 0 ^"13 M or less. Alternatively, "specific binding" refers to the ability of the FN3 domain of the invention to bind CD137 at least 5-fold above the negative control in standard ELISA assay. The isolated FN3 domain of the invention that specifically binds CD137 may, however, have cross-reactivity to other related antigens, for example to the same predetermined antigen from other species (homologs), such as Macaca Fascicularis (cynomolgous monkey, cyno) or Pan troglodytes (chimpanzee).

"Library" refers to a collection of variants. The library may be composed of polypeptide or polynucleotide variants.

"Stability" refers to the ability of a molecule to maintain a folded state under physiological conditions such that it retains at least one of its normal functional activities, for example, binding to a predetermined antigen such as CD137.

"CD137" refers to human CD137 protein having the amino acid sequence of SEQ ID NO:44.

"Tencon" refers to the synthetic fibronectin type III (FN3) domain having the sequence shown in SEQ ID NO: 1 and described in U.S. Pat. Publ. No.

2010/0216708.

A "cancer cell" or a "tumor cell" refers to a cancerous, pre-cancerous or transformed cell, either in vivo, ex vivo, and in tissue culture, that has spontaneous or induced phenotypic changes that do not necessarily involve the uptake of new genetic material. Although transformation can arise from infection with a transforming virus and incorporation of new genomic nucleic acid, or uptake of exogenous nucleic acid, it can also arise spontaneously or following exposure to a carcinogen, thereby mutating an endogenous gene. Transformation/cancer is exemplified by, e.g., morphological changes, immortalization of cells, aberrant growth control, foci formation, proliferation, malignancy, tumor specific markers levels, invasiveness, tumor growth or suppression in suitable animal hosts such as nude mice, and the like, in vitro, in vivo, and ex vivo (Freshney, Culture of Animal Cells: A Manual of Basic Technique (3rd ed. 1994)).

"Vector" refers to a polynucleotide capable of being duplicated within a biological system or that can be moved between such systems. Vector polynucleotides typically contain elements, such as origins of replication, polyadenylation signal or selection markers that function to facilitate the duplication or maintenance of these polynucleotides in a biological system. Examples of such biological systems may include a cell, virus, animal, plant, and reconstituted biological systems utilizing biological components capable of duplicating a vector. The polynucleotide comprising a vector may be DNA or RNA molecules or a hybrid of these.

"Expression vector" refers to a vector that can be utilized in a biological system or in a reconstituted biological system to direct the translation of a polypeptide encoded by a polynucleotide sequence present in the expression vector.

"Polynucleotide" refers to a synthetic molecule comprising a chain of nucleotides covalently linked by a sugar-phosphate backbone or other equivalent covalent chemistry. cDNA is a typical example of a polynucleotide.

"Polypeptide" or "protein" refers to a molecule that comprises at least two amino acid residues linked by a peptide bond to form a polypeptide. Small polypeptides of less than about 50 amino acids may be referred to as "peptides".

"Valent" refers to the presence of a specified number of binding sites specific for an antigen in a molecule. As such, the terms "monovalent", "bivalent", "tetravalent", and "hexavalent" refer to the presence of one, two, four and six binding sites, respectively, specific for an antigen in a molecule.

"Subject" includes any human or nonhuman animal. "Nonhuman animal" includes all vertebrates, e.g., mammals and non-mammals, such as nonhuman primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc.

Except when noted, the terms "patient" or "subject" are used interchangeably.

"Isolated" refers to a homogenous population of molecules (such as synthetic polynucleotides or a polypeptide such as FN3 domains) which have been substantially separated and/or purified away from other components of the system the molecules are produced in, such as a recombinant cell, as well as a protein that has been subjected to at least one purification or isolation step. "Isolated FN3 domain" refers to an FN3 domain that is substantially free of other cellular material and/or chemicals and encompasses FN3 domains that are isolated to a higher purity, such as to 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% purity. Compositions of matter

The present invention provides fibronectin type III (FN3) domains that specifically bind human CD137 protein (SEQ ID NO:44). These molecules can be used in therapeutic and diagnostic applications and in imaging. The present invention provides polynucleotides encoding the FN3 domains of the invention or complementary nucleic acids thereof, vectors, host cells, and methods of making and using them.

The invention provides an isolated FN3 domain that specifically binds CD137.

The FN3 domain of the invention may bind CD 137 with a dissociation constant (KD) of less than about lxlO ^"7 M, for example less than about lxlO ^"8 M, less than about lxlO ^"9 M, less than about lxlO ^"10 M, less than about lxlO ^"11 M, less than about lxl 0 ^"12 M, or less than about lxl 0 ^"13 M as determined by surface plasmon resonance or the Kinexa method, as practiced by those of skill in the art. The measured affinity of a particular FN3 domain-antigen interaction can vary if measured under different conditions (e.g., osmolarity, pH). Thus, measurements of affinity and other antigen-binding parameters (e.g., KD, K _on , Koff) are made with standardized solutions of protein scaffold and antigen, and a standardized buffer, such as the buffer described herein.

The FN3 domain of the invention may bind CD 137 at least 5 -fold above the signal obtained for a negative control in standard ELISA assay.

In some embodiments, the FN3 domain that specifically binds CD 137 comprises an initiator methionine (Met) linked to the N-terminus of the molecule.

In some embodiments, the FN3 domain that specifically binds CD137 comprises a cysteine (Cys) linked to a C-terminus of the FN3 domain.

The addition of the N-terminal Met and/or the C-terminal Cys may facilitate expression and/or conjugation of half-life extending molecules.

In some embodiments, the FN3 domain that specifically binds CD137 is internalized into a cell.

Internalization of the FN3 domain may facilitate delivery of a cytotoxic agent into tumor cells. In some embodiments, the FN3 domain that specifically binds CD 137 inhibits binding of the CD137 ligand (4-lBBL) to CD137.

Inhibition of binding of 4-lBBL to CD137 by the FN3 domains of the invention may be assessed using competition ELISA. In an exemplary assay, 1 μg/ml recombinant human CD137 is bound on wells of microtiter plates, the wells are washed and blocked, and 10 μg/ml of the test FN3 domain is added. Without washing, 7.5 μg/ml 4-lBBL is added into the wells and incubated for 30 min, after which 0.5 μg/ml anti-4-lBBL antibodies are added and incubated for 30 min. The plates are washed and 0.5 μg/mL neutravidin-HRP conjugate polyclonal antibody is added and incubated for 30 minutes. The plates are washed and POD

Chemiluminescence substrate added immediately prior to reading the

luminescence signal. The FN3 domains of the invention inhibit binding of 4-lBBL to CD 137 when the binding of 4-lBBL is reduced by at least about 80%, 85%, 90%, 95% or 100%.

In some embodiments, the FN3 domain that specifically binds CD 137 is a CD 137 antagonist.

In some embodiments, the FN3 domain that specifically binds CD 137 is a CD137 agonist.

"Antagonist" refers to a FN3 domain that specifically binds CD 137 that suppresses at least one activity of CD 137 function by inhibiting CD 137 binding to to its natural ligand 4-1BB1 or inhibiting CD137 binding to other molecules. A molecule is an antagonist when the at least one reaction or activity is suppressed by at least about 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% more than the at least one reaction or activity suppressed in the absence of the antagonist (e.g., negative control), or when the suppression is statistically significant when compared to the suppression in the absence of the antagonist. A typical reaction or activity that is induced by 4-lBBL binding to CD 137 is upregulation of survival genes, enhanced cell division, induced cytokine production, and prevention of activation-induced cell death in T cells.

The antagonistic FN3 domains that specifically bind CD 137 may be used in the treatment of autoimmune or inflammatory diseases and in general diseases in which suppression of T cell responses is desirable. "Agonist" refers to a FN3 domain that specifically binds CD137 that induces at least one reaction or activity that is induced by CD137. The FN3 domain is an agonist when the at least one reaction or activity is induced by at least about 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% greater than the at least one reaction or activity induced in the absence of the agonist (e.g., negative control), or when the induction is statistically significant when compared to the induction in the absence of the agonist. A typical reaction or activity that is induced by 4-lBBL binding to CD 137 is upregulation of survival genes, enhanced cell division, induced cytokine production, and prevention of activation-induced cell death in T cells.

The agonistic FN3 domains that specifically bind CD137 may be used, for example, in the treatment of cancer or viral infections and in general in treatment of diseases in which activation of T cell responses is desirable.

In some embodiments, the FN3 domain that specifically binds CD 137 does not inhibit 4-lBBL binding to CD137.

In some embodiments, the FN3 domain that specifically binds CD 137 is based on Tencon sequence of SEQ ID NO: 1 or Tencon 27 sequence of SEQ ID NO:4, optionally having substitutions at residues positions 11, 14, 17, 37, 46, 73, or 86 (residue numbering corresponding to SEQ ID NO:4).

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NOs: 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 4, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, or 224. The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:45.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:46.

The invention also provides an isolated FN3 domain that specifically binds CD 137 comprising the amino acid sequence of SEQ ID NO:47.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:48.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:49.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:50.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:51.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:52.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:53.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:54.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:55.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:56.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:57.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:58. The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:59.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:60.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:61.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:62.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:63.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:64.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:65.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:66.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:67.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:68.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:69.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:70.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:71.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:72. The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:73.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:74.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:75.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:76.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:77.

