CD71 binding fibronectin type iii domains

Abstract

To provide polypeptides, such as fibronectin type III (FN3) domains that can bind CD71, their conjugates, isolated nucleotides encoding the molecules, vectors, host-cells, and further to provide methods of making and using the same.SOLUTION: Provided are: polypeptides including specific sequences or an amino acid sequence that is at least 90% identical to an amino acid sequence of any combination thereof; or a polypeptide including specific sequences or an amino acid sequence of any combination thereof. In some embodiments, an FN3 domain does not naturally exist. In some embodiments, the FN3 domain binds to human CD71 at a site on CD71 that does not compete with transferrin binding to CD71.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 62 / 914,643 (filed October 14, 2019) and U.S. Provisional Application No. 62 / 949,020 (filed December 17, 2019), each of which is incorporated by reference in its entirety herein.

[0002] Field The present embodiments relate to a fibronectin type III domain (FN3) that specifically binds to cluster of differentiation 71 (CD71), and methods of making and using the molecule. [Background technology]

[0003] background CD71, also known as transferrin receptor 1, is a transmembrane receptor essential for iron transport into cells. It is highly expressed in many tumor types and at the blood-brain barrier, making it an important target for drug delivery. After binding to iron-loaded transferrin, CD71 is rapidly invaginated into the plasma membrane and efficiently recycled back to the cell surface. Studies with CD71 antibody-drug conjugates suggest that targeting CD71 can improve the specificity and selectivity of drug delivery and expand the therapeutic index. Furthermore, studies with anti-CD71 monoclonal antibodies indicate that binding affinity can play a key role in enabling blood-brain barrier transcellular transport. Antibodies with high affinity for CD71 are rapidly internalized and alter normal receptor trafficking so that instead of recycling, the receptor is targeted to lysosomes for degradation. In contrast, antibodies with low affinity for CD71 allow receptor recycling and higher brain exposure.

[0004] Although antibodies or antibody fragments are the most widely used class of therapeutic proteins when high affinity and specificity for a target molecule is desired, non-antibody proteins can also be engineered to bind such targets. These "alternative scaffold" proteins have advantages over traditional antibodies due to their small size, lack of disulfide bonds, high stability, ability to be expressed in prokaryotic hosts, easy purification, and they are easily conjugated to drugs / toxins, penetrate efficiently into tissues, and are easily formatted into multiselective binders. Summary of the Invention [Problem to be solved by the invention]

[0005] One such alternative scaffold is the immunoglobulin (Ig) fold. This fold is found in the variable regions of antibodies as well as thousands of non-antibody proteins. It has been shown that the tenth fibronectin type III (FN3) repeat from one such Ig protein, human fibronectin, can tolerate a large number of mutations in the surface-exposed loops while retaining the overall Ig fold structure. Therefore, what is needed is an FN3 domain that can specifically bind to CD71, and a method of using such a molecule for cancer therapy. [Means for solving the problem]

[0006] Abstract In some embodiments, an FN3 domain (e.g., a polypeptide) is provided that specifically binds to a CD71 protein. In some embodiments, the FN3 domain is isolated. In some embodiments, the FN3 domain is recombinant. In some embodiments, the FN3 domain is non-naturally occurring.

[0007] In some embodiments, an FN3 domain is provided that comprises the amino acid sequence of SEQ ID NO: 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 or 62. In some embodiments, an FN3 domain is provided that comprises the amino acid sequence of SEQ ID NO: 146, 214, 104, 259, 134, 92, 302, 235, 237, 152, 238, 136, 197, 212, 296, 226, 261, 307, 115, 112, 278, 297, 96, 222, 95, 233, 217, 252, 194, 164, 168, 174, 190, 257, 303, 284, 85, or 149. In some embodiments, an FN3 domain is provided that comprises the amino acid sequence of SEQ ID NO: 81-309. In some embodiments, the FN3 domain binds to CD71. In some embodiments, the FN3 domain binds to human CD71 at a site on CD71 that does not compete with transferrin binding to CD71. In some embodiments, an FN3 domain is provided that comprises the amino acid sequence of SEQ ID NO: 146, 214, 104, 259, 134, 92, 302, 235, 237, 152, 238, 136, 197, 212, 296, 226, 261, 307, 115, 112, 278, 297, 96, 222, 95, 233, 217, 252, 194, 164, 168, 174, 190, 257, 303, 284, 85, or 149. In some embodiments, the FN3 domain specifically binds to CD71. In some embodiments, a polypeptide is provided that comprises more than one FN3 domain connected by a linker, such as a flexible linker. In some embodiments, the polypeptide comprises two, three, or four FN3 domains connected to each other by one or more linkers between the domains.

[0008] In some embodiments, an isolated polynucleotide encoding the FN3 domain described herein is provided.

[0009] In some embodiments, a vector is provided that comprises the polynucleotides described herein.

[0010] In some embodiments, a host cell is provided that comprises a vector described herein.

[0011] In some embodiments, a method for producing an FN3 domain is provided. In some embodiments, the method comprises culturing a host cell comprising a vector encoding or expressing the FN3 domain. In some embodiments, the method further comprises purifying the FN3 domain. In some embodiments, the FN3 domain specifically binds to CD71.

[0012] In some embodiments, a pharmaceutical composition is provided that includes an FN3 domain that binds CD71 and a pharma- ceutically acceptable carrier.

[0013] In some embodiments, anti-idiotypic antibodies that bind to the FN3 domain that binds to CD71 are provided.

[0014] In some embodiments, a kit is provided that includes one or more FN3 domains.

[0015] In some embodiments, a method is provided for detecting cancer cells expressing CD71 in tumor tissue. In some embodiments, the method comprises: and determining whether CD71 protein is expressed in the tumor tissue by contacting the sample of tumor tissue with an FN3 domain that binds to a CD71 protein comprising one of the amino acid sequences of SEQ ID NOs: 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, or 81-309, and detecting binding between the CD71 protein and the FN3 domain.

[0016] In some embodiments, methods of isolating cells expressing CD71 are provided, which in some embodiments include obtaining a sample from a subject, contacting the sample with an FN3 domain that binds to a CD71 protein comprising one of the amino acid sequences of SEQ ID NOs: 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, or 81-309, and isolating cells that bind to the FN3 domain.

[0017] In some embodiments, a method for detecting cancer cells expressing CD71 in tumor tissue is provided. In some embodiments, the method includes: binding an FN3 domain that binds to a CD71 protein comprising one of the amino acid sequences of SEQ ID NOs: 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, or 81-309 to a detectable label to form a conjugate; administering the conjugate to a subject; and visualizing the cancer cells expressing CD71 to which the conjugate is bound.

[0018] In some embodiments, a method of treating cancer in a subject in need thereof is provided. In some embodiments, the method comprises administering a polypeptide that binds to CD71. In some embodiments, the polypeptide is an FN3 domain that binds to CD71. In some embodiments, the polypeptide is selected from SEQ ID NOs: 146, 214, 104, 259, 134, 92, 302, 235, 237, 152, 238, 136, 197, 212, 296, 226, 261, 307, 115, 112, 278, 297, 96, 222, 95, 233, 217, 252, 194, 164, 168, 174, 190, 257, 303, 284, 85, 149, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, or 81-309, or a polypeptide provided herein linked or conjugated to a therapeutic agent.

[0019] In some embodiments, a method for treating a neurological condition and / or a brain tumor is provided. In some embodiments, the method comprises administering a polypeptide or pharmaceutical composition provided herein to a subject. In some embodiments, the brain tumor is selected from the group consisting of non-malignant, benign, and malignant brain tumors.

[0020] In some embodiments, a method for delivering an agent of interest to a CD71 positive cell is provided. In some embodiments, the method includes contacting the cell with an agent of interest coupled to a FN3 domain that binds to CD71, such as a polypeptide provided herein. In some embodiments, the agent of interest is a chemotherapeutic agent, a drug, a growth inhibitor, a toxin, a radioisotope, an antitubulin agent, a polynucleotide, an siRNA molecule, an antisense molecule, an RNA molecule, a DNA molecule, a DNA minor groove binder, a DNA replication inhibitor, an alkylating agent, an antibiotic, an antifolate, an antimetabolite, a chemotherapy. a sensitizer, a topoisomerase inhibitor, or a vinca alkaloid.

[0021] In some embodiments, the polypeptide is an FN3 protein that binds to CD71 at a site that does not compete with or inhibit transferrin binding to CD71.

[0022] In some embodiments, methods are provided to identify FN3 proteins that bind to CD71 at a site that does not compete with or inhibit transferrin binding to CD71. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] Detailed Description of the Disclosure 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.

[0024] "Fibronectin type III (FN3) domain" (FN3 domain) refers to a domain that occurs frequently in proteins, including fibronectins, tenascins, 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, e.g., as described in U.S. Pat. No. 8,278,419. Individual FN3 domains are referred to by domain number and protein name, for example, the third FN3 domain of tenascin (TN3) or the tenth FN3 domain of fibronectin (FN10).

[0025] The term "capture agent" refers to a substance that binds to a particular type of cell and allows for isolation of that cell from other cells. Exemplary capture agents are magnetic beads, ferrofluids, encapsulation reagents, molecules that bind to specific cell types, etc.

[0026] "Sample" refers to a collection of fluids, cells, or tissues, such as those isolated from a subject, as well as fluids, cells, or tissues present within a subject. Exemplary samples are tissue biopsies, fine needle aspirates, surgically removed tissues, organ cultures, cell cultures, and biological fluids, such as blood, serum and serous fluid, plasma, lymph, urine, saliva, cyst fluid, tears, feces, sputum, mucosal secretions of secretory tissues and organs, vaginal secretions, ascites fluid, pleural, pericardial, peritoneal, abdominal and other body cavity fluids, fluids collected by bronchial lavage, synovial fluid, liquid solutions that have been in contact with a subject or biological source, such as cell and organ culture media, including cell or organ conditioned media, and lavage fluids.

[0027] "Substituting" or "substituted" or "mutating" or "mutated" refers to changing, deleting or inserting one or more amino acids or nucleotides in a polypeptide or polynucleotide sequence to generate a variant of that sequence.

[0028] "Variant" refers to a polypeptide or polynucleotide that differs from a reference polypeptide or polynucleotide by one or more modifications, such as a substitution, insertion or deletion.

[0029] "Specifically binds" or "specific binding" refers to binding to its target, such as CD71, of approximately 1 x 10 -6 M or less, e.g., about 1x10 -7 M or smaller, approx. 1x10 -8 M or smaller, approx. 1x10 -9 M or smaller, approx. 1x10 -10 M or smaller, approx. 1x10 -11 M or smaller, approx. 1x10 -12 M or less, or about 1x10-13 M or a lower dissociation constant (K D ) Alternatively, "specific binding" refers to the ability of an FN3 domain to bind its target (e.g., CD71) at least 5-fold higher than a negative control in a standard ELISA assay. In some embodiments, the negative control is an FN3 domain that does not bind to CD71. In some embodiments, an FN3 domain that specifically binds to CD71 may have cross-reactivity to other related antigens, e.g., the same given antigen (homolog) from other species, such as Macaca Fascicularis (cynomolgus monkey, cyno) or Pan troglodytes (chimpanzee).

[0030] A "library" refers to a collection of variants. A library can be composed of variants of polypeptides or polynucleotides.

[0031] "Stability" refers to the ability of a molecule to remain folded under physiological conditions so as to retain at least one of its normal functional activities, e.g., binding to a given antigen, such as CD71.

[0032] "CD71" refers to the human CD71 protein having the amino acid sequence of SEQ ID NO: 32 or 80, in some embodiments, SEQ ID NO: 32 is the full-length human CD71 protein. In some embodiments, SEQ ID NO: 80 is the extracellular domain of human CD71.

[0033] "Tencon" refers to a synthetic fibronectin type III (FN3) domain having the sequence shown in SEQ ID NO:1 and described in US Patent Publication No. 2010 / 0216708.

[0034] "Cancer cell" or "tumor cell" refers to cancerous, precancerous or transformed cells, either in vivo, ex vivo and in tissue culture, that have spontaneous or induced phenotypic changes that do not necessarily involve the incorporation of new genetic material. Transformation can result from infection with a transforming virus and incorporation of new genomic nucleic acid, or uptake of exogenous nucleic acid, but can also occur spontaneously or following exposure to carcinogens, thereby mutating endogenous genes. Transformation / cancer is exemplified, for example, by morphological changes, cellular immortalization, aberrant growth control, lesion formation, proliferation, malignant lesions, tumor-specific marker levels, invasiveness, tumor growth or inhibition in suitable animals such as nude mice, in vitro, in vivo and ex vivo (Freshney, Culture of Animals, 1999, 144:131-135). Cells: A Manual of Basic Technique (3rd edition, 1994)).

[0035] "Vector" refers to a polynucleotide capable of being replicated in a biological system or that can be transferred between such systems. Vector polynucleotides typically contain elements such as origins of replication, polyadenylation signals, or selection markers that function to facilitate the replication or maintenance of these polynucleotides in biological systems. Examples of such biological systems may include cells, viruses, animals, plants, and reconstituted biological systems that utilize biological components capable of replicating the vector. The polynucleotides that comprise the vector may be DNA or RNA molecules or hybrids thereof.

[0036] An "expression vector" refers to a vector that can be utilized in a biological system or reconstituted biological system to direct the translation of a polypeptide encoded by a polynucleotide sequence present in the expression vector.

[0037] "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.

[0038] "Polypeptide" or "protein" refers to a molecule containing at least two amino acid residues linked by a peptide bond to form the polypeptide. Small polypeptides, less than about 50 amino acids, may be referred to as "peptides."

[0039] "Valency" refers to the presence in a molecule of a particular number of specific binding sites for an antigen. Thus, the terms "monovalent," "bivalent," "tetravalent," and "hexavalent" refer to the presence in a molecule of one, two, four, and six specific binding sites for an antigen, respectively.

[0040] A "subject" includes any human or non-human animal. A "non-human animal" includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc. Except where noted, the terms "patient" or "subject" are used interchangeably.

[0041] "Isolated" refers to a homogenous population of a molecule (e.g., a synthetic polynucleotide or a polypeptide such as an FN3 domain) that has been substantially separated and / or purified from other components of the system in which it is produced, such as a recombinant cell, as well as a protein that has been subjected to at least one purification or isolation step. An "isolated FN3 domain" refers to an FN3 domain that is substantially free of other cellular material and / or chemicals, and includes FN3 domains that have been isolated to greater degrees of purity, e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% purity.

[0042] Composition of matter In some embodiments, a protein is provided that comprises a polypeptide comprising the amino acid sequence of SEQ ID NO: 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, or 81-309. In some embodiments, a protein is provided that comprises a polypeptide comprising the amino acid sequence of SEQ ID NO: 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, or 81-309 and at least 62%, 63%, 64%, 65%, 66%, 67%, 68%, 70%, 72%, 74%, 76%, 78%, 79%, 80%, 81-309. , 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 the polypeptide of claim 1. In some embodiments, the protein is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a sequence of SEQ ID NO: 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, or 81-309. , 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, or 81-309. In some embodiments, the protein is at least 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, or 81-309.

[0043] The polypeptides provided herein may be part of a larger polypeptide and may be referred to as domains. Homology or identity between two domains in different polypeptides is based on the domains that are similar as opposed to the entire polypeptide. For example, if a polypeptide includes a polypeptide that includes a FN3 domain that includes SEQ ID NO:81, and that domain is bound to an scFV antibody, another protein that has a domain that is similar but not identical to SEQ ID NO:81 may be at least 90% identical, even if the scFV does not share homology. Thus, the % identity may be based on the domain or on the entire length of the polypeptide. Methods for determining % identity are provided herein or known to those skilled in the art.