The invention also provides an isolated FN3 domain that specifically binds CD 137 comprising the amino acid sequence of SEQ ID NO: 78.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:79.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:80.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:81.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:82.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:83.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:84.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:85.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:86. The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:87.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:88.

The invention also provides an isolated FN3 domain that specifically binds CD 137 comprising the amino acid sequence of SEQ ID NO: 89.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:90.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:91.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:92.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:93.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:94.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:95.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:96.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:97.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:98.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:99.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO: 100. The invention also provides an isolated FN3 domain that specifically binds CD 137 comprising the amino acid sequence of SEQ ID NO: 101.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO: 102.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO: 103.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO: 104.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO: 105.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO: 106.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO: 107.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO: 108.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO: 109.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO: 110.

The invention also provides an isolated FN3 domain that specifically binds CD 137 comprising the amino acid sequence of SEQ ID NO: 111.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO: 1 12.

The invention also provides an isolated FN3 domain that specifically binds CD 137 comprising the amino acid sequence of SEQ ID NO: 1 13.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO: 114. The invention also provides an isolated FN3 domain that specifically binds CD 137 comprising the amino acid sequence of SEQ ID NO: 115.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO: 116.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO: 1 17.

The invention also provides an isolated FN3 domain that specifically binds CD 137 comprising the amino acid sequence of SEQ ID NO: 118.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO: 119.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO: 120.

The invention also provides an isolated FN3 domain that specifically binds CD 137 comprising the amino acid sequence of SEQ ID NO: 121.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO: 122.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO: 123.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO: 124.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO: 125.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO: 126.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO: 127.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO: 128. The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO: 129.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO: 130.

The invention also provides an isolated FN3 domain that specifically binds CD 137 comprising the amino acid sequence of SEQ ID NO: 131.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO: 132.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO: 133.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO: 134.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO: 135.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO: 136.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO: 137.

The invention also provides an isolated FN3 domain that specifically binds CD 137 comprising the amino acid sequence of SEQ ID NO: 138.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO: 139.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO: 140.

The invention also provides an isolated FN3 domain that specifically binds CD 137 comprising the amino acid sequence of SEQ ID NO: 141.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO: 142. The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO: 143.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO: 144.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO: 145.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO: 146.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO: 147.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO: 148.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO: 149.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO: 150.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO: 151.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO: 152.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO: 153.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO: 154.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO: 155.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO: 156. The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO: 157.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO: 158.

The invention also provides an isolated FN3 domain that specifically binds CD 137 comprising the amino acid sequence of SEQ ID NO: 159.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO: 160.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO: 161.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO: 162.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO: 163.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO: 164.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO: 165.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO: 166.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO: 167.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO: 168.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO: 169.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO: 170. The invention also provides an isolated FN3 domain that specifically binds CD 137 comprising the amino acid sequence of SEQ ID NO: 171.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO: 172.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO: 173.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO: 174.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO: 175.

The invention also provides an isolated FN3 domain that specifically binds CD 137 comprising the amino acid sequence of SEQ ID NO: 176.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO: 177.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO: 178.

The invention also provides an isolated FN3 domain that specifically binds CD 137 comprising the amino acid sequence of SEQ ID NO: 179.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO: 180.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO: 181.

The invention also provides an isolated FN3 domain that specifically binds CD 137 comprising the amino acid sequence of SEQ ID NO: 182.

The invention also provides an isolated FN3 domain that specifically binds CD 137 comprising the amino acid sequence of SEQ ID NO: 183.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO: 184. The invention also provides an isolated FN3 domain that specifically binds CD 137 comprising the amino acid sequence of SEQ ID NO: 185.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO: 186.

The invention also provides an isolated FN3 domain that specifically binds CD 137 comprising the amino acid sequence of SEQ ID NO: 187.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO: 188.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO: 189.

The invention also provides an isolated FN3 domain that specifically binds CD 137 comprising the amino acid sequence of SEQ ID NO: 190.

The invention also provides an isolated FN3 domain that specifically binds CD 137 comprising the amino acid sequence of SEQ ID NO: 191.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO: 192.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO: 193.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO: 194.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO: 195.

The invention also provides an isolated FN3 domain that specifically binds CD 137 comprising the amino acid sequence of SEQ ID NO: 196.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO: 197.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO: 198. The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO: 199.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:200.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:201.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:202.

The invention also provides an isolated FN3 domain that specifically binds CD 137 comprising the amino acid sequence of SEQ ID NO: 203.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:204.

The invention also provides an isolated FN3 domain that specifically binds CD 137 comprising the amino acid sequence of SEQ ID NO: 205.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:206.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:207.

The invention also provides an isolated FN3 domain that specifically binds CD 137 comprising the amino acid sequence of SEQ ID NO: 208.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:209.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:210.

The invention also provides an isolated FN3 domain that specifically binds CD 137 comprising the amino acid sequence of SEQ ID NO:211.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:212. The invention also provides an isolated FN3 domain that specifically binds CD 137 comprising the amino acid sequence of SEQ ID NO:213.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:214.

The invention also provides an isolated FN3 domain that specifically binds CD 137 comprising the amino acid sequence of SEQ ID NO:215.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:216.

The invention also provides an isolated FN3 domain that specifically binds CD 137 comprising the amino acid sequence of SEQ ID NO:217.

The invention also provides an isolated FN3 domain that specifically binds CD 137 comprising the amino acid sequence of SEQ ID NO:218.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:219.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:220.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:221.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:222.

The invention also provides an isolated FN3 domain that specifically binds CD 137 comprising the amino acid sequence of SEQ ID NO: 223.

The invention also provides an isolated FN3 domain that specifically binds CD137 comprising the amino acid sequence of SEQ ID NO:224.

In some embodiments, the isolated FN3 domain that specifically binds CD 137 comprises an initiator methionine (Met) linked to the N-terminus of the molecule.

In some embodiments, the isolated FN3 domain that specifically binds CD 137 comprises an amino acid sequence that is 62%, 63%, 64% , 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to one of the amino acid sequences of SEQ ID NOs: 45-224.

Conjugates of the FN3 domains that specifically bind CD137 of the invention

The invention also provides an isolated FN3 domain that specifically binds CD137 conjugated to a heterologous molecule(s).

In some embodiments, the heterologous molecule is a detectable label or a cytotoxic agent.

The invention also provides an FN3 domain that specifically binds CD 137 conjugated to a detectable label.

The invention also provides an FN3 domain that specifically binds CD 137 conjugated to a cytotoxic agent.

In some embodiments, the detectable label is also a cytotoxic agent.

The FN3 domains that specifically bind CD137 of the invention conjugated to a detectable label can be used to evaluate expression of CD 137 on samples such as tumor tissue in vivo or in vitro.

Detectable label includes compositions that when conjugated to the FN3 domains that specifically bind CD 137 of the invention renders the latter detectable, via spectroscopic, photochemical, biochemical, immunochemical, or chemical means.

Exemplary detectable labels include radioactive isotopes, magnetic beads, metallic beads, colloidal particles, fluorescent dyes, electron-dense reagents, enzymes (for example, as commonly used in an ELISA), biotin, digoxigenin, haptens, luminescent molecules, chemiluminescent molecules, fluorochromes, fluorophores, fluorescent quenching agents, colored molecules, radioactive isotopes, cintillants, avidin, streptavidin, protein A, protein G, antibodies or fragments thereof, polyhistidine, Ni ²⁺ , Flag tags, myc tags, heavy metals, enzymes, alkaline phosphatase, peroxidase, luciferase, electron donors/acceptors, acridinium esters, and colorimetric substrates. A detectable label may emit a signal spontaneously, such as when the detectable label is a radioactive isotope. In other cases the detectable label emits a signal as a result of being stimulated by an external field.

Exemplary radioactive isotopes may be γ-emitting, Auger-emitting, β- emitting, an alpha-emitting or positron-emitting radioactive isotope. Exemplary radioactive isotopes include ¾, ⁿ C, ¹ C, ¹⁵ N, ¹⁸ F, ¹⁹ F, ⁵⁵ Co, ⁵⁷ Co, ⁶⁰ Co, ⁶¹ Cu, ⁶² Cu, ⁶⁴ Cu, ⁶⁷ Cu, ⁶⁸ Ga, ⁷² As, ⁷⁵ Br, ⁸⁶ Y, ⁸⁹ Zr, ⁹⁰ Sr, ^94m Tc, ^99m Tc, ¹¹⁵ In, ¹²³ 1, ¹²⁴ 1, ¹²⁵ I, ¹³¹ 1, ²¹¹ At, ²¹² Bi, ²¹³ Bi, ²²³ Ra, ²²⁶ Ra, ²²⁵ Ac and ²²⁷ Ac.

Exemplary metal atoms are metals with an atomic number greater than 20, such as calcium atoms, scandium atoms, titanium atoms, vanadium atoms, chromium atoms, manganese atoms, iron atoms, cobalt atoms, nickel atoms, copper atoms, zinc atoms, gallium atoms, germanium atoms, arsenic atoms, selenium atoms, bromine atoms, krypton atoms, rubidium atoms, strontium atoms, yttrium atoms, zirconium atoms, niobium atoms, molybdenum atoms, technetium atoms, ruthenium atoms, rhodium atoms, palladium atoms, silver atoms, cadmium atoms, indium atoms, tin atoms, antimony atoms, tellurium atoms, iodine atoms, xenon atoms, cesium atoms, barium atoms, lanthanum atoms, hafnium atoms, tantalum atoms, tungsten atoms, rhenium atoms, osmium atoms, iridium atoms, platinum atoms, gold atoms, mercury atoms, thallium atoms, lead atoms, bismuth atoms, francium atoms, radium atoms, actinium atoms, cerium atoms,

praseodymium atoms, neodymium atoms, promethium atoms, samarium atoms, europium atoms, gadolinium atoms, terbium atoms, dysprosium atoms, holmium atoms, erbium atoms, thulium atoms, ytterbium atoms, lutetium atoms, thorium atoms, protactinium atoms, uranium atoms, neptunium atoms, plutonium atoms, americium atoms, curium atoms, berkelium atoms, californium atoms, einsteinium atoms, fermium atoms, mendelevium atoms, nobelium atoms, or lawrencium atoms.

In some embodiments, the metal atoms may be alkaline earth metals with an atomic number greater than twenty.

In some embodiments, the metal atoms may be lanthanides.

In some embodiments, the metal atoms may be actinides.

In some embodiments, the metal atoms may be transition metals. In some embodiments, the metal atoms may be poor metals.

In some embodiments, the metal atoms may be gold atoms, bismuth atoms, tantalum atoms, and gadolinium atoms.

In some embodiments, the metal atoms may be metals with an atomic number of 53 (i.e., iodine) to 83 (i.e., bismuth).

In some embodiments, the metal atoms may be atoms suitable for magnetic resonance imaging.

The metal atoms may be metal ions in the form of +1 , +2, or +3 oxidation states, such as Ba ²⁺ , Bi ³⁺ , Cs ⁺ , Ca ²⁺ , Cr ²⁺ , Cr ³⁺ , Cr ⁶⁺ , Co ²⁺ , Co ³⁺ , Cu ⁺ , Cu ²⁺ , Cu ³⁺ , Ga ³⁺ , Gd ³⁺ , Au ⁺ , Au ³⁺ , Fe ²⁺ , Fe ³⁺ , F ³⁺ , Pb ²⁺ , Mn ²⁺ , Mn ³⁺ , Mn ⁴⁺ , Mn ⁷⁺ , Hg ²⁺ , Ni ²⁺ , Ni ³⁺ , Ag ⁺ , Sr ²⁺ , Sn ²⁺ , Sn ⁴⁺ , and Zn ²⁺ . The metal atoms may comprise a metal oxide, such as iron oxide, manganese oxide, or gadolinium oxide.

Suitable dyes include any commercially available dyes such as, for example, 5(6)-carboxyfluorescein, IRDye 680RD maleimide or IRDye 800CW, ruthenium polypyridyl dyes, and the like.

Suitable fluorophores are fluorescein isothiocyante (FITC), fluorescein thiosemicarbazide, rhodamine, Texas Red, CyDyes (e.g., Cy3, Cy5, Cy5.5), Alexa Fluors (e.g., Alexa488, Alexa555, Alexa594; Alexa647), near infrared (NIR) (700- 900 nm) fluorescent dyes, and carbocyanine and aminostyryl dyes.

The FN3 domains that specifically bind CD137 conjugated to a detectable label may be used as an imaging agent to evaluate tumor distribution, diagnosis for the presence of tumor cells and/or, recurrence of tumor.

In some embodiments, the FN3 domains that specifically bind CD 137 of the invention are conjugated to a cytotoxic agent.

In some embodiments, the cytotoxic agent is a chemotherapeutic agent, a drug, a growth inhibitory agent, a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate).

The FN3 domains that specifically bind CD137 conjugated to a cytotoxic agent of the invention may be used in the targeted delivery of the cytotoxic agent to CD137 expressing tumor cell, and intracellular accumulation therein, wherein systemic administration of these unconjugated cytotoxic agents may result in unacceptable levels of toxicity to normal cells.

In some embodiments, the cytotoxic agent is daunomycin, doxorubicin, methotrexate, vindesine, bacterial toxins such as diphtheria toxin, ricin, geldanamycin, maytansinoids or calicheamicin. The cytotoxic agent may elict their cytotoxic and cytostatic effects by mechanisms including tubulin binding, DNA binding, or topoisomerase inhibition.

In some embodiments, the cytotoxic agent is an enzymatically active toxins such as diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.

In some embodiments, the cytotoxic agent is a radionuclide, such as ²¹² Bi, ¹³¹ I, ¹³¹ In, ⁹⁰ Y, and ¹⁸⁶ Re.

In some embodiments, the cytotoxic agent is dolastatins or dolostatin peptidic analogs and derivatives, auristatin or monomethyl auristatin

phenylalanine. Exemplary molecules are disclosed in U.S. Pat No. 5,635,483 and 5,780,588. Dolastatins and auristatins have been shown to interfere with microtubule dynamics, GTP hydrolysis, and nuclear and cellular division (Woyke et al (2001) Antimicrob Agents and Chemother. 45(12):3580-3584) and have anticancerand antifungal activity. The dolastatin or auristatin drug moiety may be attached to the FN3 domain of the invention through the N (amino) terminus or the C (carboxyl) terminus of the peptidic drug moiety (WO 02/088172), or via any cysteine engineered into the FN3 domain.

The FN3 domains that specifically bind CD 137 of the invention may be conjugated to a detectable label using known methods.

In some embodiments, the detectable label is complexed with a chelating agent.

In some embodiments, the detectable label is conjugated to the FN3 domain that specifically binds CD137 of the invention via a linker. The detectable label or the cytotoxic moiety may be linked directly, or indirectly, to the FN3 domain that specifically binds CD137 of the invention using known methods. Suitable linkers are known in the art and include, for example, prosthetic groups, non-phenolic linkers (derivatives of N-succimidyl-benzoates; dodecaborate), chelating moieties of both macrocyclics and acyclic chelators, such as derivatives of 1,4,7, 10-tetraazacyclododecane- 1,4,7, 10,tetraacetic acid (DOTA), derivatives of diethylenetriaminepentaacetic avid (DTP A), derivatives of S-2-(4- Isothiocyanatobenzyl)-l,4,7-triazacyclononane-l,4,7-triaceti c acid (NOTA) and derivatives of 1,4,8,1 l-tetraazacyclodocedan-l,4,8,l l-tetraacetic acid (TETA), N- succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HC1), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis- azido compounds (such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5- difluoro-2,4-dinitrobenzene) and other chelating moieties. Suitable peptide linkers are well known.

In some embodiment, the FN3 domain that specifically binds CD 137 is removed from the blood via renal clearance.

Isolation of CD 137 binding FN3 domains from a library based on Tencon sequence

Tencon (SEQ ID NO: 1) is a non-naturally occurring fibronectin type III (FN3) domain designed from a consensus sequence of fifteen FN3 domains from human tenascin-C (Jacobs et al, Protein Engineering, Design, and Selection, 25: 107-117, 2012; U.S. Pat. Publ. No. 2010/0216708). The crystal structure of Tencon shows six surface-exposed loops that connect seven beta-strands as is characteristic to the FN3 domains, the beta-strands referred to as A, B, C, D, E, F, and G, and the loops referred to as AB, BC, CD, DE, EF, and FG loops (Bork and Doolittle, Proc Natl Acad Sci USA 89:8990-8992, 1992; U.S. Pat. No. 6,673,901). These loops, or selected residues within each loop, may be randomized in order to construct libraries of fibronectin type III (FN3) domains that may be used to select novel molecules that bind CD137. Table 1 shows positions and sequences of each loop and beta-strand in Tencon (SEQ ID NO:l).

Library designed based on Tencon sequence may thus have randomized FG loop, or randomized BC and FG loops, such as libraries TCL1 or TCL2 as described below. The Tencon BC loop is 7 amino acids long, thus 1, 2, 3, 4, 5, 6 or 7 amino acids may be randomized in the library diversified at the BC loop and designed based on Tencon sequence. The Tencon FG loop is 7 amino acids long, thus 1, 2, 3, 4, 5, 6 or 7 amino acids may be randomized in the library diversified at the FG loop and designed based on Tencon sequence. Further diversity at loops in the Tencon libraries may be achieved by insertion and/or deletions of residues at loops. For example, the FG and/or BC loops may be extended by 1-22 amino acids, or decreased by 1-3 amino acids. The FG loop in Tencon is 7 amino acids long, whereas the corresponding loop in antibody heavy chains ranges from 4-28 residues. To provide maximum diversity, the FG loop may be diversified in sequence as well as in length to correspond to the antibody CDR3 length range of 4-28 residues. For example, the FG loop can further be diversified in length by extending the loop by additional 1, 2, 3, 4 or 5 amino acids.