[0044] In some embodiments, fibronectin type III (FN3) domains are provided that bind or specifically bind to human CD71 protein (SEQ ID NO: 32 or 80). As provided herein, FN3 domains can bind to CD71 protein. It is also provided that these domains can also specifically bind to CD71 protein, even if not explicitly stated. Thus, for example, FN3 domains that bind to CD71 would also encompass FN3 domain proteins that specifically bind to CD71. These molecules can be used, for example, in therapeutic and diagnostic applications, as well as in imaging. In some embodiments, polynucleotides encoding the FN3 domains disclosed herein or their complementary nucleic acids, vectors, host cells, and methods of making and using them are provided.

[0045] In some embodiments, an isolated FN3 domain is provided that binds or specifically binds to CD71.

[0046] In some embodiments, the FN3 domain comprises two FN3 domains connected by a linker. The linker can be a flexible linker. The linker can be a short peptide sequence, such as those described herein. For example, the linker can be a G / S linker, etc.

[0047] In some embodiments, the FN3 domain has a molecular weight of about 1×10 as determined by surface plasmon resonance or Kinexa methods, as performed by one of skill in the art. -7 Less than M, for example, about 1x10 -8 Less than M, about 1x10 -9 Less than M, about 1x10 -10 Less than M, about 1x10 -11 Less than M, about 1x10 -12 Less than M or about 1x10 -13 Dissociation constant (K D ) can bind to CD71. The measured affinity of a particular FN3 domain-antigen interaction may vary when measured under different conditions (e.g., osmolality, pH). Thus, affinity and other antigen binding parameters (e.g., K D , K on , K off ) is measured using standardized solutions of protein scaffold and antigen, and standardized buffers, such as those described herein.

[0048] In some embodiments, the FN3 domain may bind at least 5-fold higher than the signal obtained for a negative control in a standard ELISA assay.

[0049] In some embodiments, the FN3 domain that binds or specifically binds to CD71 comprises an initiator methionine (Met) linked to the N-terminus of the molecule. In some embodiments, the FN3 domain that binds or specifically binds to CD71 comprises a cysteine ​​(Cys) linked to the C-terminus of the FN3 domain. Adding an N-terminal Met and / or a C-terminal Cys can facilitate expression and / or binding of a half-life extension molecule.

[0050] The FN3 domain may also contain cysteine ​​substitutions as described in U.S. Patent No. 10,196,446, which is incorporated herein by reference in its entirety. Briefly, in some embodiments, the polypeptides provided herein may contain at least one cysteine ​​substitution at a position selected from the group consisting of residues 6, 8, 10, 11, 14, 15, 16, 20, 30, 34, 38, 40, 41, 45, 47, 48, 53, 54, 59, 60, 62, 64, 70, 88, 89, 90, 91, and 93 of the FN3 domain based on SEQ ID NO:6 or SEQ ID NO:1 of U.S. Patent No. 10,196,446, and equivalent positions in related FN3 domains. In some embodiments, the substitution is at residue 6. In some embodiments, the substitution is at residue 8. In some embodiments, the substitution is at residue 10. In some embodiments, the substitution is at residue 11. In some embodiments, the substitution is at residue 14. In some embodiments, the substitution is at residue 15. In some embodiments, the substitution is at residue 16. In some embodiments, the substitution is at residue 20. In some embodiments, the substitution is at residue 30. In some embodiments, the substitution is at residue 34. In some embodiments, the substitution is at residue 38. In some embodiments, the substitution is at residue 40. In some embodiments, the substitution is at residue 41. In some embodiments, the substitution is at residue 45. In some embodiments, the substitution is at residue 47. In some embodiments, the substitution is at residue 48. In some embodiments, the substitution is at residue 53. In some embodiments, the substitution is at residue 54. In some embodiments, the substitution is at residue 59. In some embodiments, the substitution is at residue 60. In some embodiments, the substitution is at residue 62. In some embodiments, the substitution is at residue 64. In some embodiments, the substitution is at residue 70. In some embodiments, the substitution is at residue 88. In some embodiments, the substitution is at residue 89. In some embodiments, the substitution is at residue 90. In some embodiments, the substitution is at residue 91. In some embodiments, the substitution is at residue 93.

[0051] Cysteine ​​substitution at a position in a domain or protein involves replacing an existing amino acid residue with a cysteine ​​residue. Other examples of cysteine ​​modifications can be found, for example, in US Patent Publication No. 20170362301, which is incorporated by reference in its entirety. Sequence alignment can be performed, for example, using BlastP at the NCBI website using default parameters.

[0052] In some embodiments, the FN3 domain that binds to CD71 is internalized into the cell. In some embodiments, the internalization of the FN3 domain can facilitate the delivery of a detectable label or a therapeutic agent into the cell. In some embodiments, the internalization of the FN3 domain can facilitate the delivery of a cytotoxic agent into the cell. The cytotoxic agent can act as a therapeutic agent. In some embodiments, the internalization of the FN3 domain can facilitate the delivery of any detectable label, therapeutic agent and / or cytotoxic agent disclosed herein into the cell. In some embodiments, the cell is a tumor cell. In some embodiments, the cell is a liver cell.

[0053] In some embodiments, the FN3 domain that binds to CD71 optionally is at residue position 11, 14, 17, 37, 46, 73, or 86 (residue numbers corresponding to SEQ ID NO:4). Tencon sequence of SEQ ID NO: 1 or Tencon sequence of SEQ ID NO: 4, Based on the alignment of 27 sequences.

[0054] In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

[0055] In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, or 61.

[0056] In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO:33.

[0057] In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO:34.

[0058] In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO:35.

[0059] In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO:36.

[0060] In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO:37.

[0061] In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO:38.

[0062] In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO:39.

[0063] In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO:40.

[0064] In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO:41.

[0065] In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO:42.

[0066] In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO:43.

[0067] In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO:44.

[0068] In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO:45.

[0069] In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO:46.

[0070] In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO:47.

[0071] In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO:48.

[0072] In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO:49.

[0073] In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO:50.

[0074] In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO:51.

[0075] In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO:52.

[0076] In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO:53.

[0077] In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO:54.

[0078] In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO:55.

[0079] In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO:56.

[0080] In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO:57.

[0081] In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO:58.

[0082] In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO:59.

[0083] In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO:60.

[0084] In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO:61.

[0085] In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO:62.

[0086] In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 81. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 82. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 83. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 84. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 85. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 86. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 87. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 88. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 89. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 90. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 91. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 92. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 93. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 94. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 95. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 96. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 97. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 98.In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 99. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 100. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 101. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 102. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 103. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 104. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 105. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 106. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 107. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 108. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 109. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO:110. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 111. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 112. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 113. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 114. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 115. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 116.In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 117. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 118. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 119. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 120. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 121. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 122. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 123. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 124. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 125. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 126. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 127. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 128. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 129. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 130. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 131. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 132. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 133. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 134.In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 135. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 136. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 137. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 138. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 139. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 140. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 141. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 142. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 143. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 144. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 145. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 146. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 147. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 148. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 149. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 150. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 151. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 151. comprises the amino acid sequence of SEQ ID NO: 152. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 153. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 154. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 155. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 156. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 157. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 158. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 159. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 160. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 161. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 162. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 163. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 164. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 165. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 166. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 167. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 168. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 169.In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 170. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 171. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 172. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 173. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 174. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 175. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 176. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 177. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 178. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 179. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 180. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 181. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 182. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 183. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 184. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 185. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 186. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 187.In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO:188. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 189. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 190. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 191. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 192. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 193. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 194. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 195. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 196. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 197. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 198. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 199. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 200. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 201. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 202. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 203. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 204. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 205. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 206. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 207. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 208.In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 209. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 210. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 211. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 212. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 213. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 214. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 215. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 216. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 217. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 218. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 219. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 220. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 221. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 222. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 223. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 224. comprises the amino acid sequence of SEQ ID NO: 224. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 225. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 226. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 227. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 228. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 229. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 230. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 231. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 232. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 233. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 234. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 235. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 236. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 237. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 238. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 239. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 240. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 241.In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 242. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 243. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 244. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 245. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 246. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 247. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 248. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 249. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 250. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 251. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 252. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 253. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 254. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 255. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 256. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 257. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 258. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 259.In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO:260. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 261. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 262. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 263. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 264. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 265. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 266. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 267. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 268. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 269. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 270. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 271. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 272. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 273. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 274. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 275. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 276. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 277. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 278.In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 279. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 280. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 281. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 282. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 283. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 284. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 285. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 286. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 287. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 288. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 289. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 290. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 291. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 292. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 293. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 294. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 295. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 296.In some embodiments, the ligation is to CD71. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 297. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 298. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 299. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 300. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 301. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 302. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 303. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 304. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 305. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 306. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 307. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 308. In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 309.

[0087] In some embodiments, the FN3 domain binds to human CD71 at a site on CD71 that does not compete with transferrin binding to CD71. In some embodiments, the FN3 domain comprises the sequence of SEQ ID NO: 146, 214, 104, 259, 134, 92, 302, 235, 237, 152, 238, 136, 197, 212, 296, 226, 261, 307, 115, 112, 278, 297, 96, 222, 95, 233, 217, 252, 194, 164, 168, 174, 190, 257, 303, 284, 85, or 149.

[0088] In some embodiments, the isolated FN3 domain that binds CD71 comprises an initiator methionine (Met) linked to the N-terminus of the molecule.

[0089] In some embodiments, the isolated FN3 domain that binds CD71 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: 33-50. In some embodiments, the isolated FN3 domain that binds CD71 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: 51-61 or 62. In some embodiments, the isolated FN3 domain that binds CD71 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: 81-309. In some embodiments, the isolated FN3 domain that binds to CD71 is selected from the group consisting of SEQ ID NOs: 146, 214, 104, 259, 134, 92, 302, 235, 237, 152, 238, 136, 197, 212, 296, 226, 261, 62%, 63%, 64% with one of the amino acid sequences of 307, 115, 112, 278, 297, 96, 222, 95, 233, 217, 252, 194, 164, 168, 174, 190, 257, 303, 284, 85, or 149 , 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 the amino acid sequence of the present invention.

[0090] Percent identity can be determined by aligning the two sequences using BlastP, available through the NCBI website, using default parameters.

[0091] Conjugates of FN3 domains that bind to CD71 of the present disclosure In some embodiments, an isolated FN3 domain that binds to CD71 linked to a heterologous molecule is provided.

[0092] In some embodiments, the FN3 domain is bound to an oligonucleotide. For example, the oligonucleotide can be used to inhibit the expression of a gene or mRNA transcript. The oligonucleotide can be an siRNA, miRNA, an antisense oligonucleotide, etc.

[0093] In some embodiments, peptides are attached to lipid nanoparticles, which can be used, for example, for cell-specific targeting.

[0094] In some embodiments, the protein is conjugated to a binding moiety that targets CD71 or another protein for protein degradation. For example, the protein can be conjugated to PROTACS (binding site for E3 ubiquitin ligase), thus delivering the protein to the E3 ligase. These can be linked via a linker, such as a glycine-serine linker.

[0095] The FN3 domain that binds to CD71 can also be linked or attached to another FN3 domain that binds to a different target other than CD71. This allows the peptide to be multispecific (e.g., bispecific, trispecific, etc.), thereby binding to CD71 and another, e.g., protein. In some embodiments, the CD71 FN3 binding domain is linked to another FN3 domain that binds to an antigen expressed by tumor cells (tumor antigen).

[0096] In some embodiments, the FN3 domains can be linked together by a linker to form a bivalent FN3 domain. The linker can be a flexible linker. In some embodiments, the linker is a G / S linker. In some embodiments, the linker has 1, 2, 3, or 4 G / S repeats. A G / S repeat unit is 4 glycines followed by a serine, e.g., GGGGS.

[0097] In some embodiments, the heterologous molecule is a detectable label or a therapeutic agent, for example, but not limited to, a cytotoxic agent.

[0098] In some embodiments, an FN3 domain that binds to CD71 is provided that is linked to a detectable label. Non-limiting examples of detectable labels are provided herein.

[0099] In some embodiments, a FN3 domain that binds to CD71 is provided that is linked to a therapeutic agent. Therapeutic agents, including but not limited to cytotoxic agents, are described herein. This is provided in the specification.

[0100] FN3 domains that bind to CD71 linked to a detectable label can be used to assess expression of CD71 in vivo or in vitro in samples such as tumor tissue.

[0101] Detectable labels include compositions that, when attached to an FN3 domain that binds CD71, render CD71 detectable by spectroscopic, photochemical, biochemical, immunochemical, or other chemical methods.

[0102] Exemplary detectable labels include, but are not limited to, radioisotopes, magnetic beads, metal beads, colloidal particles, fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly used in ELISA), biotin, digoxigenin, haptens, luminescent molecules, chemiluminescent molecules, fluorescent dyes, fluorophores, fluorescence quenchers, colored molecules, radioisotopes, scintillants, 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 / electron acceptors, acridinium esters, and colorimetric substrates.

[0103] A detectable label may spontaneously emit a signal, such as when the detectable label is a radioisotope, hi some embodiments, a detectable label emits a signal as a result of being stimulated by an external stimulus, such as a magnetic or electric field, or an electromagnetic field.

[0104] Exemplary radioisotopes may be gamma-emitting, Auger-emitting, beta-emitting, alpha-emitting, or positron-emitting radioisotopes. Exemplary radioisotopes include: 3 H, 11 C. 13 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 is mentioned.

[0105] Exemplary metal atoms include metals having 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, and the like. an osmium atom, an iridium atom, a platinum atom, a gold atom, a mercury atom, a thallium atom, a lead atom, a bismuth atom, a francium atom, a radium atom, an actinium atom, a cerium atom, a praseodymium atom, a neodymium atom, a promethium atom, a samarium atom, a europium atom, a gadolinium atom, a terbium atom, a dysprosium atom, a holmium atom, an erbium atom, a thulium atom, a ytterbium atom, a lutetium atom, a thorium atom, a protactinium atom, a uranium atom, a neptunium atom, a plutonium atom, americium atom, a curium atom, a berkelium atom, a californium atom, an einsteinium atom, a fermium atom, a mendelevium atom, a nobelium atom, or a lawrencium atom.

[0106] In some embodiments, the metal atom is an alkaline earth metal having an atomic number greater than 20. It may be an iron group metal.

[0107] In some embodiments, the metal atom can be a lanthanide element.

[0108] In some embodiments, the metal atom can be an actinide.

[0109] In some embodiments, the metal atom can be a transition metal.

[0110] In some embodiments, the metal atom can be a base metal.

[0111] In some embodiments, the metal atoms can be gold atoms, bismuth atoms, tantalum atoms, and gadolinium atoms.

[0112] In some embodiments, the metal atom can be a metal having an atomic number between 53 (ie, iodine) and 83 (ie, bismuth).

[0113] In some embodiments, the metal atom can be an atom suitable for magnetic resonance imaging.

[0114] The metal atom may be in the form of a metal ion in the +1, +2, or +3 oxidation state, e.g., 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 atom may include a metal oxide, such as iron oxide, manganese oxide, or gadolinium oxide.

[0115] Suitable dyes include any commercially available dye, such as 5(6)-carboxyfluorescein, IRDye 680RD maleimide or IRDye 800CW, ruthenium polypyridyl dyes, and the like.

[0116] Suitable fluorophores are fluorescein isothiocyanate (FITC), fluorescein thiosemicarbazide, rhodamine, Texas Red, CyDye (e.g., Cy3, Cy5, Cy5.5), Alexa Fluor (e.g., Alexa488, Alexa555, Alexa594; Alexa647), near infrared (NIR) (700-900 nm) fluorescent dyes, as well as carbocyanine and aminostyryl dyes.