Library designed based on Tencon sequence may also have randomized alternative surfaces that form on a side of the FN3 domain and comprise two or more beta strands, and at least one loop. One such alternative surface is formed by amino acids in the C and the F beta-strands and the CD and the FG loops (a C-CD- F-FG surface). A library design based on Tencon alternative C-CD-F-FG surface is described in U.S. Pat. Publ. No. 2013/0226834. Library designed based on Tencon sequence also includes libraries designed based on Tencon variants, such as Tencon variants having substitutions at residues positions 11, 14, 17, 37, 46, 73, or 86 (residue numbering corresponding to SEQ ID NO: 1), and which variants display improve thermal stability. Exemplary Tencon variants are described in US Pat. Publ. No. 2011/0274623, and include Tencon27 (SEQ ID NO:4) having substitutions El 1R, L17A, N46V and E86I when compared to Tencon of SEQ ID NO: l. Table 1. Tencon topology

Tencon and other FN3 sequence based libraries may be randomized at chosen residue positions using a random or defined set of amino acids. For example, variants in the library having random substitutions may be generated using NNK codons, which encode all 20 naturally occurring amino acids. In other diversification schemes, DVK codons may be used to encode amino acids Ala, Trp, Tyr, Lys, Thr, Asn, Lys, Ser, Arg, Asp, Glu, Gly, and Cys. Alternatively, NNS codons may be used to give rise to all 20 amino acid residues and simultaneously reducing the frequency of stop codons. Libraries of FN3 domains with biased amino acid distribution at positions to be diversified may be synthesized for example using Slonomics® technology

(http:_//www_sloning_com). This technology uses a library of pre-made double stranded triplets that act as universal building blocks sufficient for thousands of gene synthesis processes. The triplet library represents all possible sequence combinations necessary to build any desired DNA molecule. The codon designations are according to the well-known IUB code. The FN3 domains that specifically bind CD137 of the invention may be isolated by producing the FN3 library such as the Tencon library using cis display to ligate DNA fragments encoding the scaffold proteins to a DNA fragment encoding RepA to generate a pool of protein-DNA complexes formed after in vitro translation wherein each protein is stably associated with the DNA that encodes it (U.S. Pat. No. 7,842,476; Odegrip et al, Proc Natl Acad Sci U S A 101, 2806- 2810, 2004), and assaying the library for specific binding to PSMA by any method known in the art and described in the Example. Exemplary well known methods which can be used are ELISA, sandwich immunoassays, and competitive and noncompetitive assays (see, e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York). The identified FN3 domains that specifically bind CD 137 are further characterized for their binding to CD137, modulation of CD137 activity, internalization, stability, and other desired characteristics.

The FN3 domains that specifically bind CD 137 of the invention may be generated using any FN3 domain as a template to generate a library and screening the library for molecules specifically binding CD 137 using methods provided within. Exemplar FN3 domains that may be used are the 3rd FN3 domain of tenascin C (TN3) (SEQ ID NO:32), Fibcon (SEQ ID NO:33), and the 10 ^th FN3 domain of fibronectin (FN10) (SEQ ID NO:34). Standard cloning and expression techniques are used to clone the libraries into a vector or synthesize double stranded cDNA cassettes of the library, to express, or to translate the libraries in vitro. For example ribosome display (Hanes and Pluckthun, Proc Natl Acad Sci USA, 94, 4937- 4942, 1997), mRNA display (Roberts and Szostak, Proc Natl Acad Sci USA, 94, 12297-12302, 1997), or other cell-free systems (U.S. Pat. No. 5,643,768) can be used. The libraries of the FN3 domain variants may be expressed as fusion proteins displayed on the surface for example of any suitable bacteriophage. Methods for displaying fusion polypeptides on the surface of a bacteriophage are well known (U.S. Pat. Publ. No. 2011/0118144; Int. Pat. Publ. No. WO2009/085462; U.S. Pat. No. 6,969,108; U.S. Pat. No. 6,172,197; U.S. Pat. No. 5,223,409; U.S. Pat. No.

6,582,915; U.S. Pat. No. 6,472,147). In some embodiments, the FN3 domain that specifically binds CD 137 is based on Tencon sequence of SEQ ID NO: 1 or Tencon27 sequence of SEQ ID NO:4, the SEQ ID NO: 1 or the SEQ ID NO:4, optionally having substitutions at residues positions 11, 14, 17, 37, 46, 73, and/or 86.

The FN3 domains that specifically bind CD 137 of the invention may be modified to improve their properties such as improve thermal stability and reversibility of thermal folding and unfolding. Several methods have been applied to increase the apparent thermal stability of proteins and enzymes, including rational design based on comparison to highly similar thermostable sequences, design of stabilizing disulfide bridges, mutations to increase alpha-helix propensity, engineering of salt bridges, alteration of the surface charge of the protein, directed evolution, and composition of consensus sequences (Lehmann and Wyss, Curr Opin Biotechnol, 12, 371-375, 2001). High thermal stability may increase the yield of the expressed protein, improve solubility or activity, decrease immunogenicity, and minimize the need of a cold chain in manufacturing.

Residues that may be substituted to improve thermal stability of Tencon (SEQ ID NO: l) are residue positions 11, 14, 17, 37, 46, 73, or 86, and are described in US Pat. Publ. No. 2011/0274623. Substitutions corresponding to these residues may be incorporated to the FN3 domain containing molecules of the invention.

Measurement of protein stability and protein lability can be viewed as the same or different aspects of protein integrity. Proteins are sensitive or "labile" to denaturation caused by heat, by ultraviolet or ionizing radiation, changes in the ambient osmolarity and pH if in liquid solution, mechanical shear force imposed by small pore-size filtration, ultraviolet radiation, ionizing radiation, such as by gamma irradiation, chemical or heat dehydration, or any other action or force that may cause protein structure disruption. The stability of the molecule can be determined using standard methods. For example, the stability of a molecule can be determined by measuring the thermal melting ("T _m ") temperature, the temperature in ° Celsius (°C) at which half of the molecules become unfolded, using standard methods. Typically, the higher the T _m , the more stable the molecule. In addition to heat, the chemical environment also changes the ability of the protein to maintain a particular three dimensional structure. In one embodiment, the FN3 domain that specifically binds CD 137 of the invention may exhibit increased stability by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more compared to the same domain prior to engineering measured by the increase in the TV

Chemical denaturation can likewise be measured by a variety of methods. Chemical denaturants include guanidinium hydrochloride, guanidinium thiocyanate, urea, acetone, organic solvents (DMF, benzene, acetonitrile), salts (ammonium sulfate, lithium bromide, lithium chloride, sodium bromide, calcium chloride, sodium chloride); reducing agents (e.g. dithiothreitol, beta- mercaptoethanol, dinitrothiobenzene, and hydrides, such as sodium borohydride), non-ionic and ionic detergents, acids (e.g. hydrochloric acid (HC1), acetic acid (CH3COOH), halogenated acetic acids), hydrophobic molecules (e.g.

phosopholipids), and targeted denaturants. Quantitation of the extent of denaturation can rely on loss of a functional property, such as ability to bind a target molecule, or by physiochemical properties, such as tendency to aggregation, exposure of formerly solvent inaccessible residues, or disruption or formation of disulfide bonds.

The FN3 domain that specifically binds CD 137 may be generated as monomers, dimers, or multimers, for example, as a means to increase the valency and thus the avidity of target molecule binding, or to generate bi- or multispecific scaffolds simultaneously binding two or more different target molecules. The dimers and multimers may be generated by linking monospecific, bi- or multispecific protein scaffolds, for example, by the inclusion of an amino acid linker, for example a linker containing poly-glycine, glycine and serine, or alanine and proline. Exemplary linker include (GS) ₂ , (SEQ ID NO: 35), (GGGS) ₂ (SEQ ID NO:36), (GGGGS) ₅ (SEQ ID NO:37), (AP) ₂ (SEQ ID NO:38), (AP)s (SEQ ID NO: 39), (AP)io (SEQ ID NO: 40), (AP) ₂ o (SEQ ID NO:41) and A(EAAAK) ₅ AAA (SEQ ID NO:42). The dimers and multimers may be linked to each other in a N-to C-direction. The use of naturally occurring as well as artificial peptide linkers to connect polypeptides into novel linked fusion polypeptides is well known in the literature (Hallewell et al, J Biol Chem 264, 5260-5268, 1989; Alfthan et al, Protein Eng. 8, 725-731, 1995; Robinson & Sauer, Biochemistry 35, 109-116, 1996; U.S. Pat. No. 5,856,456).

Half-life extending moieties

The FN3 domains that specifically bind CD 137 may incorporate other subunits for example via covalent interaction. In one aspect of the invention, the FN3 domains that specifically bind CD 137 further comprise a half-life extending moiety. Exemplary half-life extending moieties are albumin, albumin variants, albumin-binding proteins and/or domains, transferrin and fragments and analogues thereof, and Fc regions. An exemplary albumin variant is shown in SEQ ID NO: 43. Amino acid sequences of the human Fc regions are well known, and include IgGl, IgG2, IgG3, IgG4, IgM, IgA and IgE Fc regions.

All or a portion of an antibody constant region may be attached to the FN3 domain that specifically binds CD 137 to impart antibody -like properties, especially those properties associated with the Fc region, such as Fc effector functions such as Clq binding, complement dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, down regulation of cell surface receptors (e.g., B cell receptor; BCR), and may be further modified by modifying residues in the Fc responsible for these activities (for review; see Strohl, Curr Opin Biotechnol. 20, 685-691, 2009).

Additional moieties may be incorporated into the FN3 domains that specifically bind CD137 such as polyethylene glycol (PEG) molecules, such as PEG5000 or PEG20,000, fatty acids and fatty acid esters of different chain lengths, for example laurate, myristate, stearate, arachidate, behenate, oleate, arachidonate, octanedioic acid, tetradecanedioic acid, octadecanedioic acid, docosanedioic acid, and the like, polylysine, octane, carbohydrates (dextran, cellulose, oligo- or polysaccharides) for desired properties. These moieties may be direct fusions with the protein scaffold coding sequences and may be generated by standard cloning and expression techniques. Alternatively, well known chemical coupling methods may be used to attach the moieties to recombinantly produced molecules of the invention. A pegyl moiety may for example be added to the FN3 domain that specifically binds CD137 by incorporating a cysteine residue to the C-terminus of the molecule, or engineering cysteines into residue positions that face away from the CD 137 binding face of the molecule, and attaching a pegyl group to the cysteine using well known methods.

FN3 domains that specifically bind CD 137 incorporating additional moieties may be compared for functionality by several well-known assays. For example, altered properties due to incorporation of Fc domains and/or Fc domain variants may be assayed in Fc receptor binding assays using soluble forms of the receptors, such as the FcyRI, FcyRII, FcyRIII or FcRn receptors, or using well known cell-based assays measuring for example ADCC or CDC, or evaluating pharmacokinetic properties of the molecules of the invention in in vivo models.

Polynucleotides, vectors, host cells

The invention also provides nucleic acids encoding the FN3 domains specifically binding CD137 as isolated polynucleotides or as portions of expression vectors or as portions of linear DNA sequences, including linear DNA sequences used for in vitro transcription/translation, vectors compatible with prokaryotic, eukaryotic or filamentous phage expression, secretion and/or display of the compositions or directed mutagens thereof. Certain exemplary polynucleotides are disclosed herein, however, other polynucleotides which, given the degeneracy of the genetic code or codon preferences in a given expression system, encode the FN3 domains of the invention are also within the scope of the invention.

The invention also provides an isolated polynucleotide encoding the FN3 domain specifically binding CD137 comprising the amino acid sequence of SEQ ID NOs: 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, or 224.

The polynucleotides of the invention may be produced by chemical synthesis such as solid phase polynucleotide synthesis on an automated polynucleotide synthesizer and assembled into complete single or double stranded molecules. Alternatively, the polynucleotides of the invention may be produced by other techniques such as PCR followed by routine cloning. Techniques for producing or obtaining polynucleotides of a given known sequence are well known in the art.

The polynucleotides of the invention may comprise at least one non-coding sequence, such as a promoter or enhancer sequence, intron, polyadenylation signal, a cis sequence facilitating RepA binding, and the like. The polynucleotide sequences may also comprise additional sequences encoding additional amino acids that encode for example a marker or a tag sequence such as a histidine tag or an HA tag to facilitate purification or detection of the protein, a signal sequence, a fusion protein partner such as RepA, Fc or bacteriophage coat protein such as pIX or pill.

The invention also provides a vector comprising at least one polynucleotide of the invention. Such vectors may be plasmid vectors, viral vectors, vectors for baculovirus expression, transposon based vectors or any other vector suitable for introduction of the polynucleotides of the invention into a given organism or genetic background by any means. Such vectors may be expression vectors comprising nucleic acid sequence elements that can control, regulate, cause or permit expression of a polypeptide encoded by such a vector. Such elements may comprise transcriptional enhancer binding sites, RNA polymerase initiation sites, ribosome binding sites, and other sites that facilitate the expression of encoded polypeptides in a given expression system. Such expression systems may be cell- based, or cell-free systems well known in the art.

The invention also provides a host cell comprising the vector of the invention. The FN3 domain that specifically bind CD 137 may be optionally produced by a cell line, a mixed cell line, an immortalized cell or clonal population of immortalized cells, as well known in the art. See, e.g., Ausubel, et al, ed., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NY, NY (1987- 2001); Sambrook, et al, Molecular Cloning: A Laboratory Manual, 2 ^nd Edition, Cold Spring Harbor, NY (1989); Harlow and Lane, Antibodies, a Laboratory Manual, Cold Spring Harbor, NY (1989); Colligan, et al, eds., Current Protocols in Immunology, John Wiley & Sons, Inc., NY (1994-2001); Colligan et al, Current Protocols in Protein Science, John Wiley & Sons, NY, NY, (1997-2001).

The host cell chosen for expression may be of mammalian origin or may be selected from COS-1, COS-7, HEK293, BHK21, CHO, BSC-1, He G2, SP2/0, HeLa, myeloma, lymphoma, yeast, insect or plant cells, or any derivative, immortalized or transformed cell thereof. Alternatively, the host cell may be selected from a species or organism incapable of glycosylating polypeptides, e.g. a prokaryotic cell or organism, such as BL21, BL21(DE3), BL21-GOLD(DE3), XL 1 -Blue, JM109, HMS 174, HMS 174(DE3), and any of the natural or engineered E. coli spp, Klebsiella spp., or Pseudomonas spp strains.

The invention also provides a method of producing the isolated FN3 domain that specifically binds CD 137, comprising culturing the isolated host cell of the invention under conditions such that the isolated FN3 domain that specifically binds CD 137 is expressed, and purifying the FN3 domain.

The FN3 domains that specifically bind CD 137 may be purified from recombinant cell cultures by well-known methods, for example by protein A purification, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite

chromatography and lectin chromatography, or high performance liquid chromatography (HPLC).

Anti-idiotypic antibodies

The present invention also provides an anti-idiotypic antibody binding to the FN3 domain. The invention also provides an anti-idiotypic antibody that specifically binds the FN3 domain comprising the amino acid sequences of one of SEQ ID NOs: 45-224.

Kits

The invention also provides a kit comprising the FN3 domain that specifically binds CD137.

The kit may be used for therapeutic uses and as a diagnostic kit.

In some embodiments, the kit comprises the FN3 domain that specifically binds CD 137 and reagents for detecting the FN3 domain. The kit can include one or more other elements including: instructions for use; other reagents, e.g., a label, an agent useful for chelating, or otherwise coupling, a radioprotective composition; devices or other materials for preparing the FN3 domain that specifically binds CD137 for administration for imaging, diagnostic or therapeutic purpose;

pharmaceutically acceptable carriers; and devices or other materials for administration to a subject.

In some embodiments, the kit comprises the FN3 domain that specifically binds CD137 comprising the amino acid sequences of one of SEQ ID NOs: 45- 224.

Uses of CD 137 binding FN3 domains of the invention

The FN3 domains that specifically bind CD 137 may be used to diagnose, monitor, modulate, treat, alleviate, help prevent the incidence of, or reduce the symptoms of human disease or specific pathologies in cells, tissues, organs, fluid, or, generally, a host. The FN3 domains that specifically bind CD 137 may also be used in imaging CD 137 positive tumor tissue in a subject. The methods of the invention may be used with an animal patient belonging to any classification. Examples of such animals include mammals such as humans, rodents, dogs, cats and farm animals.

The invention provides a method of diagnosing a subject having, or who is likely to develop cancer of a tissue based on the expression of CD 137 by cells of the cancer tissue, methods of predicting success of immunotherapy, methods of prognosis, and methods of treatment.

The invention also provides a method of detecting CD137-expressing cancer cells in a tumor tissue, comprising

obtaining a sample of the tumor tissue from a subject;

detecting whether CD 137 is expressed in the tumor tissue by contacting toe sample of the tumor tissues with the FN3 domain that specifically binds CD 137 comprising the amino acid sequence of one of SEQ ID NOs: 45-224 and detecting the binding between CD 137 and the FN3 domain.

The tissue can be tissue of any organ or anatomical system, for example lung, epithelial, connective, vascular, muscle, neural, skeletal, lymphatic, prostate, cervical, breast, spleen, gastric, intestinal, oral, esophageal, uterine, ovarian, renal or testicular tissue.

CD137 expression may be evaluated using known methods, such as immunohistochemistry or ELISA.

The invention also provides a method of isolating CD137 expressing cells, comprising

obtaining a sample from a subject;

contacting the sample with the FN3 domain that specifically binds CD 137 comprising the amino acid sequence of one of SEQ ID NOs: 45-224, and

isolating the cells bound to the FN3 domains.

The invention also provides a method of detecting CD137-expressing cancer cells in a tumor tissue, comprising

conjugating the FN3 domain that specifically binds CD 137 comprising the amino acid sequence of one of SEQ ID NOs: 45-224 to a detectable label to form a conjugate;

administering the conjugate to a subject; and

visualizing the CD137 expressing cancer cells to which the conjugate is bound. The invention also provides a method of treating a subject having cancer, comprising administering to the subject a FN 3 domain that specifically binds CD 137 of the invention.

In some embodiments, he subject has a solid tumor.

In some embodiments, he subject has a hematological malignancy.

In some embodiments, he solid tumor is a melanoma.

In some embodiments, he solid tumor is a lung cancer.

In some embodiments, he solid tumor is a non-small cell lung cancer (NSCLC).

In some embodiments, the solid tumor is a squamous non-small cell lung cancer (NSCLC).

In some embodiments, the solid tumor is a non-squamous NSCLC.

In some embodiments, the solid tumor is a lung adenocarcinoma.

In some embodiments, the solid tumor is a renal cell carcinoma (RCC).

In some embodiments, the solid tumor is a mesothelioma.

In some embodiments, the solid tumor is a nasopharyngeal carcinoma

(NPC).

In some embodiments, the solid tumor is a colorectal cancer.

In some embodiments, the solid tumor is a prostate cancer.