[0117] FN3 domains that specifically bind to CD71 linked to a detectable label can be used, for example, as imaging agents to diagnose tumor distribution, the presence of tumor cells, and / or to assess tumor recurrence.

[0118] In some embodiments, an FN3 domain that specifically binds to CD71 is conjugated to a therapeutic agent, such as, but not limited to, a cytotoxic agent.

[0119] In some embodiments, the therapeutic 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 a fragment thereof), or a radioactive isotope (i.e., a radioconjugate).

[0120] The FN3 domain that binds to CD71 conjugated to a therapeutic agent disclosed herein is These conjugated drugs may be used in the targeted delivery of, and intracellular accumulation of, therapeutic agents to cells (e.g., tumor cells) that express .71. Without being bound to any particular theory, this type of delivery may be useful in cases where systemic administration of these unconjugated drugs may result in unacceptable levels of toxicity to normal cells.

[0121] In some embodiments, therapeutic agents can induce their cytotoxic and / or cytostatic effects by mechanisms such as, but not limited to, tubulin binding, DNA binding, topoisomerase inhibition, DNA cross-linking, chelation, spliceosome inhibition, NAMPT inhibition, and HDAC inhibition.

[0122] In some embodiments, the therapeutic agent is a spliceosome inhibitor, a NAMPT inhibitor, or an HDAC inhibitor. In some embodiments, the agent is an immune system agonist, such as TLR7, 8, 9, RIG-I (dsRNA), and STING (CpG) agonists. In some embodiments, the agent is daunomycin, doxorubicin, methotrexate, vindesine, a bacterial toxin, such as diphtheria toxin, castor toxin, geldanamycin, maytansinoid, or calicheamicin.

[0123] In some embodiments, the therapeutic agent is an enzymatically active toxin such as diphtheria A chain, a nonbinding active fragment of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), castor toxin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii protein, dianthin protein, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, or trichothecenes.

[0124] In some embodiments, the therapeutic agent is a radionuclide, e.g. 212 Bi, 131 I, 131 In, 90 Y, or 186 Re.

[0125] In some embodiments, the therapeutic agent is a dolastatin or dolostatin peptide analogs and derivatives, auristatin or monomethylauristatin phenylalanine. Exemplary molecules are disclosed in U.S. Patent Nos. 5,635,483 and 5,780,588. Dolastatins and auristatins interfere with microtubule dynamics, GTP hydrolysis, and nuclear and cell division (Woyke et al. (2001) Antimicrob Agents and Chemother. 45(12):3580-3584), and have been shown to have anticancer and antifungal activity. The dolastatin or auristatin drug moiety can be attached to the FN3 domain via the N (amino) or C (carboxyl) terminus of the peptide drug moiety (WO 02 / 088172), or via any cysteine ​​engineered into the FN3 domain.

[0126] In some embodiments, the therapeutic agent can be, for example, auristatins, camptothecins, duocarmycins, etoposide, maytansine and maytansinoids, taxanes, benzodiazepines or benzodiazepine-containing drugs (e.g., pyrrolo[1,41-benzodiazepines (PBD), indolinobenzodiazepines, and oxazolidinobenzodiazepines), or vinca alkaloids.

[0127] An FN3 domain that specifically binds to CD71 can be conjugated to a detectable label using known methods.

[0128] In some embodiments, the detectable label is conjugated to a chelator.

[0129] In some embodiments, the detectable label is attached to the FN3 domain that binds CD71 via a linker.

[0130] A detectable label, therapeutic compound, or cytotoxic compound can be linked directly or indirectly to the FN3 domain that binds CD71 using known methods. Suitable linkers are known in the art and include, for example, prosthetic groups, non-phenolic linkers (derivatives of N-succinimidyl benzoate; dodecaborate), chelating moieties of both macrocyclic and acyclic chelators, such as derivatives of 1,4,7,10-tetraazacyclododecane-1,4,7,10,tetraacetic acid (DOTA), derivatives of diethylenetriaminepentaacetic acid (DTPA), derivatives of S-2-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), and derivatives of 1,4,8,11-tetraazacyclododecane-1,4,8,11-tetraacetic acid (TETA), N-succinimidyl- These include 3-(2-pyridyldithiol)propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (e.g., dimethyl adipimidate HCl), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bis-azide compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (e.g., bis(p-diazoniumbenzoyl)ethylenediamine), diisocyanates (e.g., toluene 2,6-diisocyanate), and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene) and other chelating moieties. Suitable peptide linkers are well known.

[0131] In some embodiments, the FN3 domain that binds to CD71 is cleared from the blood via renal clearance.

[0132] Isolation of CD71-binding FN3 domains from a Tencon sequence-based library Tencon (SEQ ID NO: 1) is a non-naturally occurring fibronectin type III (FN3) domain designed from the consensus sequence of 15 FN3 domains from human tenascin-C (Jacobs et al., Protein Engineering, Design, and Selection, 25:107-117, 2012; U.S. Patent Publication No. 2010 / 0216708). The crystal structure of Tencon shows six surface-exposed loops connecting the seven beta-strands characteristic of FN3 domains, the beta strands designated A, B, C, D, E, F, and G, and the loops designated AB, BC, CD, DE, EF, and FG loops (Bork and Doolittle, Proc Natl Acad Sci USA 89:8990-8992, 1992; U.S. Patent No. 6,673,901). These loops, or selected residues within each loop, can be randomized to construct a library of fibronectin type III (FN3) domains that can be used to select novel molecules that bind to CD71. Table 1 shows the location and sequence of each loop and beta strand in Tencon (SEQ ID NO:1).

[0133] Thus, libraries designed based on Tencon sequences can have a randomized FG loop, or randomized BC and FG loops, for example libraries TCL1 or TCL2 as described below. The Tencon BC loop is 7 amino acids long, therefore 1, 2, 3, 4, 5, 6 or 7 amino acids can be diversified in the BC loop and randomized in libraries designed based on Tencon sequences. The Tencon FG loop is 7 amino acids long, therefore 1, 2, 3, 4, 5, 6 or 7 amino acids can be diversified in the FG loop and randomized in libraries designed based on the Tencon sequence. Further diversity in the loops in the Tencon library can be achieved by insertion and / or deletion of residues in the loop. For example, the FG and / or BC loops can be extended by 1-22 amino acids or reduced by 1-3 amino acids. The FG loop in Tencon is 7 amino acids long, while the corresponding loops in antibody heavy chains range from 4-28 residues. To provide maximum diversity, the FG loop can be diversified in sequence and even length corresponding to the range of antibody CDR3 lengths from 4 to 28 residues. For example, the FG loop can be further diversified in length by extending the loop by an additional 1, 2, 3, 4 or 5 amino acids.

[0134] Libraries designed based on Tencon sequences may also have randomized alternative surfaces formed on the sides of the FN3 domain and may include two or more beta strands and at least one loop. One such alternative surface is formed by amino acids in the C and F beta strands and the CD and FG loops (C-CD-F-FG surface). Library designs based on Tencon alternative C-CD-F-FG surfaces are described in US Patent Publication No. 2013 / 0226834. Libraries designed based on Tencon sequences also include libraries designed based on Tencon variants, such as those with substitutions at residue positions 11, 14, 17, 37, 46, 73, or 86 (residue numbering corresponding to SEQ ID NO:1), and these variants show improved thermostability. Exemplary Tencon variants are described in US Patent Publication No. 2011 / 0274623 and include Tencon27 (SEQ ID NO:4), which has the substitutions E11R, L17A, N46V and E86I when compared to the Tencon of SEQ ID NO:1.

[0135] [Table 1]

[0136] Libraries based on Tencon and other FN3 sequences can be randomized at selected residue positions using a random or defined set of amino acids. For example, variants in libraries with random substitutions can be generated using the NNK codon, which codes for all 20 naturally occurring amino acids. In other diversification schemes, DVK codons can be used to code for the amino acids Ala, Trp, Tyr, Lys, Thr, Asn, Lys, Ser, Arg, Asp, Glu, Gly, and Cys. Alternatively, NNS codons can be used to generate all 20 amino acid residues and simultaneously reduce the frequency of stop codons. Libraries of FN3 domains with biased amino acid distributions at the positions to be diversified can 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 required to construct any desired DNA molecule. Codon designation follows the well-known IUB code.

[0137] FN3 domains that specifically bind to CD71 can be isolated by using cis display to link a DNA fragment encoding a scaffold protein to a DNA fragment encoding RepA, generating a pool of protein-DNA complexes formed after in vitro translation in which each protein is stably linked to the DNA that encodes it (U.S. Patent No. 7,842,476; Odegrip et al., Proc Natl Acad Sci USA 101, 2806-2810, 2004), producing an FN3 library, such as the Tencon library, and assaying the library for specific binding to PSMA by any method known in the art and described in the Examples. Exemplary well-known methods that can be used are ELISA, sandwich assay, or the like. These include immunoassays, as well as competitive and noncompetitive assays (e.g., Ausubel et al., eds., 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York). Identified FN3 domains that specifically bind to CD71 are further characterized for their binding to CD71, modulation of CD71 activity, internalization, stability, and other desirable characteristics.

[0138] FN3 domains that specifically bind to CD71 can be generated by using any FN3 domain as a template to generate a library and screening the library for molecules that specifically bind to CD71 using the methods shown. Exemplary FN3 domains that can be used are the third FN3 domain of tenascin-C (TN3), Fibcon, and the tenth FN3 domain of fibronectin (FN10). Accordingly, PCT applications WO2010 / 051274, WO 2011 / 137319, and WO 2013 / 049275 are incorporated herein in their entirety. Standard cloning and expression techniques are used to clone the library into a vector or to synthesize double-stranded cDNA cassettes of the library, and to express or translate the library 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. Patent No. 5,643,768) can be used. The library of FN3 domain variants can be expressed, for example, as a fusion protein displayed on the surface of any suitable bacteriophage. Methods for displaying fusion polypeptides on the surface of bacteriophage are well known (U.S. Patent Publication No. 2011 / 0118144; International Patent Publication No. WO2009 / 085462; U.S. Patent No. 6,969,108; U.S. Patent No. 6,172,197; U.S. Patent No. 5,223,409; U.S. Patent No. 6,582,915; U.S. Patent No. 6,472,147).

[0139] In some embodiments, the FN3 domain that binds to CD71 is based on the Tencon sequence of SEQ ID NO: 1 or the Tencon27 sequence of SEQ ID NO: 4, where SEQ ID NO: 1 or SEQ ID NO: 4 optionally have substitutions at residue positions 11, 14, 17, 37, 46, 73, and / or 86.

[0140] In some embodiments, the FN3 protein or polypeptide binds to human CD71 at a site on CD71 that does not compete with transferrin binding to CD71. As used herein, a site on CD71 that does not compete with transferrin binding to CD71 refers to an epitope or portion of CD71 where binding of the FN3 protein at that site does not compete with or inhibit transferrin binding to CD71. The competition or lack of competition can be complete or partial. In some embodiments, the binding also does not inhibit internalization of transferrin into cells via its interaction with CD71.

[0141] In some embodiments, a method is provided for identifying FN3 proteins that bind to CD71 at a site that does not compete or inhibit transferrin binding to CD71. In some embodiments, the method comprises contacting CD71 with a test FN3 protein in the presence of transferrin or an agent that binds to the CD71 transferrin binding site; and identifying a test FN3 protein that binds to CD71 in the presence of transferrin or an agent that binds to the CD71 transferrin binding site. In some embodiments, the method comprises isolating a test FN3 protein that binds to CD71 in the presence of transferrin or an agent that binds to the CD71 transferrin binding site. In some embodiments, the method comprises sequencing a test FN3 protein that binds to CD71 in the presence of transferrin or an agent that binds to the CD71 transferrin binding site. In some embodiments, the method comprises producing or obtaining a nucleic acid sequence that encodes a test FN3 protein that binds to CD71 in the presence of transferrin or an agent that binds to the CD71 transferrin binding site. In some embodiments, the method comprises expressing a test FN3 protein that binds to CD71 in the presence of transferrin or an agent that binds to the CD71 transferrin binding site from a nucleic acid sequence that encodes a test FN3 protein that binds to CD71 in the presence of transferrin or an agent that binds to the CD71 transferrin binding site. In some embodiments, the test FN3 protein is expressed in a cell. In some embodiments, the method comprises isolating and / or purifying the expressed test FN3 protein.

[0142] In some embodiments, an FN3 protein identified according to any of the methods provided herein is provided.

[0143] FN3 domains that specifically bind to CD71 can be modified to improve their properties, such as improving 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 with highly similar thermostable sequences, design to stabilize disulfide bridges, mutations that increase alpha-helical propensity, engineering salt bridges, altering the surface charge of the protein, directed evolution, and constructing consensus sequences (Lehmann and Wyss, Curr. Opin. Biotechnol., 12, 371-375, 2001). High thermal stability can increase the yield of expressed proteins, improve solubility or activity, reduce immunogenicity, and minimize the need for cold chain in production. Residues that can be substituted to improve the thermal stability of Tencon (SEQ ID NO:1) are at residue positions 11, 14, 17, 37, 46, 73, or 86, and are described in U.S. Patent Publication No. 2011 / 0274623. Substitutions corresponding to these residues can be incorporated into the FN3 domain-containing molecules disclosed herein.

[0144] Measures of protein stability and protein instability can be assessed as the same or different aspects of protein integrity. Proteins are sensitive or "unstable" to denaturation caused by heat, ultraviolet light or ionizing radiation, ambient osmolarity and pH when in liquid solution, mechanical shear forces exerted by small pore filtration, ultraviolet light, ionizing radiation, e.g. gamma irradiation, chemical or thermal dehydration, or any other action or force that can cause protein structure disruption. Molecular stability can be determined by standard methods. For example, molecular stability can be measured by thermal melting ("T m ") temperature, the temperature in degrees Celsius (°C) at which half of the molecule becomes unfolded, can be determined using standard methods. Typically, T m The higher the A, the more stable the molecule. In addition to heat, the chemical environment also alters the ability of a protein to maintain a particular three-dimensional structure.

[0145] In some embodiments, the FN3 domain that binds to CD71 is m In some embodiments, the domain may exhibit at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more increased stability compared to the same domain prior to engineering as measured by an increase in

[0146] Chemical modification can also be measured by a variety of methods. Chemical modifications include guanidine hydrochloride, guanidine thiocyanate, urea, acetone, organic solvents (DMF, benzene, acetonitrile), salts (ammonium sulfate, lithium bromide, lithium chloride, sodium bromide), etc. reducing agents (e.g., dithiothreitol, beta-mercaptoethanol, dinitrothiobenzene, and hydrides such as sodium borohydride), non-ionic and ionic surfactants, acids (e.g., hydrochloric acid (HCl), acetic acid (CH 3 Modifications include amines, amines, amine derivatives, amines with amine groups, amine groups ...

[0147] FN3 domains that bind to CD71 may be generated as monomers, dimers, or multimers, for example, as a means to increase the valency and, consequently, the avidity of target molecule binding, or to generate bispecific or multispecific scaffolds that bind two or more different target molecules simultaneously. Dimers and multimers can be generated by linking monospecific, bispecific, or multispecific protein scaffolds, for example, by including amino acid linkers, such as linkers containing polyglycine, glycine and serine, or alanine and proline. Exemplary linkers include (GS) 2 , (SEQ ID NO: 63), (GGGS) 2(SEQ ID NO:64), (GGGGS) 5 (SEQ ID NO: 65), (AP) 2 (SEQ ID NO: 66), (AP) 5 (SEQ ID NO: 67), (AP) 10 (SEQ ID NO: 68), (AP) 20 (SEQ ID NO: 69) and A(EAAAK) 5 AAA (SEQ ID NO: 70). Dimers and multimers can be linked together in the 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. Patent No. 5,856,456).