In some embodiments, the solid tumor is castration-resistant prostate cancer.

In some embodiments, the solid tumor is a stomach cancer.

In some embodiments, the solid tumor is an ovarian cancer.

In some embodiments, the solid tumor is a gastric cancer.

In some embodiments, the solid tumor is a liver cancer.

In some embodiments, the solid tumor is pancreatic cancer.

In some embodiments, the solid tumor is a thyroid cancer.

In some embodiments, the solid tumor is a squamous cell carcinoma of the head and neck.

In some embodiments, the solid tumor is a carcinomas of the esophagus or gastrointestinal tract.

In some embodiments, he solid tumor is a breast cancer. In some embodiments, the solid tumor is a fallopian tube cancer.

In some embodiments, the solid tumor is a brain cancer.

In some embodiments, the solid tumor is an urethral cancer.

In some embodiments, the solid tumor is a genitourinary cancer.

In some embodiments, the solid tumor is an endometriosis.

In some embodiments, the solid tumor is a cervical cancer.

In some embodiments, the solid tumor is a metastatic lesion of the cancer.

In some embodiments, the hematological malignancy is a lymphoma, a myeloma or a leukemia.

In some embodiments, the hematological malignancy is a B cell lymphoma.

In some embodiments, the hematological malignancy is Burkitt's lymphoma.

In some embodiments, the hematological malignancy is Hodgkin's lymphoma.

In some embodiments, the hematological malignancy is a non-Hodgkin's lymphoma.

In some embodiments, the hematological malignancy is a myelodysplastic syndrome.

In some embodiments, the hematological malignancy is an acute myeloid leukemia (AML).

In some embodiments, the hematological malignancy is a chronic myeloid leukemia (CML).

In some embodiments, the hematological malignancy is a chronic myelomoncytic leukemia (CMML).

In some embodiments, the hematological malignancy is a multiple myeloma (MM).

In some embodiments, the hematological malignancy is a plasmacytoma. In some embodiments, the cancer is kidney cancer.

"Treat" or "treatment" refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) an undesired physiological change or disorder, such as the development or spread of cancer. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the condition or disorder as well as those prone to have the condition or disorder or those in which the condition or disorder is to be prevented.

A "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result. A therapeutically effective amount of the FN3 domains that specifically bind CD 137 of the invention may vary according to factors such as the disease state, age, sex, and weight of the individual. Exemplary indicators of an effective FN3 domain that specifically binds CD 137 is improved well-being of the patient, decrease or shrinkage of the size of a tumor, arrested or slowed growth of a tumor, and/or absence of metastasis of cancer cells to other locations in the body.

Administration/ Pharmaceutical Compositions

The invention provides for pharmaceutical compositions of the FN3 domains that specifically bind CD 137, optionally conjugated to a detectable label or a cytotoxic drug of the invention and a pharmaceutically acceptable carrier. For therapeutic use, the FN3 domains that specifically bind CD137of the invention may be prepared as pharmaceutical compositions containing an effective amount of the domain or molecule as an active ingredient in a pharmaceutically acceptable carrier. "Carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the active compound is administered. Such vehicles can be liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. For example, 0.4% saline and 0.3% glycine can be used. These solutions are sterile and generally free of particulate matter. They may be sterilized by conventional, well-known sterilization techniques (e.g., filtration). The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, stabilizing, thickening, lubricating and coloring agents, etc. The concentration of the molecules of the invention in such pharmaceutical formulation can vary widely, i.e., from less than about 0.5%, usually at least about 1% to as much as 15 or 20% by weight and will be selected primarily based on required dose, fluid volumes, viscosities, etc., according to the particular mode of administration selected.

Suitable vehicles and formulations, inclusive of other human proteins, e.g., human serum albumin, are described, for example, in e.g. Remington: The Science and Practice of Pharmacy, 21 ^st Edition, Troy, D.B. ed., Lipincott Williams and Wilkins, Philadelphia, PA 2006, Part 5, Pharmaceutical Manufacturing pp 691-1092, See especially pp. 958-989.

The mode of administration for therapeutic use of the FN3 domains of the invention may be any suitable route that delivers the agent to the host, such as parenteral administration, e.g., intradermal, intramuscular, intraperitoneal, intravenous or subcutaneous, pulmonary; transmucosal (oral, intranasal, intravaginal, rectal), using a formulation in a tablet, capsule, solution, powder, gel, particle; and contained in a syringe, an implanted device, osmotic pump, cartridge, micropump; or other means appreciated by the skilled artisan, as well known in the art. Site specific administration may be achieved by for example intrarticular, intrabronchial, intraabdominal, intracapsular, intracartilaginous, intracavitary, intracelial, intracerebellar, intracerebroventricular, intracolic, intracervical, intragastric, intrahepatic, intracardial, intraosteal, intrapelvic, intrapericardiac, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravascular, intravesical, intralesional, vaginal, rectal, buccal, sublingual, intranasal, or transdermal delivery.

Pharmaceutical compositions can be supplied as a kit comprising a container that comprises the pharmaceutical composition as described herein. A pharmaceutical composition can be provided, for example, in the form of an injectable solution for single or multiple doses, or as a sterile powder that will be reconstituted before injection. Alternatively, such a kit can include a dry-powder disperser, liquid aerosol generator, or nebulizer for administration of a pharmaceutical composition. Such a kit can further comprise written information on indications and usage of the pharmaceutical composition.

While having described the invention in general terms, the embodiments of the invention will be further disclosed in the following examples that should not be construed as limiting the scope of the claims.

EXAMPLES

EXAMPLE 1. Construction of Tencon libraries with randomized loops

Tencon (SEQ ID NO: l) is an immunoglobulin-like scaffold, fibronectin type III (FN3) domain, designed from a consensus sequence of fifteen FN3 domains from human tenascin-C (Jacobs et al, Protein Engineering, Design, and Selection, 25: 107-117, 2012; U.S. Pat. No. 8,278,419). The crystal structure of Tencon shows six surface-exposed loops that connect seven beta-strands. These loops, or selected residues within each loop, can be randomized in order to construct libraries of fibronectin type III (FN3) domains that can be used to select novel molecules that bind to specific targets.

Tencon:

LPAPKNLVVSEVTEDSLRLSWTAPDAAFDSFLIQYQESEKVGEAINLTVPGS ERSYDLTGLKPGTEYTVSIYGVKGGHRSNPLSAEFTT (SEQ ID NO 1):

Various libraries were generated using the tencon scaffold and various design strategies. In general, libraries TCL1 and TCL2 produced good binders.

Generation of TCL1 and TCL2 libraries are described in detail in Int. Pat. Publ. No. WO/2014081944A2.

Construction of TCL1 library

A library designed to randomize only the FG loop of Tencon (SEQ ID NO:l), TCL1, was constructed for use with the cw-display system (Jacobs et al, Protein Engineering, Design, and Selection, 25: 107-117, 2012). In this system, a single-strand DNA incorporating sequences for a Tac promoter, Tencon library coding sequence, RepA coding sequence, cis-element, and ori element is produced. Upon expression in an in vitro transcription/translation system, a complex is produced of the Tencon-RepA fusion protein bound in cis to the DNA from which it is encoded. Complexes that bind to a target molecule are then isolated and amplified by polymerase chain reaction (PCR), as described below.

Construction of the TCLl library for use with cw-display was achieved by successive rounds of PCR to produce the final linear, double-stranded DNA molecules in two halves; the 5' fragment contains the promoter and Tencon sequences, while the 3' fragment contains the repA gene and the cis- and ori elements. These two halves are combined by restriction digest in order to produce the entire construct. The TCLl library was designed to incorporate random amino acids only in the FG loop of Tencon, KGGHRSN (SEQ ID NO:55). NNS codons were used in the construction of this library, resulting in the possible incorporation of all 20 amino acids and one stop codon into the FG loop. The TCLl library contains six separate sub-libraries, each having a different randomized FG loop length, from 7 to 12 residues, in order to further increase diversity.

TCLl library (SEQ ID NO:2)

LPAPKNLVVSEVTEDSLRLSWTAPDAAFDSFLIQYQESEKVGEAINLTVPGS ERS YDLTGLKP GTEYTV SI YGVX7- 12PL S AEFTT;

wherein

Construction of TCL2 Library

TCL2 library was constructed in which both the BC and the FG loops of Tencon were randomized and the distribution of amino acids at each position was strictly controlled. Table 3 shows the amino acid distribution at desired loop positions in the TCL2 library. The designed amino acid distribution had two aims. First, the library was biased toward residues that were predicted to be structurally important for Tencon folding and stability based on analysis of the Tencon crystal structure and/or from homology modeling. For example, position 29 was fixed to be only a subset of hydrophobic amino acids, as this residue was buried in the hydrophobic core of the Tencon fold. A second layer of design included biasing the amino acid distribution toward that of residues preferentially found in the heavy chain HCDR3 of antibodies, to efficiently produce high-affinity binders (Birtalan et al, J Mol Biol 377: 1518-28, 2008; Olson et al, Protein Sci 16:476-84, 2007). Towards this goal, the "designed distribution" in Table 2 refers to the distribution as follows: 6% alanine, 6% arginine, 3.9% asparagine, 7.5% aspartic acid, 2.5% glutamic acid, 1.5% glutamine, 15% glycine, 2.3% histidine, 2.5% isoleucine, 5% leucine, 1.5% lysine, 2.5% phenylalanine, 4% proline, 10% serine, 4.5% threonine, 4% tryptophan, 17.3% tyrosine, and 4% valine. This distribution is devoid of methionine, cysteine, and STOP codons.

*residue numbering is based on Tencon sequence of SEQ ID NO: 1

Subsequently, these libraries were improved by various ways, including building of the libraries on a stabilized Tencon framework (U.S. Pat. No.

8,569,227) that incorporates substitutions E11R/L17A/N46V/E86I (Tencon27; SEQ ID NO:4) when compared to the wild type tencon as well as altering of the positions randomized in the BC and FG loops. Tencon27 is described in Int. Pat. Appl. No. WO2013049275. From this, new libraries designed to randomize only the FG loop of Tencon (library TCL9), or a combination of the BC and FG loops (library TCL7) were generated. These libraries were constructed for use with the cis-display system (Odegrip et al, Proc Natl Acad Sci U S A 101 : 2806-2810, 2004). The details of this design are shown below:

For library construction, DNA fragments encoding randomized BC loops (lengths 6-9 positions) or FG loops (lengths 7-12 positions) were synthesized using Slonomics technology (Sloning Biotechnology GmbH) so as to control the amino acid distribution of the library and to eliminate stop codons. Two different sets of DNA molecules randomizing either the BC loop or the FG loops were synthesized independently and later combined using PCR to produce the full library product.

Construction of FG loop libraries (TCL9)

A set of synthetic DNA molecules consisting of a 5' Tac promoter followed by the complete gene sequence of Tencon with the exception of randomized codons in the FG loop was produced (SEQ ID NOs: 26-31). For FG loop randomization, all amino acids except cysteine and methionine were encoded at equal percentages. The lengths of the diversified portion are such that they encode for 7, 8, 9, 10, 11, or 12 amino acids in the FG loop. Sub-libraries of each length variation were synthesized individually at a scale of 2ug and then amplified by PCR using oligos Sloning-FOR (SEQ ID NO:9) and Sloning-Rev (SEQ ID NO: 10). The 3' fragment of the library is a constant DNA sequence containing elements for display, including a PspOMI restriction site, the coding region of the repA gene, and the cis- and ori elements. PCR reactions were performed to amplify this fragment using a plasmid (pCR4Blunt) (Invitrogen) as a template with M13 Forward and M13 Reverse primers. The resulting PCR products were digested by PspOMI overnight and gel -purified. To ligate the 5' portion of library DNA to the 3' DNA containing rep A gene, 2 pmol (~540ng to 560ng) of 5' DNA was ligated to an equal molar (-1.25 μg) of 3' repA DNA in the presence of Notl and PspOMI enzyme and T4 ligase at 37°C overnight. The ligated library product was amplified by using 12 cycles of PCR with oligos POP2250 (SEQ ID NO: 11) and DigLigRev (SEQ ID NO: 12). For each sub-library, the resulting DNA from 12 PCR reactions were combined and purified by Qiagen spin column. The yield for each sub-library of TCL9 ranged from 32-34 μg.

Construction of FG/BC Loop libraries (TCL7)

The TCL7 library provides for a library with randomized Tencon BC and FG loops. In this library, BC loops of lengths 6-9 amino acids were mixed combinatorially with randomized FG loops of 7-12 amino acids in length.

Synthetic Tencon fragments BC6, BC7, BC8, and BC9 (SEQ ID NOs: 13-16, respectively) were produced to include the Tencon gene encoding for the N- terminal portion of the protein up to and including residue VX such that the BC loop is replaced with either 6, 7, 8, or 9 randomized amino acids. These fragments were synthesized prior to the discovery of L17A, N46V and E83I mutations (CEN5243) but these mutations were introduced in the molecular biology steps described below. In order to combine this fragment with fragments encoding for randomized FG loops, the following steps were taken.

First, a DNA fragment encoding the Tac promoter and the 5' sequence of Tencon up to the nucleotide encoding for amino acid A17 (130mer-L17A, SEQ ID NO: 17 ) was produced by PCR using oligos POP2222ext (SEQ ID NO: 18 ) and LSI 114 (SEQ ID NO: 19). This was done to include the L17A mutation in the library (CEN5243). Next, DNA fragments encoding for Tencon residues R18- V75 including randomized BC loops were amplified by PCR using BC6, BC7, BC8, or BC9 as a template and oligos LSI 115 (SEQ ID NO:20) and LSI 117 (SEQ ID NO:21). This PCR step introduced a Bsal site at the 3' end. These DNA fragments were subsequently joined by overlapping PCR using oligos POP2222ext and LS 1117 as primers. The resulting PCR product of 240bp was pooled and purified by Qiagen PCR purification kit. The purified DNA was digested with Bsal-HF and gel purified.

Fragments encoding the FG loop were amplified by PCR using FG7, FG8, FG9, FG10, FG11, and FG12 as templates with oligonucleotides SDG10 (SEQ ID NO:22) and SDG24 (SEQ ID NO:23) to incorporate a Bsal restriction site and N46V and E86I variations (CEN5243).

The digested BC fragments and FG fragments were ligated together in a single step using a 3-way ligation. Four ligation reactions in the 16 possible combinations were set up, with each ligation reaction combining two BC loop lengths with 2 FG loop lengths. Each ligation contained -300 ng of total BC fragment and 300 ng of the FG fragment. These 4 ligation pools were then amplified by PCR using oligos POP2222 (SEQ ID NO:24) and SDG28 SEQ ID NO:25). 7.5 μg of each reaction product were then digested with Notl and cleaned up with a Qiagen PCR purification column. 5.2 μg of this DNA, was ligated to an equal molar amount of RepA DNA fragment (-14 μg) digested with PspOMI and the product amplified by PCR using oligos POP2222.

EXAMPLE 2: Generation of Tencon libraries having alternative binding surfaces

The choice of residues to be randomized in a particular library design governs the overall shape of the interaction surface created. X-ray crystallographic analysis of an FN3 domain containing scaffold protein selected to bind maltose binding protein (MBP) from a library in which the BC, DE, and FG loops were randomized was shown to have a largely curved interface that fits into the active site of MBP (Koide et al., Proc Natl Acad Sci U S A 104: 6632-6637, 2007). In contrast, an ankyrin repeat scaffold protein that was selected to bind to MBP was found to have a much more planar interaction surface and to bind to the outer surface of MBP distant from the active (Binz et al, Nat Biotechnol 22: 575-582, 2004). These results suggest that the shape of the binding surface of a scaffold molecule (curved vs. flat) may dictate what target proteins or specific epitopes on those target proteins are able to be bound effectively by the scaffold. Published efforts around engineering protein scaffolds containing FN3 domains for protein binding has relied on engineering adjacent loops for target binding, thus producing curved binding surfaces. This approach may limit the number of targets and epitopes accessible by such scaffolds.

Tencon and other FN3 domains contain two sets of CDR-like loops lying on the opposite faces of the molecule, the first set formed by the BC, DE, and FG loops, and the second set formed by the AB, CD, and EF loops. The two sets of loops are separated by the beta-strands that form the center of the FN3 structure. If the image of the Tencon is rotated by 90 degrees, an alternative surface can be visualized. This slightly concave surface is formed by the CD and FG loops and two antiparallel beta-strands, the C and the F beta-strands, and is herein called the C-CD-F-FG surface. The C-CD-F-FG surface can be used as a template to design libraries of protein scaffold interaction surfaces by randomizing a subset of residues that form the surface. Beta-strands have a repeating structure with the side chain of every other residue exposed to the surface of the protein. Thus, a library can be made by randomizing some or all surface exposed residues in the beta strands. By choosing the appropriate residues in the beta-strands, the inherent stability of the Tencon scaffold should be minimally compromised while providing a unique scaffold surface for interaction with other proteins.

Library TCL14 (SEQ ID NO:7), was designed into Tencon27 scaffold (SEQ ID NO:4).

A full description of the methods used to construct this library is described in US. Pat. Publ. No. 2013/0226834.

The two beta strands forming the C-CD-F-FG surface in Tencon27 have a total of 9 surface exposed residues that could be randomized; C-strand: S30, L32, Q34, Q36; F-strand: E66, T68, S70, Y72, and V74, while the CD loop has 6 potential residues: S38, E39, K40, V41, G42, and E43 and the FG loop has 7 potential residues: K75, G76, G77, H78, R79, S80, and N81. Select residues were chosen for inclusion in the TCL14 design due to the larger theoretical size of the library if all 22 residues were randomized.

Thirteen positions in Tencon were chosen for randomizing: L32, Q34 and Q36 in C-strand, S38, E39, K40 and V41 in CD-loop, T68, S70 and Y72 in F- strand, H78, R79, and N81 in FG-loop. In the C and F strands S30 and E66 were not randomized as they lie just beyond the CD and FG loops and do not appear to be as apparently a part of the C-CD-F-FG surface. For the CD loop, G42 and E43 were not randomized as glycine, providing flexibility, can be valuable in loop regions, and E43 lies at the junction of the surface. The FG loop had K75, G76, G77, and S80 excluded. The glycines were excluded for the reasons above while careful inspection of the crystal structures revealed S80 making key contacts with the core to help form the stable FG loop. K75 faces away from the surface of the C-CD-F-FG surface and was a less appealing candidate for randomization.