[0148] Half-life extension moiety An FN3 domain that specifically binds to CD71 may incorporate other subunits, for example, through common binding interactions. In some embodiments, an FN3 domain that specifically binds to CD71 further comprises a half-life extending moiety. Exemplary half-life extending moieties are albumin, albumin variants, albumin binding proteins and / or domains, transferrin and fragments and analogs thereof, and Fc regions. The amino acid sequences of human Fc regions are well known and include IgG1, IgG2, IgG3, IgG4, IgM, IgA, and IgE Fc regions. In some embodiments, an FN3 domain that specifically binds to CD71 may incorporate a second FN3 domain that binds to a molecule that extends the half-life of the entire molecule, such as, but not limited to, any of the half-life extending moieties described herein. In some embodiments, the second FN3 domain binds to albumin, albumin variants, albumin binding proteins and / or domains, and fragments and analogs thereof.

[0149] All or a portion of the antibody constant region may be attached to an FN3 domain that binds CD71 to confer antibody-like properties, particularly those properties associated with the Fc region, e.g., Fc effector functions such as C1q 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 that are responsible for these activities (for review see; Strohl, Curr Opin Biotechnol. 20, 685-691, 2009).

[0150] For desired properties, polyethylene glycol (PEG) molecules, such as PEG 5000 or PEG 20,000, fatty acids and fatty acid esters of various chain lengths, such as lauric acid Additional moieties such as esters, myristic acid esters, stearic acid esters, arachidic acid esters, behenic acid esters, oleic acid esters, arachidonic acid esters, octanedioic acid, tetradecanedioic acid, octadecanedioic acid, docosanedioic acid, etc., polylysine, octane, carbohydrates (dextran, cellulose, oligosaccharides or polysaccharides) can be incorporated into the FN3 domain that specifically binds to CD71. These moieties can be directed to fusion with sequences encoding protein scaffolds and can be produced by standard cloning and expression techniques. Alternatively, well-known chemical coupling methods can be used to link these moieties to recombinantly produced molecules disclosed herein.

[0151] A pegylation moiety can be added to an FN3 domain that binds CD71, for example, by incorporating a cysteine ​​residue at the C-terminus of the molecule or by engineering a cysteine ​​at a residue position that faces away from the CD71-binding face of the molecule and attaching a pegylated group to the cysteine ​​using well-known methods.

[0152] FN3 domains that specifically bind to CD71 incorporating additional moieties can be compared for functionality by several well-known assays. For example, altered properties due to the incorporation of an Fc domain and / or an Fc domain variant can be assayed in Fc receptor binding assays, using soluble forms of receptors such as FcγRI, FcγRII, FcγRIII or FcRn receptors, or using well-known cell-based assays measuring, for example, ADCC or CDC, or evaluating the pharmacokinetic properties of the molecules disclosed herein in in vivo models.

[0153] Polynucleotides, Vectors, and Host Cells In some embodiments, nucleic acids encoding FN3 domains that specifically bind CD71 are provided as isolated polynucleotides or as part of an expression vector or as part of a linear DNA sequence, 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 composition or directed mutagenesis thereof. However, certain exemplary polynucleotides are disclosed herein, and taking into account the degeneracy of the genetic code or codon preference in a given expression system, other polynucleotides encoding the FN3 domains disclosed herein are also within the scope of the disclosure.

[0154] In some embodiments, the isolated polynucleotide encodes an FN3 domain that specifically binds CD71, comprising the amino acid sequence of SEQ ID NO: 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, or 81-309.

[0155] The polynucleotides disclosed herein can be produced by chemical synthesis, such as solid phase polynucleotide synthesis on an automated polynucleotide synthesizer, and assembled into complete single-stranded or double-stranded molecules. Alternatively, the polynucleotides disclosed herein can be produced by other techniques, such as PCR followed by conventional cloning. Techniques for producing or obtaining polynucleotides of a given known sequence are well known in the art.

[0156] The polynucleotides disclosed herein may contain at least one non-coding sequence, such as a promoter or enhancer sequence, an intron, a polyadenylation signal, a Cis sequence that facilitates RepA binding, etc. The polynucleotide sequence may also contain, for example, a marker that facilitates purification or detection of the protein, or a tag sequence, such as a histidine tag or an FA tag, a signal sequence, a fusion protein partner, such as RepA, an Fc or may also contain additional sequences encoding additional amino acids that code for a bacteriophage coat protein such as pIX or pIII.

[0157] In some embodiments, a vector is provided that comprises at least one polynucleotide disclosed herein. Such vectors can be plasmid vectors, viral vectors, vectors for baculovirus expression, transposon-based vectors, or any other vector suitable for introducing the polynucleotides disclosed herein into a given organism or genetic background by any means. Such vectors can be expression vectors that contain nucleic acid sequence elements that can control, regulate, cause, or enable the expression of the polypeptide encoded by such vector. Such elements can include transcription enhancer binding sites, RNA polymerase initiation sites, ribosome binding sites, and other sites that facilitate the expression of the encoded polypeptide in a given expression system. Such expression systems can be cell-based or cell-free systems well known in the art.

[0158] In some embodiments, a host cell comprising the vector is provided. The FN3 domain that specifically binds to CD71 can be produced by cell lines, mixed cell lines, immortalized cells, or clonal populations of immortalized cells, as known in the art. See, for example, Ausubel et al., eds., Current Protocols in Molecular Biology, John Wiley&Sons, Inc., NY, NY (1987-2001); Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., 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).

[0159] The host cell selected 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 any species or organism that is unable to glycosylate polypeptides, e.g., prokaryotic cells or organisms such as BL21, BL21(DE3), BL21-GOLD(DE3), XL1-Blue, JM109, HMS174, HMS174(DE3), and any naturally occurring or engineered strain of E. coli, Klebsiella, or Pseudomonas.

[0160] In some embodiments, a method of producing an isolated FN3 domain that binds CD71 comprises culturing an isolated host cell under conditions such that the isolated FN3 domain that binds CD71 is expressed, and purifying the FN3 domain.

[0161] FN3 domains that bind CD71 can be purified from recombinant cell cultures by well-known methods, such as protein A purification, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography and lectin chromatography, or high performance liquid chromatography (HPLC).

[0162] Anti-idiotype antibodies In some embodiments, the anti-idiotypic antibody binds to an FN3 domain.

[0163] In some embodiments, an anti-idiotypic antibody that binds to an FN3 domain comprises the amino acid sequence of one of SEQ ID NOs: 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, or 81-309.

[0164] kit In some embodiments, a kit is provided that includes an FN3 domain that binds CD71.

[0165] The kits can be used for therapeutic applications and as diagnostic kits.

[0166] In some embodiments, the kit comprises a FN3 domain that binds CD71 and a reagent for detecting the FN3 domain. In some embodiments, the kit comprises a bivalent FN3 domain. The kit may include one or more other elements, including: instructions for use; other reagents, such as agents useful for labeling, chelating or otherwise coupling, radioprotective compositions; devices or other materials for preparing the FN3 domain that binds CD71 for administration for imaging, diagnostic or therapeutic purposes; a pharma- ceutically acceptable carrier; and other devices or materials for administration to a subject.

[0167] In some embodiments, the kit comprises a CD71-binding FN3 domain comprising the amino acid sequence of one of SEQ ID NOs: 33-50.

[0168] In some embodiments, the kit comprises a CD71-binding FN3 domain comprising the amino acid sequence of one of SEQ ID NOs: 51-61.

[0169] In some embodiments, the kit comprises a CD71-binding FN3 domain comprising the amino acid sequence of one of SEQ ID NOs: 81-309.

[0170] In some embodiments, the kit comprises a CD71-binding FN3 domain comprising the amino acid sequence of one of SEQ ID NOs: 146, 214, 104, 259, 134, 92, 302, 235, 237, 152, 238, 136, 197, 212, 296, 226, 261, 307, 115, 112, 278, 297, 96, 222, 95, 233, 217, 252, 194, 164, 168, 174, 190, 257, 303, 284, 85, or 149.

[0171] Use of CD71-binding FN3 domains FN3 domains that specifically bind CD71 or conjugates thereof can be used to diagnose, monitor, regulate, treat, alleviate, help prevent the occurrence of, or reduce symptoms of human diseases or specific pathologies in cells, tissues, organs, fluids, or generally in the host.

[0172] In some embodiments, FN3 domain or its conjugate that specifically binds to CD71 can also be used in imaging CD71-positive tumor tissue in a subject.The methods disclosed herein can be used in any category of animal patients.Examples of such animals include mammals such as humans, rodents, dogs, cats and livestock.

[0173] In some embodiments, methods are provided for diagnosing, predicting immunotherapeutic success, prognostic, and treatment of a subject who has or is likely to develop cancer of a tissue based on expression of CD71 by cells of the cancerous tissue.

[0174] In some embodiments, a method of detecting cancer cells expressing CD71 in tumor tissue is provided, the method comprising: obtaining a tumor tissue sample from a subject; contacting the toe sample of the tumor tissue with a CD71-binding FN3 domain comprising one of the amino acid sequences of SEQ ID NOs: 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, or 81-309, and detecting binding between CD71 and the FN3 domain, thereby detecting whether CD71 is expressed in the tumor tissue.

[0175] In some embodiments, the CD71 cells are cells involved in CNS diseases, inflammatory / immune diseases, such as MS of the brain, and infectious diseases.

[0176] In some embodiments, the tissue can be tissue of any organ or anatomical system that expresses CD71.

[0177] In some embodiments, CD71 expression may be assessed using known methods such as immunohistochemistry or ELISA.

[0178] In some embodiments, a method for isolating cells expressing CD71 is provided, the method comprising: obtaining a sample from a subject; contacting the sample with an FN3 domain that binds to CD71 comprising an amino acid sequence of one of SEQ ID NOs: 33-50, and isolating cells that bind to the FN3 domain.

[0179] In some embodiments, a method for isolating cells expressing CD71 is provided, the method comprising: obtaining a sample from a subject; contacting the sample with an FN3 domain that binds to CD71 comprising an amino acid sequence of one of SEQ ID NOs: 51-61, and isolating cells that bind to the FN3 domain.

[0180] In some embodiments, a method for detecting cancer cells expressing CD71 in tumor tissue is provided, the method comprising: conjugating a CD71-binding FN3 domain comprising one of the amino acid sequences of SEQ ID NOs: 33-50 to a detectable label to form a conjugate; administering the conjugate to a subject; and visualizing the CD71-expressing cancer cells bound by the conjugate. In some embodiments, a method for detecting cancer cells expressing CD71 in tumor tissue is provided, the method comprising: conjugating a CD71-binding FN3 domain comprising one of the amino acid sequences of SEQ ID NOs: 51-62 or 81-309 to a detectable label to form a conjugate; administering the conjugate to a subject; and visualizing the CD71-expressing cancer cells to which the conjugate is bound.

[0181] In some embodiments, methods of treating a subject having cancer are provided, the methods comprising administering to the subject an FN3 domain that binds to CD71. In some embodiments, the FN3 domain is conjugated to a therapeutic agent (e.g., a cytotoxic agent, an oligonucleotide, an FN3 domain that binds to another target, etc.).

[0182] In some embodiments, the subject has a solid tumor.

[0183] In some embodiments, the solid tumor is a melanoma.

[0184] In some embodiments, the solid tumor is lung cancer. In some embodiments, the solid tumor is non-small cell lung cancer (NSCLC). In some embodiments, the solid tumor is squamous non-small cell lung cancer (NSCLC). In some embodiments, the solid tumor is non-squamous NSCLC. In some embodiments, the solid tumor is lung adenocarcinoma.

[0185] In some embodiments, the solid tumor is renal cell carcinoma (RCC).

[0186] In some embodiments, the solid tumor is mesothelioma.

[0187] In some embodiments, the solid tumor is nasopharyngeal carcinoma (NPC).

[0188] In some embodiments, the solid tumor is colorectal cancer.

[0189] In some embodiments, the solid tumor is prostate cancer. In some embodiments, the solid tumor is castration-resistant prostate cancer.

[0190] In some embodiments, the solid tumor is stomach cancer.

[0191] In some embodiments, the solid tumor is ovarian cancer.

[0192] In some embodiments, the solid tumor is gastric cancer.

[0193] In some embodiments, the solid tumor is liver cancer.

[0194] In some embodiments, the solid tumor is pancreatic cancer.

[0195] In some embodiments, the solid tumor is thyroid cancer.

[0196] In some embodiments, the solid tumor is head and neck squamous cell carcinoma.

[0197] In some embodiments, the solid tumor is a cancer of the esophagus or gastrointestinal tract.

[0198] In some embodiments, the solid tumor is breast cancer.

[0199] In some embodiments, the solid tumor is fallopian tube cancer.

[0200] In some embodiments, the solid tumor is brain cancer.

[0201] In some embodiments, the solid tumor is a urethral cancer.

[0202] In some embodiments, the solid tumor is a genitourinary cancer.

[0203] In some embodiments, the solid tumor is endometriosis.

[0204] In some embodiments, the solid tumor is cervical cancer.

[0205] In some embodiments, the solid tumor is a cancer metastasis.

[0206] In some embodiments, the subject has a hematological malignancy.

[0207] In some embodiments, the hematological malignancy is lymphoma, myeloma, or leukemia. In some embodiments, the hematological malignancy is 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 non-Hodgkin's lymphoma.

[0208] In some embodiments, the hematological malignancy is a myelodysplastic syndrome.

[0209] In some embodiments, the hematological malignancy is acute myeloid leukemia (AML). In some embodiments, the hematological malignancy is chronic myelogenous leukemia (CML). In some embodiments, the hematological malignancy is chronic myelomonocytic leukemia (CMML).

[0210] In some embodiments, the hematological malignancy is multiple myeloma (MM).

[0211] In some embodiments, the hematological malignancy is plasmacytoma.

[0212] In some embodiments, the compositions or pharmaceutical compositions provided herein may be administered alone or in combination with other therapeutic agents, i.e., simultaneously or sequentially. In some embodiments, the other or additional therapeutic agents are other anti-tumor or therapeutic agents. Different tumor types and tumor stages may require the use of different supplementary compounds useful in the treatment of cancer. For example, the compositions provided herein may be used in combination with different chemotherapeutic agents, such as taxol, tyrosine kinase inhibitors, leucovorin, fluorouracil, irinotecan, phosphatase inhibitors, MEK inhibitors, among others. The compositions may also be used in combination with drugs that modulate the immune response against tumors, such as anti-PD-1 or anti-CTLA-4, among others. The additional treatment may be an agent that modulates the immune system, such as an antibody that targets PD-1 or PD-L1.

[0213] In some embodiments, the FN3 domain or its conjugates that specifically bind to CD71 can be used to diagnose, monitor, regulate, treat, alleviate, or help prevent the occurrence of or reduce symptoms of human diseases or specific pathologies in cells, tissues, organs, fluids, or in general in a host. They also exhibit the property of being able to cross the blood-brain barrier. The blood-brain barrier (BBB) ​​prevents most macromolecules (e.g., DNA, RNA, and polypeptides) and many small molecules from entering the brain. The BBB is mainly composed of specialized endothelial cells with highly restrictive tight junctions, and as a result, the passage of large and small substances from the blood to the central nervous system is controlled by the BBB. This structure makes it difficult to treat and manage patients with neurological diseases and disorders (e.g., brain cancer), since many therapeutic agents cannot be delivered across the BBB with the desired efficacy. Further conditions involving disruption of the BBB include: stroke, diabetes, epileptic seizures, hypertensive encephalopathy, acquired immune deficiency syndrome, traumatic brain injury, multiple sclerosis, Parkinson's disease (PD) and Alzheimer's disease. This ability is particularly useful for treating brain cancers, including, for example: astrocytoma, medulloblastoma, glioma, ependymoma, germinoma (pinealoma), glioblastoma multiforme, oligodendroglioma, schwannoma, retinoblastoma, and congenital tumors; or cancers of the spinal cord, such as neurofibroma, meningioma, glioma, and sarcoma. In a particular embodiment, a CD71-specific antibody comprising one of the amino acid sequences of SEQ ID NOs: 33-50 is used. An FN3 domain or conjugate thereof that specifically binds to CD71 is useful, for example, for delivering a therapeutic or cytotoxic agent across the blood-brain barrier. In certain embodiments, an FN3 domain or conjugate thereof that specifically binds to CD71, comprising one of the amino acid sequences of SEQ ID NOs: 51-61, is useful, for example, for delivering a therapeutic or cytotoxic agent across the blood-brain barrier. In some embodiments, the protein comprises one of the amino acid sequences of SEQ ID NOs: 146, 214, 104, 259, 134, 92, 302, 235, 237, 152, 238, 136, 197, 212, 296, 226, 261, 307, 115, 112, 278, 297, 96, 222, 95, 233, 217, 252, 194, 164, 168, 174, 190, 257, 303, 284, 85, 149, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, or 81 to 309.