Although the above mentioned residues were not randomized in the original TCL14 design, they could be included in subsequent library designs to provide additional diversity for de novo selection or for example for an affinity maturation library on a select TCL14 target specific hit.

Subsequent to the production of TCL14, 3 additional Tencon libraries of similar design were produced. These two libraries, TCL19, TCL21 and TCL23, are randomized at the same positions as TCL14 (see above) however the distribution of amino acids occurring at these positions is altered (Table 3).

TCL19 and TCL21 were designed to include an equal distribution of 18 natural amino acids at every position (5.55% of each), excluding only cysteine and methionine. TCL23 was designed such that each randomized position approximates the amino acid distribution found in the HCDR3 loops of functional antibodies (Birtalan et al, J Mol Biol 377: 1518-1528, 2008) as described in Table 3. As with the TCL21 library, cysteine and methionine were excluded.

A third additional library was built to expand potential target binding surface of the other libraries library. In this library, TCL24, 4 additional Tencon positions were randomized as compared to libraries TCL14, TCL19, TCL21 , and TCL23. These positions include N46 and T48 from the D strand and S84 and 186 from the G strand. Positions 46, 48, 84, and 86 were chosen in particular as the side chains of these residues are surface exposed from beta-strands D and G and lie structurally adjacent to the randomized portions of the C and F strand, thus increasing the surface area accessible for binding to target proteins. The amino acid distribution used at each position for TCL24 is identical to that described for TCL19 and TCL21 in Table 3.

Table 3. Amino acid frequency (%) at each randomized position for TCL21, TCL23, and TCL24.

Generation of TCL21, TCL23, and TCL24 libraries

The TCL21 library was generated using Colibra library technology (Isogenica) in order to control amino acid distributions. TCL19, TCL23, and TCL24 gene fragments were generated using Slonomics technology (Morphosys) to control amino acid distributions. PCR was used to amplify each library following initial synthesis followed by ligation to the gene for RepA in order to be used in selections using the CIS-display system (Odegrip et al, Proc Natl Acad Sci U S A 101 : 2806-2810, 2004) as described above for the loop libraries. EXAMPLE 3: Selection of fibronectin type III (FN3) domains that bind CD137

Panning

FN3 domains specific for human CD137 were selected via CIS-Display (Odegrip et al 2004) using recombinant biotinylated CD137 protein (Fc-fusion protein, R&D Systems 838-4B). For in vitro transcription and translation (ITT), 3 μg of DNA from Centyrin libraries TCL18, TCL19, TCL21, TCL23, and TCL24 (See accompanying library description document) were incubated at 30°C with 0.1 mM complete amino acids, IX S30 premix components, and 15 of S30 extract (Isogenica) in a total volume of 50 μL. After 1 hour, 375 of blocking solution (2% BSA in PBS, Invitrogen) was added and reactions were incubated on a cold block for 15 minutes. Unbound library members were removed by successive washes with TBST and TBS. After washing, DNA was eluted from the target protein by heating to 75°C for 10 minutes and amplified by PCR using KOD polymberase for further rounds of panning. High affinity binders were isolated by successively lowering the concentration of target CD 137 during each round from 400 nM to 100 nM and increasing the washing stringency.

Outputs from the fifth round panning were subjected to four additional rounds of off-rate selection. Library transcription and translation was performed as described above after which, the ITT reactions were incubated with biotinylated recombinant CD 137 proteins and captured on neutravidin or streptavidin coated magnetic beads, before being washed in TBST extensively then subsequently washed in 5 uM cold recombinant CD 137 protein for 1 hour. The biotinylated target antigen concentration was reduced from 25 nM in rounds 6 and 7 to 2.5 nM in rounds 8 and 9.

Following panning, genes encoding the selected FN3 domains were amplified by PCR, subcloned into a pET vector modified to include a ligase independent cloning site, and transformed into BL21 (DE3) (Stratagene) cells for soluble expression in E. coli using standard molecular biology techniques. A gene sequence encoding a C-terminal poly-histidine tag was added to each Centyrin to enable purification and detection. Cultures were grown to an optical density of 0.6-0.8 in TB medium supplemented with 100 μg/mL carbenicillin in 1 mL 96-well blocks at 37°C before the addition of IPTG to 1 mM, at which point the temperature was reduced to 30°C. Cells were harvested approximately 16 hours later by centrifugation and frozen at -20°C. Cell lysis was achieved by incubating each pellet in 0.6 mL of BugBuster® HT lysis buffer (Novagen EMD Biosciences) supplemented with 0.2 mg/mL lysozyme with shaking at room temperature for 30 minutes.

Biochemical Screening for FN3 domains that Recombinant CD 137

Streptavidin-coated Maxisorp plates (Nunc catalog 436110) were blocked for lh in Starting Block T20 (Pierce) and then coated with biotinylated CD 137 (using same antigen as in panning) or negative controls (an unrelated Fc-fused recombinant protein and human serum albumin) for lh. Plates were rinsed with TBST and diluted lysate was applied to plates for lh. Following additional rinses, wells were treated with HRP-conjugated anti-Centyrin antibody (PAB25) for lh and then assayed with POD (Roche catalog 11582950001). The DNA from Centyrin lysates with signals at least 10-fold ELISA signal above that of Fc and HSA controls were sequenced resulting in 78 (Table 1) and 102 (Table 2) unique, readable Centyrin sequences isolated from Round 5 and Round 9 screening respectively.

High-throughput Expression of anti-CD 137 FN3 domains

102 Isolated clones from unique hits identified by biochemical binding ELISA from Round 9 were combined for growth into 96-well block plate; clones grew in 1 mL cultures (LB media supplemented with kanamycin for selection) at 37°C overnight with shaking. For protein expression in 96-block plates, 1 mL TB media supplemented with kanamycin was inoculated with 50 uL of the overnight culture and grown at 37°C with continual shaking at 300rpm until OD600 = 0.6-1. Once the target OD was reached, protein expression was induced with addition of IPTG to 1 mM; plates were transferred to 30°C (300 rpm) for overnight growth. Overnight cultures were centrifuged to harvest the cells; bacterial pellets were stored at -80°C until ready for use. Pellets were lysed with BugBuster® HT lysis buffer (Novagen EMD Biosciences) and His-tagged FN3 domains purified from the clarified lysates with His MultiTrap™ HP plates (GE Healthcare) and eluted in buffer containing 20 mM sodium phosphate, 500 mM sodium chloride, and 250 mM imidazole at pH 7.4. Purified samples were exchanged into PBS pH 7.4 for analysis using PD MultiTrap™ G-25 plates (GE Healthcare).

Size Exclusion Chromatography Analysis

Size exclusion chromatography was used to determine the aggregation state of anti-CD137 FN3 domains. Aliquots (10 μL) of each purified Centyrin were injected onto a Superdex 75 5/150 column (GE Healthcare) at a flow rate of 0.3 mL/min in a mobile phase of PBS pH 7.4. Elution from the column was monitored by absorbance at 280 nm. Tencon protein was included in each run as a control. Agilent ChemStation software was used to analyse the elution profiles. 46 anti- CD 137 FN3 domains demonstrated a retention time between 5.2 and 6.4 minutes and only a single SEC peak indicative of monomeric protein.

Table 4. Summary of Round 5 Screening Hits

Table 5. Summary of Round 9 Screening Hits EXAMPLE 4: Characterization of fibronectin type III (FN3) domains that bind CD 137

Fluorescence-activated cell sorting (FACS)

Cell surface binding was analyzed via flow cytometry. Cenyrins were prepared at a maximal concentration of 500 nM in the presence of 125 nM anti His-mlgGl antibody and then serially diluted in PBS/1% FCS buffer. Samples were then applied to approx 100 000 CHO-K1 cells expressing the extracellular domain of human CD 137 on their surface. Unbound Centyrin/ Antibody complexes were washed away and bound FN3 domains were detected by the addition of goat anti mouse-FITC labeled antibody. Samples were then aquired by flow cytometry. Subsequently, Mean flurescence intensity was plotted against the log Centyrin concentration and EC50 values were calculated by nonlinear regression using GraphPad Prism.

Binding analysis with Biacore

For selected FN3 domains, binding to recombinant CD137-Fc was evaluated using surface plasmon resonance (Biacore T100). For each cycle approximately 500 RU of recombinant human CD137-Fc-IgGl was captured via an anti human IgG immobilized on the surface of a CM5 chip. Once CD137-Fc protein was captured, increasing concentrations of Centyrin candidates were injected for 120s and dissociation was then analyzed for 240s. A flow cell immobilized with just the anti human IgGl antibody served as reference flow cell. Kd values were subsequently extrapolated using a 1 : 1 kinetic binding model (BiaEvaluation software).

Table 6. Centyrin binding analysis by FACS and Biacore

SEQUENCES

SEQ ID NO: l= Original Tencon Sequence

LPAPKNLVVSEVTEDSLRLSWTAPDAAFDSFLIQYQESEKVGEAINLTVPGSERSY DLTGLKPGTEYTVSIYGVKGGHRSNPLSAEFTT

SEQ ID NO:2= TCL1 library

LPAPKNLVVSEVTEDSLRLSWTAPDAAFDSFLIQYQESEKVGEAINLTVPGSERSY DLTGLKPGTEYTVSIYGV(X)v ₂ PLSAEFTT;