[0214] In some embodiments, a polypeptide capable of facilitating transport of a therapeutic agent across the BBB is a protein comprising the sequence of SEQ ID NO: 146, 214, 104, 259, 134, 92, 302, 235, 237, 152, 238, 136, 197, 212, 296, 226, 261, 307, 115, 112, 278, 297, 96, 222, 95, 233, 217, 252, 194, 164, 168, 174, 190, 257, 303, 284, 85, or 149.

[0215] "Treat" or "treatment" refers to therapeutic treatment and prophylactic measures, where the objective is to prevent or slow (reduce) undesirable physiological changes or disorders, such as the development or spread of cancer. In some embodiments, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, whether detectable or undetectable, reduction in the extent of disease, a stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of a disease state, and remission (whether partial or complete). "Treatment" can also mean prolonging survival compared to expected survival in the absence of treatment. Those in need of treatment include those already with the condition or disorder, as well as those prone to having the condition or disorder, or those in which the condition or disorder is to be prevented.

[0216] A "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic outcome. The therapeutically effective amount of a FN3 domain that specifically binds to CD71 may vary depending on factors such as the disease state, age, sex, and weight of the individual. Exemplary indicators of an effective CD71-binding FN3 domain are improved health of the patient, reduction or shrinkage in size of the tumor, halt or slowed growth of the tumor, and / or lack of metastasis of cancer cells to other locations in the body.

[0217] Administration / Pharmaceutical Compositions In some embodiments, a pharmaceutical composition is provided of a FN3 domain that specifically binds to CD71, as disclosed herein, optionally conjugated to a detectable label, a therapeutic agent, or a cytotoxic agent, and a pharma- ceutical acceptable carrier. For therapeutic use, the FN3 domain that specifically binds to CD71 can be prepared as a pharmaceutical composition containing an effective amount of the domain or molecule as an active ingredient in a pharma- ceutical acceptable carrier. A "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which an 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 can be sterilized by conventional, well-known sterilization techniques (e.g., filtration). The compositions can be prepared in a manner that is necessary to approximate physiological conditions. Depending on the composition, the pharmaceutical composition may contain pharma- ceutically acceptable auxiliary substances, such as pH adjusting and buffering agents, stabilizers, thickeners, lubricants, and colorants. The concentration of the molecules disclosed herein in such pharmaceutical formulations may 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 is selected in accordance with the particular mode of administration selected, based primarily on the required dose, fluid volumes, viscosities, and the like. Suitable vehicles and formulations include those described, for example, in Remington: See, for example, The Science and Practice of Pharmacy, 21st ed., Troy, D.B. Ed., Lipincott Williams and Wilkins, Philadelphia, PA 2006, Part 5, Pharmaceutical Manufacturing, pp. 691-1092, in particular pp. 958-989.

[0218] Modes of administration for therapeutic uses of the FN3 domains disclosed herein can be any suitable route that delivers an agent to a host, as is well known in the art, for example, parenteral administration, e.g., intradermal, intramuscular, intraperitoneal, intravenous or subcutaneous, pulmonary; mucosal (oral, intranasal, intravaginal, rectal) using formulations in tablets, capsules, liquids, powders, gels, particles; and contained in syringes, implanted devices, osmotic pumps, cartridges, micropumps; or other means understood by those of skill in the art. Site-specific administration can be achieved, for example, by intra-articular, intrabronchial, intra-abdominal, intracapsular, intrachondral, intracavitary, intracelial, intracerebellar, intraventricular, intracolonic, intracervical, intragastric, intrahepatic, intracardiac, intraosteal, intrapelvic, intrapericardial, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravascular, intravesical, intralesional, vaginal, rectal, buccal, sublingual, intranasal, or transdermal delivery.

[0219] The pharmaceutical composition may be provided as a kit comprising a container containing the pharmaceutical composition described herein.The pharmaceutical composition may be provided, for example, in the form of an injectable liquid for single or multiple doses, or as a sterile powder to be reconstituted before injection.Alternatively, such a kit may comprise a dry powder disperser, liquid aerosol generator, or nebulizer for administration of the pharmaceutical composition.Such a kit may further comprise written information regarding the indications and use of the pharmaceutical composition. EXAMPLES

[0220] The following examples serve to illustrate the embodiments disclosed herein. These examples are provided for illustrative purposes only, and the embodiments should not be construed in any way as being limited to these examples, but rather as embracing any and all variations that become evident as a result of the teachings provided herein. Those skilled in the art will readily recognize a variety of non-critical parameters that can be changed or modified to obtain essentially similar results.

[0221] Example 1. Construction of Tencon libraries containing randomized loops Tencon (SEQ ID NO: 1) is an immunoglobulin-like scaffold fibronectin type III (FN3) domain designed from the consensus sequence of 15 FN3 domains from human tenascin-C (Jacobs et al., Protein Engineering, Design, and Selection, 25:107-117, 2012; U.S. Patent 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 to construct a library of fibronectin type III (FN3) domains that can be used to select novel molecules that bind to specific targets.

[0222] Tencon: LPAPKNLVVSEVTEDSLRLSWTAPDAAFDSFLIQYQESEKVGEAINLTVPGSERSYDLTGLKPGTEYTVSIYGVKGGHRSNPLSAEFTT (SEQ ID NO: 1) Various libraries were generated using the Tencon scaffold and various design strategies. In general, libraries TCL1 and TCL2 yielded good binders. The generation of the TCL1 and TCL2 libraries is described in detail in International Patent Publication No. WO / 2014081944A2.

[0223] Construction of the TCL1 library TCL1, a library designed to randomize only the FG loop of Tencon (SEQ ID NO:1), was constructed for use with a cis-display system (Jacobs et al., Protein Engineering, Design, and Selection, 25:107-117, 2012). In this system, single-stranded DNA was generated that incorporated sequences for the Tac promoter, the Tencon library coding sequence, the RepA coding sequence, the cis-element, and the ori element. Upon expression in an in vitro transcription / translation system, a complex of the Tencon-RepA fusion protein bound in cis to the DNA for which it is encoded was generated. Complexes that bound to the target molecule were then isolated and amplified by polymerase chain reaction (PCR), as described below.

[0224] Construction of the TCL1 library for use with cis display was accomplished by multiple sequential rounds of PCR to generate two halves of the final linear double stranded DNA molecule; the 5' fragment contains the promoter and Tencon sequences, while the 3' fragment contains the repA gene and cis and ori elements. These two halves were combined by restriction digestion to generate the entire construct. The TCL1 library was designed to incorporate random amino acids only into the FG loop of Tencon. The NNS codon was 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 TCL1 library contains six separate sub-libraries, each with different randomized FG loop lengths ranging from 7 to 12 residues to further increase diversity.

[0225] TCL1 library (SEQ ID NO:2) LPAPKNLVVSEVTEDSLRLSWTAPDAAFDSFLIQYQESEKVGEAINLTVPGSERSYDLTGLKPGTEYTVSIYGVX 7-12 PLSAEFTT; where X 1 , X2 , X 3 , X 4 , X 5 , X 6 , X 7 is any amino acid; and X 8 , X 9 , X 10 , X 11 and X 12 is any amino acid or is deleted.

[0226] Building the TCL2 library A TCL2 library was constructed in which both the BC and FG loops of Tencon were randomized and the distribution of amino acids at each position was tightly controlled. Table 2 shows the amino acid distribution at the desired loop positions in the TCL2 library. The designed amino acid distribution had two purposes. First, the library was biased towards residues 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 only a subset of hydrophobic amino acids because the residue at position 29 is buried in the hydrophobic core of the Tencon fold. The second layer of the design involved biasing the amino acid distribution towards that of residues preferentially found in the heavy chain HCDR3 of antibodies to efficiently generate high affinity binders (Birtalan et al., J Mol Biol 377:1518-1520). 28, 2008; Olson et al., Protein Sci 16:476-84, 2007). For this purpose, the "designed distribution" in Table 2 refers to a 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 lacks methionine, cysteine, and a stop (STOP) codon.

[0227] TCL2 library (SEQ ID NO:3) LPAPKNLVVSEVTEDSLRLSWX 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 SFLIQYQESEKVGEAINLTVPGSERSYDLTGLKPGTEYTVSIYGVX 9 X 10 X 11 X 12 X 13 SX 14 X 15 LSAEFTT; where X 1 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr, or Val; 2 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr, or Val; 3 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr, or Val; 4 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr, or Val; 5 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr, or Val; 6 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr, or Val; 7 is Phe, Ile, Leu, Val or Tyr; X 8 is Asp, Glu or Thr; X9 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr, or Val; 10 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val; X 11 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val; X 12 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val; X 13 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val; X 14 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val; and X 15 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val.

[0228] [Table 2]

[0229] These libraries were then improved in various ways, including building libraries on a stabilized Tencon framework (US Pat. No. 8,569,227) incorporating substitutions E11R / L17A / N46V / E86I (Tencon27; SEQ ID NO: 4) when compared to wild-type tencon, as well as altering randomized positions in the BC and FG loops. Tencon27 is described in International Patent Application No. WO2013049275. From this, new libraries were generated that were designed to randomize only the FG loop of Tencon (library TCL9) or a combination of the BC and FG loops (library TCL7). These libraries were constructed for use with cis-display systems (Odegrip et al., Proc. Natl. Acad. Sci. USA 101:2806-2810, 2004). The details of this design are given below:

[0230] Stabilized Tencon (Tencon27) (SEQ ID NO: 4) LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFLIQYQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVKGGHRSNPLSAIFTT

[0231] TCL7 (randomized FG and BC loops) (SEQ ID NO:5) LPAPKNLVVSRVTEDSARLSWX 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 FDSFLIQYQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVX 10 X 11 X 12 X 13 X 14 X 15 X 16 X 17 X 18 X 19SNPLSAIFTT; Where: X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , X 10 , X 11 , X 12 , X 13、 X 14、 X 15 and X 16 is A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W or Y; and X 7 , X 8 , X 9 , X 17 , X 18 and X 19 is A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, Y or deleted.

[0232] TCL9 (randomized FG loop) (SEQ ID NO:6) LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFLIQYQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIYGV X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 X 11 X 12 SNPLSAIFTT; X 1 , X 2 , X 3 , X 4 , X 5 , X 6 and X 7 is A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W or Y; and X 8、 X 9、 X 10、 X 11 and X12 is A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, Y or deleted.

[0233] For library construction, DNA fragments encoding randomized BC loops (length 6-9 positions) or FG loops (length 7-12 positions) were synthesized using Slonomics technology (Sloning Biotechnology GmbH) to control the amino acid distribution of the library and to exclude stop codons. Two different sets of DNA molecules randomizing either the BC or FG loop were synthesized independently and then combined using PCR to produce the complete library product.

[0234] Construction of FG loop library (TCL9) A set of synthetic DNA molecules was produced consisting of the 5'Tac promoter followed by the complete gene sequence of Tencon except for the randomized codons in the FG loop (SEQ ID NO: 26-31). For the FG loop randomization, all amino acids were coded for in equal percentages except for cysteine ​​and methionine. The lengths of the diversified parts were such that they coded for 7, 8, 9, 10, 11, or 12 amino acids in the FG loop. Sublibraries of each length variation were synthesized individually at the 2ug scale and then amplified by PCR using oligos Sloning-FOR (SEQ ID NO: 9) and Sloning-Rev (SEQ ID NO: 10).

[0235] 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 cis and ori elements. A PCR reaction was performed using the plasmid (pCR4Blunt) (Invitrogen) as a template with M13 forward and M13 reverse primers to amplify this fragment. The resulting PCR product was digested overnight with PspOMI and gel purified. To ligate the 5' portion of the library DNA to the 3' DNA containing the repA gene, 2 pmol (~540ng-560ng) of 5' DNA was ligated to an equimolar concentration (~1.25μg) of 3' repA DNA in the presence of NotI and PspOMI enzymes and T4 ligase overnight at 37°C. The ligated library product was amplified by using 12 cycles of PCR with oligo POP2250 (SEQ ID NO:11) and DigLigRev (SEQ ID NO:12). For each sublibrary, the resulting DNA from 12 PCR reactions was combined and purified by Qiagen spin columns. Yields for each TCL9 sublibrary ranged from 32 to 34 μg.

[0236] Construction of FG / BC loop library (TCL7) The TCL7 library contains randomized Tencon BC and FG loops. The present invention comprises a library in which BC loops of 6-9 amino acids in length were combinatorically mixed 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 made containing a Tencon gene encoding the N-terminal portion of the protein up to and including residue VX, such that the BC loop was replaced with either 6, 7, 8, or 9 randomized amino acids, which are represented by the string of "N"s in the sequences provided herein. These fragments were synthesized prior to the discovery of the L17A, N46V, and E83I mutations (CEN5243), which were introduced in the molecular biology steps described below. To combine this fragment with a fragment encoding a randomized FG loop, the following steps were taken.

[0237] First, a DNA fragment (130mer-L17A, SEQ ID NO:17) encoding the Tac promoter and the 5' sequence of Tencon up to the nucleotide encoding amino acid A17 was generated by PCR using oligos POP2222ext (SEQ ID NO:18) and LS1114 (SEQ ID NO:19). This was done to include the L17A mutation in the library (CEN5243). Next, a DNA fragment encoding Tencon residues R18 to V75, including the randomized BC loop, was amplified by PCR using BC6, BC7, BC8, or BC9 as template and oligos LS1115 (SEQ ID NO:20) and LS1117 (SEQ ID NO:21). This PCR step introduced a BsaI site at the 3' end. These DNA fragments were then joined by overlap PCR using oligos POP2222ext and LS1117 as primers. The resulting 240 bp PCR products were pooled and purified by Qiagen PCR purification kit. The purified DNA was digested with BsaI-HF and gel purified.

[0238] The fragment encoding the FG loop was amplified using PCR using FG7, FG8, FG9, FG10, FG11, and FG12 as templates with oligonucleotides SDG10 (SEQ ID NO:22) and SDG24 (SEQ ID NO:23), incorporating a BsaI restriction site and the N46V and E86I mutations (CEN5243).

[0239] The digested BC and FG fragments were ligated together in a single step using 3-way ligation. Four ligation reactions in 16 possible combinations were set up, with each ligation reaction combining two BC and two FG loop lengths. Each ligation contained approximately 300 ng of total BC fragments and 300 ng of FG fragments. These four 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 was then digested with Not1 and cleaned up using a Qiagen PCR purification column. 5.2 μg of this DNA was ligated to an equimolar amount of RepA DNA fragment (approximately 14 μg), digested with PspOMI, and the products were amplified by PCR using oligos POP2222.

[0240] Example 2: Generation of Tencon libraries with alternative binding surfaces The choice of residues to be randomized in a particular library design influences the overall shape of the interaction surface generated. X-ray crystallography of the 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 highly curved interface that fits into the active site of MBP (Koide et al., Proc. Natl. Acad. Sci. USA 104: 6632-6637, 2007). In contrast, the ankyrin repeat scaffold protein selected to bind MBP was found to have a much flatter interaction surface and bind to the outer surface of MBP, away from the active site (Binz et al., Nat. Biotechnol. 22: 575-582, 2004). These The results suggest that the shape of the binding surface of the scaffold molecule (curved vs. flat) may determine which target proteins or specific epitopes on those target proteins can be efficiently bound by the scaffold. Published efforts centered on engineering protein scaffolds containing FN3 domains for protein binding have relied on engineering adjacent loops for target binding, thus resulting in curved binding surfaces. This approach may limit the number of targets and epitopes accessible by such scaffolds.

[0241] Tencon and other FN3 domains contain two sets of CDR-like loops on 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 a beta strand that forms the center of the FN3 structure. If the image of Tencon is rotated 90 degrees, another surface can be visualized. This slightly concave surface is formed by the CD and FG loops and two antiparallel beta strands, the C and F beta strands, and is referred to herein as 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 the residues that form the surface. The beta strands have a repeating structure with the side chains of all other residues exposed to the surface of the protein. Thus, libraries can be created by randomizing some or all of the surface-exposed residues in the beta strands. By selecting 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.

[0242] Library TCL14 (SEQ ID NO:7) was designed on the Tencon27 scaffold (SEQ ID NO:4).

[0243] A full description of the methods used to construct this library is described in U.S. Patent Publication No. 2013 / 0226834.

[0244] TCL14 library (SEQ ID NO:7): LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFX 1 IX 2 YX 3 EX 4 X 5 X 6 X 7 GEAIVLTVPGSERSYDLTGLKPGTEYX 8 VX 9 IX 10 GVKGGX 11 X 12SX 13 PLSAIFTT; Where: X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , X 7 , X 8 , X 9 , X 10 , X 11 , X 12 and X 13 is A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, Y, C or M.

[0245] The two beta strands that form the C-CD-F-FG surface in Tencon27 have a total of nine surface-exposed residues that can be randomized; C strand: S30, L32, Q34, Q36; F strand: E66, T68, S70, Y72, and V74, while the CD loop has six potential residues: S38, E39, K40, V41, G42, and E43, and the FG loop has seven potential residues: K75, G76, G77, H78, R79, S80, and N81. Selected residues were chosen for inclusion in the TCL14 design due to the larger theoretical size of the library if all 22 residues were randomized.

[0246] Thirteen positions in Tencon were chosen for randomization: L32, Q34, and Q36 in the C strand, S38, E39, K40, and V41 in the CD loop, T68, S70, and Y72 in the F strand, and H78, R79, and N81 in the FG loop. In the C and F strands, S30 and E66 are just beyond the CD and FG loops, and As these residues do not appear to be part of the C-CD-F-FG surface, they were not randomized. For the CD loop, G42 and E43 were not randomized because glycines providing mobility may be beneficial in the loop region, and E43 is at the interface of the surface. The FG loop excluded K75, G76, G77, and S80. Although glycines were excluded for the reasons mentioned above, careful examination of the crystal structure showed that S80 makes a key contact with the core and helps to form a stable FG loop. K75 points away from the surface of the C-CD-F-FG surface and was not a very attractive candidate for randomization. Although the above-mentioned residues were not randomized in the original TCL14 design, they may be included in subsequent library designs to generate additional diversity for de novo selection or, for example, affinity maturation libraries for hits specific to the selected TCL14 target.

[0247] After the production of TCL14, three additional Tencon libraries of similar design were produced. These two libraries, TCL19, TCL21 and TCL23, were randomized at the same positions as TCL14 (see above), but the distribution of amino acids present at these positions was altered (Table 3). TCL19 and TCL21 were designed to contain equal distributions of the 18 natural amino acids (5.55% each) at all positions, with the only exception of cysteine ​​and methionine. TCL23 was designed such that each randomized position approximated the amino acid distribution found in the HCDR3 loop of a functional antibody (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.

[0248] A third additional library was constructed to expand the potential target binding surface of the other libraries. In this library, TCL24, four additional Tencon positions were randomized compared to libraries TCL14, TCL19, TCL21, and TCL23. These positions include N46 and T48 from the D strand, and S84 and I86 from the G strand. These residues were specifically selected because their side chains are surface-exposed from the beta strands D and G, and are structurally adjacent to the randomized positions of the C and F strands, thus increasing the surface area accessible for binding to the target protein. The amino acid distribution used at each position for TCL24 was the same as that described for TCL19 and TCL21 in Table 3.

[0249] TCL24 library (SEQ ID NO:8) LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFX 1 IX 2 YX 3 EX 4 X 5 X 6 X 7 GEAIX 8 LX 9 VPGSERSYDLTGLKPGTEYX 10 VX 11 IX 12 GVKGGX 13 X 14 SX 15 PLX 16 AX 17 FTT; Where: X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , X 10 , X 11 , X 12 , X 13、 X 14 , X 15 , X 16 and X 17is A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, Y or W.

[0250] [Table 3]

[0251] Generation of TCL21, TCL23, and TCL24 libraries The TCL21 library was generated using Colibra library technology (Isogenica) to control the amino acid distribution. The TCL19, TCL23, and TCL24 gene fragments were generated using Slonomics technology (Morphosys) to control the amino acid distribution. Each library was amplified after the initial synthesis using PCR and subsequently linked to the gene for RepA to be used in selection using the CIS-display system (Odegrip et al., Proc. Natl. Acad. Sci. USA 101:2806-2810, 2004), as described above for the loop library.

[0252] Example 3: Selection of Fibronectin type III (FN3) domains that bind to CD71 Panning and biochemical screening FN3 domains specific for human CD71 were selected by CIS-display (Odegrip et al., 2004) using recombinant biotinylated CD71 extracellular domain with an N-terminal 6His tag (Sino Biologics). For in vitro transcription and translation (ITT), 3 μg of DNA from FN3 domain libraries TCL18, TCL19, TCL21, TCL23, and TCL24 were used and unbound library members were removed by washing. DNA was eluted from the target protein by heating and amplified by PCR using KOD polymerase for further successive rounds of panning. The concentration of target CD71 was serially increased from 400 nM to 100 nM during each round. High affinity binders were isolated by decreasing the concentration of IgG and increasing the washing stringency. The output from the fifth round of panning was subjected to four additional rounds of off-rate selection. The biotinylated target antigen concentration was decreased from 25 nM in rounds 6 and 7 to 2.5 nM in rounds 8 and 9.

[0253] Following panning, genes encoding selected FN3 domains were amplified by PCR, subcloned into a pET vector modified to contain 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 polyhistidine tag was added to each FN3 domain to allow for purification and detection.

[0254] To screen for FN3 domains that specifically bind to CD71, streptavidin-coated Maxisorp plates (Nunc catalog 436110) were blocked in Starting Block T20 (Pierce) for 1 hour and then coated with biotinylated CD71 (using the same antigen as in panning) or negative controls (irrelevant Fc-fusion recombinant protein and human serum albumin) for 1 hour. Plates were rinsed with TBST and diluted lysates were applied to the plates for 1 hour. After further rinsing, wells were treated with HRP-conjugated anti-V5 tag antibody (Abcam, ab1325) for 1 hour and then assayed with POD (Roche, 11582950001). DNA from FN3 domain lysates with ELISA signals at least 10-fold higher than the signal of the streptavidin control was sequenced and 23 different readable FN3 domain sequences were isolated from round 9 screening (Table 4).

[0255] [Table 4]

[0256] Size exclusion chromatography analysis Size exclusion chromatography was used to determine the aggregation state of the anti-CD71 FN3 domains. Aliquots (10 μL) of each purified FN3 domain were purified using Superdex A 75 5 / 150 column (GE Healthcare) was injected in a mobile phase of PBS pH 7.4 at a flow rate of 0.3 mL / min. Elution from the column was monitored by absorbance at 280 nm. Tencon protein was included in each run as a control. Elution profiles were analyzed using Agilent ChemStation software. Selected SEC parameters for the 18 identified FN3 domains are listed in Table 5.

[0257] [Table 5]

[0258] High-throughput expression and conjugation Identified clones were grown in duplicate 5 mL cultures in 24-well deep block plates. Briefly, 5 mL of TB medium supplemented with 50 μg / mL kanamycin was added. L / well were inoculated with 150 μL of overnight culture and grown for approximately 3 hours at 37°C with shaking at 220 rpm (OD600 approx. 1). Cultures were induced with IPTG to a final concentration of 1 mM for an additional 4 hours at 37°C, 220 rpm. Bacterial pellets were collected by centrifugation at 2250xg for 15 minutes. 600 μL / well BugBuster HT (Novagen) supplemented with 0.2 mg / mL lysozyme (Sigma) was added to each well; the pellet was dissociated by pipetting and then shaken vigorously on a shaking platform for approximately 30 minutes until the pellet was dissolved. The plate was spun at 2250xg for 15 minutes to clear the lysate, and two 600 μL aliquots were combined for each sample. The His-tagged FN3 domain was purified on HisTrap plates (GE) according to the manufacturer's instructions, followed by buffer exchange into TBS using Zeba Spin 7K desalting plates (Thermo Scientific). Protein concentration was assessed by Nanodrop. For binding to GlyGly-VC-MMAF, FN3 domain (30 μM) was mixed with 150 μM GlyGlyVC-MMAF (Concortis) and 1 μM Sortase A in a total volume of 200 μL. Binding was allowed to proceed for 1.5 h at room temperature and then incubated for 2 h at 4°C for 1 h at 4°C for 1 h at 4°C. Binding was then allowed to proceed for 1.5 h at room temperature and then incubated for 2 h at 4°C for 1 ... Purification was again performed using HP plates according to the manufacturer's instructions. Buffer exchange into PBS was achieved using Zeba desalting plates, followed by sterile filtration using Multiscreen HTS GV plates (Durapore) with centrifugation at 3000xg for 2 min. Concentration was assessed by Nanodrop.

[0259] Identification of SK-BR3-binding FN3 domains SK-BR-3 cells were cultured in McCoy's 5a medium + 10% fetal bovine serum. FN3 dilutions were prepared in FACS buffer. 50,000 SK-BR-3 cells were added to each well; the medium was aspirated after centrifugation, and the cells were resuspended in 100 μL of FACS buffer containing HiLyte-labeled FN3 domain. The cells were incubated for 2 hours at 37°C, 5% CO2. The cells were rinsed three times with FACS buffer, and finally resuspended in 100 μL of FACS buffer. Fluorescence was detected by Intellictye. Cell populations were identified by FSC-SSC dot plots, followed by recording of FL4 MFI. Data were normalized to the average of eight unstained cells, and dose-response curves were fitted using GraphPad.

[0260] Binding of selected clones by dose-response ELISA Selected clones were analyzed by ELISA to determine EC50 values ​​for binding. Briefly, Maxisorb plates were coated with streptavidin at 5 μg / ml overnight at 4C. Plates were then blocked with StartingBlock (ThermoFisher) for 1 h at room temperature and then washed with TBS-Tween. Biotinylated CD71 (2 μg / ml) was captured on the streptavidin plate and serially diluted Centyrins were added to appropriate wells for 1 h at room temperature. After washing, bound Centyrins was detected using an anti-V5 tag antibody conjugated to HRP and POD substrates and a luminescence plate reader. Luminescence values ​​were plotted as a function of concentration and fitted to a dose response using PRISM to determine EC50 values ​​for binding.

[0261] Identification of internalized FN3 domains via toxin conjugates. FN3 domains were conjugated to the cytotoxic tubulin inhibitor monomethyl auristatin F (MMAF) via an enzyme-cleavable Val-Cit linker or a non-cleavable PEG4 linker (VC-MMAF) using methods described for NEM conjugation. Cell killing was assessed by measuring the viability of SKBR-3 cells after exposure to cysteine ​​variant-cytotoxin conjugates. Cells were plated at 3000 / well in white well, opaque bottom, tissue culture treated plates (Fisher, PI15042) in 50 μL / well of phenol red RPMI medium (Gibco, 11875093) containing 10% fetal bovine serum (Gibco). Cells The cells were allowed to attach overnight at 37°C in a humidified 5% CO2 atmosphere. Cells were treated with 25uL fresh medium and 25uL 4x inhibitor made up in fresh medium. Cell viability was determined by end-point assay using Cell TiterGlo (Promega) at 72 hours. IC50 values ​​were determined by fitting the data to an equation for a sigmoidal dose response with variable slope using GraphPad Prism (GraphPad Software). The results are shown in Table 6 and demonstrate that FN3 domains that bind to CD71 were internalized and cytotoxic.

[0262] [Table 6]

[0263] Bivalent FN3 protein Bivalent FN3 proteins are produced using two FN3 domains connected by a 4-repeat G / S linker. Bivalent FN3 proteins are conjugated to VC-MMAF as described and evaluated for cytotoxicity in SK-BR3 cells. The IC50 values ​​for the bivalent molecules are found to be superior.

[0264] Competition for transferrin binding and internalization FN3 domain VCMMAF conjugates were screened for competition with human transferrin using the cytotoxicity assay described above. FN3 domains were screened in the absence or presence of 0.6uM holo-human transferrin (T0665-100MG). IC50 values ​​for FN3 domain toxin conjugates against SK-BR3 cells screened in the absence or presence of competitor are shown in Table 7.

[0265] [Table 7]

[0266] SEQ ID NO:1 = original Tencon sequence LPAPKNLVVSEVTEDSLRLSWTAPDAAFDSFLIQYQESEKVGEAINLTVPGSERSYDLTGLKPGTEYTVSIYGVKGGHRSNPLSAEFTT

[0267] SEQ ID NO:2 = TCL1 library LPAPKNLVVSEVTEDSLRLSWTAPDAAFDSFLIQYQESEKVGEAINLTVPGSERSYDLTGLKPGTEYTVSIYGV(X) 7-12 PLSAEFTT; Where: X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , X 7 is any amino acid; and X 8 , X 9 , X 10 , X 11 and X 12 is any amino acid or is deleted.

[0268] SEQ ID NO:3 = TCL2 library LPAPKNLVVSEVTEDSLRLSWX 1 X 2 X 3 X4 X 5 X 6 X 7 X 8 SFLIQYQESEKVGEAINLTVPGSERSYDLTGLKPGTEYTVSIYGVX 9 X 10 X 11 X 12 X 13 SX 14 X 15 LSAEFTT; Where: X 1 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr, or Val; 2 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr, or Val; 3 Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val; X 4 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr, or Val; 5 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr, or Val; 6 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr, or Val; 7 is Phe, Ile, Leu, Val or Tyr; X 8 is Asp, Glu or Thr; X 9 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr, or Val;10 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val; X 11 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val; X 12 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val; X 13 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val; X 14 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val; and X 15 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val.

[0269] SEQ ID NO:4 = stabilized Tencon LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFLIQYQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVKGGHRSNPLSAIFTT

[0270] SEQ ID NO:5 = TCL7 (FG and BC loops) LPAPKNLVVSRVTEDSARLSWX 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9FDSFLIQYQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVX 10 X 11 X 12 X 13 X 14 X 15 X 16 X 17 X 18 X 19 SNPLSAIFTT; Where: X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , X 10 , X 11 , X 12 , X 13、 X 14、 X 15 and X 16 is A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W or Y; and X 7 , X 8 , X 9 , X 17 , X 18 and X 19 is A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, Y or missing.

[0271] SEQ ID NO:6 = TCL9 (FG loop) LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFLIQYQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIYGV X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 X 11 X 12 SNPLSAIFTT; Where: X 1 , X 2, X 3 , X 4 , X 5 , X 6 and X 7 is A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W or Y; and X 8、 X 9、 X 10、 X 11 and X 12 is A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, Y or missing.

[0272] TCL14 library (SEQ ID NO:7) LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFX 1 IX 2 YX 3 EX 4 X 5 X 6 X 7 GEAIVLTVPGSERSYDLTGLKPGTEYX 8 VX 9 IX 10 GVKGGX 11 X 12 SX 13 PLSAIFTT; Where: X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , X 7 , X 8 , X 9 , X 10 , X 11 , X 12 and X 13 is A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, Y, C or M.

[0273] TCL24 library (SEQ ID NO:8) LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFX 1 IX 2 YX 3EX 4 X 5 X 6 X 7 GEAIX 8 LX 9 VPGSERSYDLTGLKPGTEYX 10 VX 11 IX 12 GVKGGX 13 X 14 SX 15 PLX 16 AX 17 FTT; Where: X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , X 10 , X 11 , X 12 , X 13、 X 14 , X 15 , X 16 and X 17 is A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, Y or W.

[0274] SEQ ID NO:9 = Sloning-FOR GTGACACGGCGGTTAGAAC

[0275] SEQ ID NO:10 = Sloning-REV GCCTTTGGGAAGCTTCTAAG

[0276] Sequence number 11 = POP2250 CGGCGGTTAGAACGCGGCTACAATTAATAC

[0277] SEQ ID NO:12 = DigLigRev CATGATTACGCCAAGCTCAGAA

[0278] SEQ ID NO:13=BC9 GTGACACGGCGGTTAGAACGCGGCTACAATTAATACATAACCCCATCCCCCTGTTGACAATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGGATCTACCATGCTGCCGGCGCCGAAAAACCTGGTTGTTTCTGAAGTTACCGAAGACTCTCTGCGTCTGTCTTGGNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNTTYGACTCTTTCCTGATCCAGTACCAGGAATCTGAAAAAGTTGGTGAAGCGATCAACCTGACCGTTCCGGGTTCTGAACGTTCTTACGACCTGACCGGTCTGAAACCGGGTACCGAATACACCGTTTCTATCTACGGTGTTCTTAGAAGCTTCCCAAAGGC (where N is any base)

[0279] SEQ ID NO:14=BC8 GTGACACGGCGGTTAGAACGCGGCTACAATTAATACATAACCCCATCCCCCTGTTGACAATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGGATCTACCATGCTGCCGGCGCCGAAAAACCTGGTTGTTTCTGAAGTTACCGAAGACTCTCTGCGTCTGTCTTGGNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNTTYGACTCTTTCCTGATCCAGTACCAGGAATCTGAAAAAGTTGGTGAAGCGATCAACCTGACCGTTCCGGGTTCTGAACGTTCTTACGACCTGACCGGTCTGAAACCGGGTACCGAATACACCGTTTCTATCTACGGTGTTCTTAGAAGCTTCCCAAAGGC (where N is any base)

[0280] SEQ ID NO:15=BC7 GTGACACGGCGGTTAGAACGCGGCTACAATTAATACATAACCCCATCCCCCTGTTGACAATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGGATCTACCATGCTGCCGGCGCCGAAAAACCTGGTTGTTTCTGAAGTTACCGAAGACTCTCTGCGTCTGTCTTGGNNNNNNNNNNNNNNNNNNNNNNNNNNTTYGACTCTTTCCTGATCCAGTACCAGGAATCTGAAAAAGTTGGTGAAGCGATCAACCTGACCGTTCCGGGTTCTGAACGTTCTTACGACCTGACCGGTCTGAAACCGGGTACCGAATACACCGTTTCTATCTACGGTGTTCTTAGAAGCTTCCCAAAGGC (where N is any base)

[0281] SEQ ID NO:16=BC6 GTGACACGGCGGTTAGAACGCGGCTACAATTAATACATAACCCCATCCCCCTGTTGACAATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGGATCTACCATGCTGCCGGCGCCGAAAAACCTGGTTGTTTCTGAAGTTACCGAAGACTCTCTGCGTCTGTCTTGGNNNNNNNNNNNNNNNNNNNNNNTTYGACTCTTTCCTGATCCAGTACC AGGAATCTGAAAAAGTTGGTGAAGCGATCAACCTGACCGTTCCGGGTTCTGAACGTTCTTACGACCTGACCGGTCTGAAACCGGGTACCGAATACACCGTTTCTATCTACGGTGTTCTTAGAAGCTTCCCAAAGGC (where N is any base)

[0282] SEQ ID NO:17=130mer-L17A CGGCGGTTAGAACGCGGCTACAATTAATACATAACCCCATCCCCCTGTTGACAATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGGATCTACCATGCTG

[0283] Enclosure 18=POP222ext CGG CGG TTA GAA CGC GGC TAC YOU ARE TAC

[0284] Enclosure 19=LS1114 CCA AGA CAG ACG GGC AGA GTC TTC GGT AAC GCG AGA AAC AAC CAG GTT TTT CGG CGC CGG CAG CAT GGT AGA TCC TGT TTC

[0285] range 20=LS1115 CCG AAG ACT CTG CCC GTC TGT CTT GG

[0286] Enclosure 21=LS1117 CAG TGG TCT CAC GGA TTC CTG GTA CTG GAT CAG GAA AGA GTC GAA

[0287] Dimensions 22=SDG10 CATGCGGTCTCTTCCGAAAAAGTTGGTGAAGCGATCGTCCTGACCGTTCCGGGT

[0288] range 23=SDG24 GGTGGTGAAGATCGCAGACAGCGGGTTAG

[0289] Enclosure 24=POP2222 CGGCGGTTAGAACGCGGCTAC

[0290] Dimensions 25=SDG28 AAGATCAGTTGCGGCCGCTAGACTAGAACCGCTGCCACCGCCGGTGGTGAAGATCGCAGAC

[0291] SEQ ID NO:26=FG12 GTGACACGGCGGTTAGAACGCGGCTACAATTAATACATAACCCCATCCCCCTGTTGACAATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGGATCTACCATGCTGCCGGCGCCGAAAAACCTGGTTGTTTCTCGCGTTACCGAAGACT CTGCGCGTCTTGTCTTGGACCGCGCCGGACGCGGCGTTCGACTCTTTCTGATCCAGTACCAGGAATCTGAAAAAGTTGGTGAAGCGATCGTGCTGACCGTTCCGGGTTCTGAACGTTCTTACGACCTGACCGGTCTGAAACCGGGTACCGAATACACCGTTTCTATCTACGGTGTTNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNTCTAACCCGCTGTCTGCGATCTTCACCACCGGCGGTCACCATCACCATCACCATGGCAGCGGTTCTAGTCTAGCGGCCGCAACTGATCTTGGC (where N is any base)

[0292] SEQ ID NO:27=FG11 GTGACACGGCGGTTAGAACGCGGCTACAATTAATACATAACCCCATCCCCCTGTTGACAATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGGATCTACCATGCTGCCGGCGCCGAAAAACCTGGTTGTTTCTCGCGTTACCGAAGACTCTGCGCGTCTGTCTTGGACCGCGCCGGACGGCGTTCGACTCTTTCCTGATCCAGTACCAGGAATCTGAAAAAGTTGGTGAAGCGATCGTGCTGACCGTTCCGGGTTCTGAACGTTCTTACGACCTGACCGGTCTGAAACCGGGTACCGAATACACCGTTTCTATCTACGGTGTTNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNTCTAACCCGCTGTCTGCGATCTTCACCACCGGCGGTCACCATCACCATCACCATGGCAGCGGTTCTAGTCTAGCGGCCGCAACTGATCTTGGC (where N is any base)

[0293] SEQ ID NO:28=FG10 GTGACACGGCGGTTAGAACGCGGCTACAATTAATACATAACCCCATCCCCCTGTTGACAATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGGATCTACCATGCTGCCGGCGCCGAAAAACCTGGTTGTTTCTCGCGTTACCGAAGACTCTGCGCGTCTGTCTTGGACCGCGCCGGACGGCGTTCGACTCTTTCCTGATCCAGTACCAGGAATCTGAAAAAGTTGGTGAAGCGATCGTGCTGACCGTTCCGGGTTCTGAACGTTCTTACGACCTGACCGGTCTGAAACCGGGTACCGAATACACCGTTTCTATCTACGGTGTTNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNTCTAACCCGCTGTCTGCGATCTTCACCACCGGCGGTCACCATCACCATCACCATGGCAGCGGTTCTAGTCTAGCGGCCGCAACTGATCTTGGC (where N is any base)

[0294] SEQ ID NO:29=FG9 GTGACACGGCGGTTAGAACGCGGCTACAATTAATACATAACCCCATCCCCCTGTTGACAATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGGATCTACCATGCTGCCGGCGCCGAAAAACCTGGTTGTTTCTCGCGTTACCGAAGACTCTGCGCGTCTGTCTTGGACCGCGCCGGACGGCGTTCGACTCTTTCCTGATCCAGTACCAGGAATCTGAAAAAGTTGGTGAAGCGATCGTGCTGACCGTTCCGGGTTCTGAACGTTCTTACGACCTGACCGGTCTGAAACCGGGTACCGAATACACCGTTTCTATCTACGGTGTTNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNTCTAACCCGCTGTCTGCGATCTTCACCACCGGCGGTCACCATCACCATCACCATGGCAGCGGTTCTAGTCTAGCGGCCGCAACTGATCTTGGC (where N is any base)

[0295] SEQ ID NO:30=FG8 GTGACACGGCGGTTAGAACGCGGCTACAATTAATACATAACCCCATCCCCCTGTTGACAATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGGATCTACCATGCTGCCGGCGCCGAAAAACCTGGTTGTTTCTCGCGTTACCGAAGACTCTGCGCGTCTGTCTTGGACCGCGCCGGACGGCGTTCGACTCTTTCCTGATCCAGTACCAGGAATCTGAAAAAGTTGGTGAAGCGATCGTGCTGACCGTTCCGGGTTCTGAACGTTCTTACGACCTGACCGGTCTGAAACCGGGTACCGAATACACCGTTTCTATCTACGGTGTTNNNNNNNNNNNNNNNNNNNNNNNNNNNNTCTAACCCGCTGTCTGCGATCTTCACCACCGGCGGTCACCATCACCATCACCATGGCAGCGGTTCTAGTCTAGCGGCCGCAACTGATCTTGGC (where N is any base)

[0296] SEQ ID NO:31=FG7 GTGACACGGCGGTTAGAACGCGGCTACAATTAATACATAACCCCATCCCCCTGTTGACAATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGGATCTACCATGCTGCCGGCGCCGAAAAACCTGGTTGTTTCTCGCGTTACCGAAGACTCTGCGCGTCTGTCTTGGACCGCGCCGGACGGCGTTCGACTCTTTCCTGATCCAGTACCAGGAATCTGAAAAAGTTGGTGAAGCGATCGTGCTGACCGTTCCGGGTTCTGAACGTTCTTACGACCTGACCGGTCTGAAACCGGGTACCGAATACACCGTTTCTATCTACGGTGTTNNNNNNNNNNNNNNNNNNNNNNNTCTAACCCGCTGTCTGCGATCTTCACCACCGGCGGTCACCATCACCATCACCATGGCAGCGGTTCTAGTCTAGCGGCCGCAACTGATCTTGGC (where N is any base)

[0297] SEQ ID NO:32 = human mature CD71 MTKEYQDLQHLDNEESDHHQLRKGPPPPQPLLQRLCSGPRLLLLSLGLSLLLLVVVCVIGSQNSQLQEELRGLRETFSNFTASTEAQVKGLSTQGGNVGRKMKSLESQLEKQQKDLSEDHSSLLLHVKQFVSDLRSLSCQMAALQ GNGSERTCCPVNWVEHERSCYWFSRSGKAWADADNYCRLEDAHLVVVTSWEEQKFVQHHIGPVNTWMGLHDQNGPWKWVDGTDYETGFKNWRPEQPDDWYGHGLGGGEDCAHFTDDGRWNDDVCQRPYRWVCETELDKASQEPPLL

[0298] SEQ ID NO:80 = human mature CD71 extracellular domain QNSQLQEELRGLRETFSNFTASTEAQVKGLSTQGGNVGRKMKSLESQLEKQQKDLSEDHSSLLLHVKQFVSDLRSLSCQMAALQGNGSERTCCPVNWVEHERSCYWFSRSGKAWA DADNYCRLEDAHLVVTSWEEQKFVQHHIGPVNTWMGLHDQNGPWKWVDGDTDYETGFKNWRPEQPDDWYGHGLGGGEDCAHFTDDGRWNDDVCQRPYRWVCETELDKASQEPPLL

[0299] [Table 8-1] [Table 8-2]

[0300] Example 4: Selection of Fibronectin Type III (FN3) Domains that Bind to CD71 Panning and biochemical screening methods to identify Fn3 domains that bind to CD71 and do not inhibit transferrin binding to CD71. To screen for FN3 domains that specifically bind to CD71 and do not inhibit transferrin binding to CD71, streptavidin-coated Maxisorp plates (Nunc catalog 436110) were blocked in Starting Block T20 (Pierce) for 1 hour, and then coated with biotinylated CD71 (using the same antigen as in panning) or negative controls (irrelevant Fc fusion recombinant protein and serum albumin) for 1 hour in the presence of transferrin or in the presence of FN3 protein that binds to the CD71 transferrin binding site. The concentration of transferrin is up to 35 μM. Without being bound to any particular theory, the inclusion of transferrin or FN3 protein that binds to the CD71 transferrin binding site advances the selection of FN3 domains to those that do not compete with or inhibit transferrin binding to CD71. Plates were rinsed with TBST and diluted lysates were applied to the plates for 1 hour. After further rinsing, wells were treated with HRP-conjugated anti-V5 tag antibody (Abcam, ab1325) for 1 hour and then assayed using POD (Roche, 11582950001). DNA from FN3 domain lysates with ELISA signals at least 10-fold higher than the ELISA signal of the streptavidin control were sequenced, yielding the FN3 domain sequences isolated from the screen.

[0301] Example 5: Selection of Fibronectin Type III (FN3) Domains that Bind to CD71 and Do Not Compete with Transferrin A biased CIS display strategy was designed to identify CD71-binding FN3 domains that do not or only minimally compete with transferrin. Briefly, the output recovered after five rounds of panning on the ECD of human CD71 is used (Example 3). Further rounds of off-rate selection were performed as described in Example 3, adding either 1) a wash step using human holo-transferrin to elute centyrins bound at the same site as transferrin before the final elution step, or 2) elution of FN3 domain binders using monoclonal antibody OKT9. FN3 domains recovered from the transferrin wash strategy and the OKT9 elution strategy were PCR amplified and cloned into pET vectors as previously described (Example 3). 228 FN3 domains that specifically bound to huCD71 were confirmed by ELISA for binding to huCD71 ECD (Table 8). A subset of unique binders were analyzed by SEC (Table 9), bound to MMAF, and assessed for internalization by cell viability assay in SKBR-3 cells + / - holo human transferrin (Table 10). The polypeptides were found to be internalized by the receptor. The data and sequences of the hits are identified in the table below.

[0302] [Table 9-1] [Table 9-2] [Table 9-3] [Table 9-4] [Table 9-5] [Table 9-6]

[0303]

Table 10-1

Table 10-2

Table 10-3

Table 10-4

Table 10-5

Table 10-6

[0304]

Table 11-1

Table 11-2

[0305]

Table 12-1

Table 12-2

Table 12-3

Table 12-4

Table 12-5

Table 12-6

Table 12-7

Table 12-8

[0306] Example 6. Knockdown of mRNA in muscle cells using CD71 FN3 domain-oligonucleotide conjugates. The muCD71-binding FN3 domain was conjugated to siRNA oligonucleotides or antisense oligonucleotides (ASOs) using the maleimide chemistry described above. Cysteine ​​substitutions can be as shown herein and also in U.S. Patent Publication No. 20150104808, which is incorporated herein by reference in its entirety. The siRNA or ASO was modified using standard chemical modifications and confirmed to allow targeted knockdown of mRNA in vitro. The FN3 domain-oligonucleotide conjugates were administered intravenously to mice at doses up to 10 mg / kg oligonucleotide payload. Mice were sacrificed at various times following administration; skeletal muscle, cardiac muscle, and various other tissues were harvested and analyzed using RNAlater. TM (Sigma Aldrich) and stored until needed. Target gene knockdown was assessed using standard qPCR ΔΔC T Using the method and primers specific for the target and control genes, the target genes were found to be knocked down in muscle, and such knockdown was enhanced by binding siRNA or ASO to the CD71 FN3-binding domain.

[0307] Example 7. General Methods Standard methods in molecular biology are described in Sambrook, Fritsch, and Maniatis (1982 and 1989, 2nd ed., 2001, 3rd ed.) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Sambrook and Russell (2001) Molecular Cloning, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Wu (1993) Recombinant DNA, Vol. 217, Academic Press, San Diego, CA). Standard methods are also found in Ausbel et al. (2001) Current Protocols in Molecular Biology, Vols. 1-4, John Wiley and Sons, Inc. New York, NY, which describe cloning and DNA mutagenesis in bacterial cells (Vol. 1), cloning in mammalian cells and yeast (Vol. 2), glycoconjugates and protein expression (Vol. 3), and bioinformatics (Vol. 4).

[0308] Protein purification methods including immunoprecipitation, chromatography, electrophoresis, centrifugation, and crystallization are described (Coligan et al. (2000) Current Protocols in Protein Science, Vol. 1, John Wiley and Sons, Inc., New York). Chemical analysis, chemical alterations, post-translational modifications, production of fusion proteins, and glycosylation of proteins are described (see, e.g., Coligan et al. (2000) Current Protocols in Protein Science, Vol. 2, John Wiley and Sons, Inc., New York; Ausubel et al. (2001) Current Protocols in Molecular Biology, Vol. 3, John Wiley and Sons, Inc., NY, NY, pp. 16.0.5-16.22.17; Sigma-Aldrich, Co. (2001) Products for Life Science Research, St. Louis, MO; pp. 45-89; Amersham Pharmacia Biotech (2001) BioDirectory, Piscataway, NJ, pp. 384-391). The production, purification, and fragmentation of polyclonal and monoclonal antibodies are described (Coligan et al. (2001) Current Protocols in Immunology, Vol. 1, John Wiley and Sons, Inc., New York; Harlow and Lane (1999) Using Antibodies, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Harlow and Lane, supra). Standard techniques are available for this purpose (see, e.g., Coligan et al. (2001) Current Protocols in Immunology, Vol. 4, John Wiley, Inc., New York).

[0309] All references cited herein are incorporated by reference to the same extent as if each individual publication, database entry (e.g., Genbank sequence or GeneID entry), patent application, or patent was specifically and individually indicated to be incorporated by reference. This statement of incorporation by reference is intended by the applicants in accordance with 37 CFR § 1.57(b)(1) to relate to each and every individual publication, database entry (e.g., Genbank sequence or GeneID entry), patent application, or patent (each of which is expressly identified in accordance with 37 CFR § 1.57(b)(2)), even if such citation is not immediately adjacent to the dedicated statement of incorporation by reference. The inclusion of dedicated statements of incorporation by reference, if any, within the specification does not in any way weaken this general statement of incorporation by reference. The citation of a reference herein is not intended as an admission that the reference is related to the prior art, and does not constitute any admission as to the contents or dates of these publications or documents.

[0310] The present embodiments should not be limited in scope by the specific embodiments described herein. Indeed, various modifications of the embodiments in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to be within the scope of the appended claims.

[0311] The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice the embodiments. Various modifications of the embodiments in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims.

Claims

1. SEQ ID NOs: 146, 214, 104, 259, 134, 92, 302, 235, 237, 152, 238, 136, 197, 212, 296, 226, 261, 307, 115, 112, 278, 297, 96, 222, 95, 233, 217, 252, 194, 164, 168, 174, 190, 257, 303, 284, 85, 149, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, or 81-309, or any combination thereof. or a polypeptide comprising SEQ ID NOs: 146, 214, 104, 259, 134, 92, 302, 235, 237, 152, 238, 136, 197, 212, 296, 226, 261, 307, 115, 112, 278, 297, 96, 222, 95, 233, 217, 252, 194, 164, 168, 174, 190, 257, A polypeptide comprising an amino acid sequence of 303, 284, 85, 149, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, or 81-309, or any combination thereof.

2. SEQ ID NOs: 146, 214, 104, 259, 134, 92, 302, 235, 237, 152, 238, 136, 197, 212, 296, 226, 261, 307, 115, 112, 278, 297, 96, 222, 95, 233, 217, 252, 194, 164, 168, 174, 190, 257, 303, 284, 85, 149, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, or two of 81 to 309.

3. A polypeptide that binds to human CD71 at a site on CD71 that does not compete with transferrin binding to CD71.

4. 4. The polypeptide of claim 3, comprising an amino acid sequence that is at least 90% identical to a polypeptide having the sequence of SEQ ID NO: 146, 214, 104, 259, 134, 92, 302, 235, 237, 152, 238, 136, 197, 212, 296, 226, 261, 307, 115, 112, 278, 297, 96, 222, 95, 233, 217, 252, 194, 164, 168, 174, 190, 257, 303, 284, 85, or 149.

5. 4. The polypeptide of claim 3, comprising the sequence of SEQ ID NO: 146, 214, 104, 259, 134, 92, 302, 235, 237, 152, 238, 136, 197, 212, 296, 226, 261, 307, 115, 112, 278, 297, 96, 222, 95, 233, 217, 252, 194, 164, 168, 174, 190, 257, 303, 284, 85, or 149.

6. The polypeptide of any one of claims 1 to 5, wherein the polypeptide is conjugated to a detectable label, an oligonucleotide, a therapeutic agent, or any combination thereof.

7. 7. The polypeptide of claim 6, wherein the detectable label is a radioisotope, a magnetic bead, a metallic bead, a colloidal particle, a fluorescent dye, an electron-dense reagent, an enzyme, biotin, digoxigenin, or a hapten.

8. Detectable labels include auristatin, monomethylauristatin phenylalanine, doxorubicin, 8. The polypeptide of claim 6 or 7, which is a chemotherapeutic agent, a drug, a growth inhibitor, a toxin, or a radioisotope.

9. 7. The polypeptide of claim 6, wherein the therapeutic agent is a chemotherapeutic agent, a drug, an antibody, a growth inhibitor, a toxin, a radioisotope, an antitubulin agent, a polynucleotide, an siRNA molecule or a sense or antisense strand thereof, an antisense molecule or a strand thereof, an RNA molecule, a DNA molecule, a DNA minor groove binder, a DNA replication inhibitor, an alkylating agent, an antibiotic, an antifolate, an antimetabolite, a chemotherapy sensitizer, a topoisomerase inhibitor, or a vinca alkaloid.

10. The polypeptide of claim 6, wherein the therapeutic agent is capable of inducing one or more cytotoxic effects by modulating gene expression, RNA expression or levels, tubulin binding, DNA binding, topoisomerase inhibition, DNA cross-linking, chelation, spliceosome inhibition, NAMPT inhibition, or HDAC inhibition.

11. The polypeptide of any one of claims 1 to 10, further comprising a methionine at the N-terminus of the polypeptide.

12. The polypeptide of any one of claims 1 to 11, wherein the polypeptide is coupled to a half-life extending moiety.

13. 13. The polypeptide of claim 12, wherein the half-life extending moiety is an albumin binding molecule, polyethylene glycol (PEG), albumin, an albumin variant, or at least a portion of an Fc region of an immunoglobulin.

14. 14. The polypeptide of claim 13, wherein the albumin binding molecule is a second polypeptide that binds to albumin or an albumin variant.

15. An isolated polynucleotide encoding the polypeptide of any one of claims 1 to 14.

16. A vector comprising the polynucleotide of claim 15.

17. A host cell comprising the vector of claim 16.

18. 20. A method for producing a polypeptide that binds to CD71, comprising culturing an isolated host cell according to claim 17 under conditions in which the polypeptide is expressed, and purifying the polypeptide.

19. A pharmaceutical composition comprising the polypeptide of any one of claims 1 to 14 and a pharma- ceutically acceptable carrier.

20. An anti-idiotype antibody that binds to the polypeptide of any one of claims 1 to 14.

21. A kit comprising the polypeptide according to any one of claims 1 to 14.

22. 1. A method of treating cancer in a subject in need thereof, comprising administering to a subject a compound selected from the group consisting of SEQ ID NOs: 146, 214, 104, 259, 134, 92, 302, 235, 237, 152, 238, 136, 197, 212, 296, 226, 261, 307, 115, 112, 278, 280, 282, 284, 286, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 309, 310, 311, 312, 313, 314, 315, 316, , 297, 96, 222, 95, 233, 217, 252, 194, 164, 168, 174, 190, 257, 303, 284, 85, 149, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, or 81-309, or a polypeptide described in claim 3, in combination with a therapeutic agent to a subject.

23. 23. The method of claim 22, wherein the cancer is brain cancer.

24. 10. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject a polypeptide described herein, such as SEQ ID NOs: 33-62 or 81-309, or a polypeptide according to claim 3, associated with an antiviral agent, an immune system modulating agent, or a nucleic acid molecule.

25. 25. The method of claim 24, wherein the cancer disease is glioblastoma.

26. 1. A method for detecting cancer cells expressing CD71 in a tumor tissue, comprising: a) obtaining a sample of tumor tissue from a subject; and b) Tumor tissue samples were cultured with the following SEQ ID NOs: 146, 214, 104, 259, 134, 92, 302, 235, 237, 152, 238, 136, 197, 212, 296, 226, 261, 307, 115, 112, 278, 297, 96, 222, 95, 233, 217, 252, 194, 164, 168, 174, 190, 257, 303, 284, 85, 149, 33, 34, 35, 3 6, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, or 81-309, and detecting binding between CD71 and the polypeptide. The above method.

27. 1. A method for isolating cells expressing CD71, comprising the steps of: a) obtaining a sample from a subject; b) SEQ ID NOs: 146, 214, 104, 259, 134, 92, 302, 235, 237, 152, 238, 136, 197, 212, 296, 226, 261, 307, 115, 112, 278, 297, 96, 222, 95, 233, 217, 252, 194, 164, 168, 174, 190, 257, 303, 2 contacting the sample with a polypeptide comprising one of the amino acid sequences of 84, 85, 149, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, or 81-309; and c) isolating cells that bind the polypeptide. The above method.

28. 1. A method for detecting cancer cells expressing CD71 in a tumor tissue, comprising: a) coupling a peptide comprising one of the amino acid sequences of SEQ ID NOs: 33-62 or 81-309 to a detectable label to form a conjugate; b) administering the conjugate to a subject; and c) Visualizing CD71-expressing cancer cells to which the conjugate is bound. The above method.

29. 20. The method of claim 19, further comprising administering to a subject a pharmaceutical composition according to claim 19, for treating a neurological condition and / or A method for treating brain tumors.

30. 30. The method of claim 29, wherein the brain tumor is selected from the group consisting of a non-malignant brain tumor, a benign brain tumor, and a malignant brain tumor.

31. 31. The method of claim 30, wherein the malignant brain tumor is selected from the group consisting of astrocytoma, medulloblastoma, glioma, ependymoma, germinoma (pinealoma), glioblastoma multiforme, oligodendroglioma, schwannoma, retinoblastoma, or cancer of the spinal cord, e.g., neurofibroma, meningioma, glioma, and sarcoma.

32. 31. The method of claim 30, wherein the brain tumor is a congenital tumor.

33. 30. The method of claim 29, wherein the neurological condition is selected from the group consisting of stroke, diabetes, epileptic seizures, hypertensive encephalopathy, acquired immune deficiency syndrome, traumatic brain injury, multiple sclerosis, Parkinson's disease (PD) and Alzheimer's disease.

34. 15. A method of delivering an agent of interest to a CD71 positive cell, comprising contacting the cell with the agent of interest coupled to a CD71-binding FN3 domain, such as a polypeptide according to any one of claims 1 to 14.

35. The method of claim 34, wherein the agent of interest is internalized into the cell via a CD71-mediated interaction.

36. The method of claim 34, wherein the FN3 domain does not compete with transferrin binding to CD71.

37. 37. The method of any one of claims 32 to 36, wherein the agent of interest is a chemotherapeutic agent, a drug, a growth inhibitor, a toxin, a radioisotope, an antitubulin agent, a polynucleotide, an siRNA molecule, an antisense molecule, an RNA molecule, a DNA molecule, a DNA minor groove binder, a DNA replication inhibitor, an alkylating agent, an antibiotic, an antifolate, an antimetabolite, a chemotherapy sensitizer, a topoisomerase inhibitor, or a vinca alkaloid.

38. The FN3 domain is represented by SEQ ID NOs: 146, 214, 104, 259, 134, 92, 302, 235, 237, 152, 238, 136, 197, 212, 296, 226, 261, 307, 115, 112, 278, 297, 96, 222, 95, 233, 217, 252, 194, 164, 168, 174, 190, 257, 303, 2 38. The method of any one of claims 34 to 37, comprising a sequence of 84, 85, 149, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, or 81 to 309 or a polypeptide according to claim 3.

39. The method of any one of claims 34 to 38, wherein the cell is a muscle cell.

40. 39. The method of any one of claims 34 to 38, wherein the cell is a brain cell or a cell inside the blood-brain barrier.

41. 1. A method for identifying an FN3 protein that binds to CD71 at a site that does not compete with or inhibit transferrin binding to CD71, the method comprising: In the presence of transferrin or a drug that binds to the CD71 transferrin binding site, contacting CD71 with a test FN3 protein; and Identifying a test FN3 protein that binds to CD71 in the presence of transferrin or an agent that binds to the CD71 transferrin binding site. The above method.

42. 41. The method of claim 40, further comprising isolating the test FN3 protein that binds to CD71 in the presence of transferrin or an agent that binds to the CD71 transferrin binding site.

43. 42. The method of claim 40 or 41, further comprising sequencing the test FN3 protein that binds to CD71 in the presence of transferrin or an agent that binds to the CD71 transferrin binding site.

44. 44. The method of any one of claims 41 to 43, further comprising producing or obtaining a nucleic acid sequence encoding a test FN3 protein that binds to CD71 in the presence of transferrin or an agent that binds to the CD71 transferrin binding site.

45. 45. The method of any one of claims 41-44, further comprising expressing a test FN3 protein that binds to CD71 in the presence of transferrin or an agent that binds to the CD71 transferrin binding site from a nucleic acid sequence encoding a test FN3 protein that binds to CD71 in the presence of transferrin or an agent that binds to the CD71 transferrin binding site.

46. 46. ​​The method of claim 45, wherein the test FN3 protein is expressed in a cell.

47. 46. ​​The method of claim 45, further comprising isolating and / or purifying the expressed test FN3 protein.

48. 48. An FN3 protein identified according to any one of claims 41 to 47.

49. A pharmaceutical composition comprising the FN3 protein of claim 48.

50. 49. An isolated polynucleotide encoding the polypeptide of claim 48.

51. A vector comprising the polynucleotide of claim 49.

52. A host cell comprising the vector of claim 51.

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