Fibronectin type III domains that bind CD71

Abstract

The present disclosure relates to polypeptides that can bind CD71, such as the fibronectin type III (FN3) domain, their conjugates, isolated nucleotides encoding molecules, vectors, host cells, and methods of making and using the same. In some embodiments, the FN3 domain is not naturally occurring. In some embodiments, the FN3 domain binds to human CD71 at a site on CD71 that does not compete for binding to CD71 with transferrin.

Description

Technical Field

[0001] The present embodiments relate to fibronectin type III domains (FN3) that specifically bind cluster of differentiation 71 (CD71) and methods of making and using these molecules. Background Art

[0002] CD71 (also known as transferrin receptor 1) is transmembrane and is essential for the transport of iron into cells. It is highly expressed on many tumor types and at the blood-brain barrier and has therefore become an important target for drug delivery. After binding to iron-loaded transferrin, CD71 is rapidly endocytosed and efficiently recycled back to the cell surface. Studies of CD71 antibody-drug conjugates have shown that targeting CD71 can improve the specificity and selectivity of drug delivery and expand the therapeutic index. In addition, studies using anti-CD71 monoclonal antibodies have shown that binding affinity may play an important role in achieving blood-brain barrier transcytosis. Antibodies with high affinity for CD71 are rapidly internalized and alter normal receptor trafficking, so that the receptor is not recycled but targeted to lysosomes for degradation. In contrast, antibodies with low affinity for CD71 allow receptor recycling and higher brain exposure.

[0003] While antibodies or antibody fragments are the most widely used type of therapeutic protein when high affinity and specificity for a target molecule is required, non-antibody proteins can also be designed to bind to these 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, ease of purification, and their ease of conjugation to drugs / toxins, effective penetration into tissues and ease of formatting into multi-specific binders.

[0004] One such alternative scaffold is the immunoglobulin (Ig) fold. This fold is found in the variable regions of antibodies as well as thousands of non-antibody proteins. It has been shown that one such Ig protein, the tenth fibronectin type III (FN3) repeat from human fibronectin, can tolerate many mutations in surface-exposed loops while retaining the overall Ig fold structure. Therefore, there is a need for FN3 domains that can specifically bind CD71, as well as methods of using such molecules for cancer therapy. Summary of the invention

[0005] In some embodiments, an FN3 domain (e.g., a polypeptide) that specifically binds to a CD71 protein is provided. 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 present.

[0006] In some embodiments, an FN3 domain 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, or 62 is provided. In some embodiments, a FN3 domain comprising an 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 is provided. In some embodiments, a FN3 domain comprising an amino acid sequence of SEQ ID NO: 81-309 is provided: 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 for binding to CD71. In some embodiments, an FN3 domain is provided that comprises an 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

[0007] In some embodiments, the FN3 domain specifically binds to CD71. In some embodiments, a polypeptide comprising more than one FN3 domain connected by a linker (e.g., a flexible linker) is provided. In some embodiments, the polypeptide comprises 2, 3 or 4 FN3 domains, which are connected to each other by one or more linkers between the domains.

[0008] In some embodiments, isolated polynucleotides encoding the FN3 domains described herein are provided.

[0009] In some embodiments, a vector comprising a polynucleotide described herein is provided.

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

[0011] In some embodiments, a method of producing the 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 comprising an FN3 domain that binds to CD71 and a pharmaceutically acceptable carrier is provided.

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

[0014] In some embodiments, kits comprising one or more FN3 domains are provided.

[0015] In some embodiments, a method for detecting cancer cells expressing CD71 in a tumor tissue is provided. In some embodiments, the method comprises obtaining a sample of the tumor tissue from a subject and binding the sample of the tumor tissue to an amino acid sequence comprising one 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.

[0016] The FN3 domain of the CD71 protein is contacted with the FN3 domain of the CD71 protein and the binding between the CD71 protein and the FN3 domain is detected to detect whether the CD71 protein is expressed in the tumor tissue.

[0017] In some embodiments, a method of isolating cells expressing CD71 is provided. In some embodiments, the method comprises obtaining a sample from a subject; contacting the sample with the FN3 domain that binds to a CD71 protein comprising an amino acid sequence of one 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 isolating the cell bound to the FN3 domain.

[0018] In some embodiments, a method for detecting cancer cells expressing CD71 in tumor tissue is provided. In some embodiments, the method comprises conjugating a CD71 protein-binding FN3 domain comprising an amino acid sequence of one 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 to a detectable label to form a conjugate; administering the conjugate to a subject; and visualizing cancer cells expressing CD71 bound to the conjugate.

[0019] 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 includes a polypeptide such as 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, 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,

[0020] 57, 58, 59, 60, 61, 62 or 81-309 sequence or a polypeptide as provided herein.

[0021] In some embodiments, a method for treating a neurological disorder and / or a brain tumor is provided. In some embodiments, the method comprises administering to the subject a polypeptide provided herein or a pharmaceutical composition. In some embodiments, the brain tumor is selected from the group consisting of non-malignant, benign and malignant brain tumors.

[0022] In some embodiments, a method of delivering a target agent to a CD71-positive cell is provided. In some embodiments, the method comprises contacting a cell with a target agent coupled to an FN3 domain that binds to CD71 (e.g., a polypeptide provided herein). In some embodiments, the target agent is a chemotherapeutic agent, a drug, a growth inhibitor, a toxin, a radioisotope, an anti-tubulin 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 chemosensitizer, a topoisomerase inhibitor, or a vinca alkaloid.

[0023] In some embodiments, the polypeptide is a FN3 protein that binds to CD71 at a site that does not compete for or inhibit the binding of transferrin to CD71.

[0024] In some embodiments, methods are provided for identifying an FN3 protein that binds to CD71 at a site that does not compete for or inhibit the binding of transferrin to CD71. DETAILED DESCRIPTION

[0025] 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 combinations of two or more cells, and the like.

[0026] "Fibronectin type III (FN3) domain" (FN3 domain) refers to a domain that frequently occurs in proteins including fibronectin, tenascin, intracellular cytoskeletal proteins, cytokine receptors, and prokaryotic enzymes (Bork and Doolittle, Proc Nat Acad Sci USA 89:8990-8994, 1992; Meinke et al., J Bacteriol 175:1910-1918, 1993; Watanabe et al., J Biol Chem 265:15659-15665, 1990). Exemplary FN3 domains are the 15 different FN3 domains present in human tenascin C, the 15 different FN3 domains present in human fibronectin (FN), and non-natural synthetic FN3 domains, as described, for example, in U.S. Pat. No. 8,278,419. Individual FN3 domains are indicated by the domain number and protein name, for example, the third FN3 domain of tenascin (TN3), or the tenth FN3 domain of fibronectin (FN10).

[0027] The term "capture agent" refers to a substance that binds to a specific type of cell and is able to separate that cell from other cells. Exemplary capture agents are magnetic beads, ferrofluids, encapsulation agents, molecules that bind to a specific cell type, and the like.

[0028] "Sample" refers to a collection of similar fluids, cells or tissues isolated from a subject, as well as fluids, cells or tissues present in a subject. Exemplary samples are tissue biopsies, fine needle aspirations, surgically removed tissues, organ cultures, cell cultures and biological fluids such as blood, serum and serum, plasma, lymph, urine, saliva, cystic fluid, tears, feces, sputum, mucosal secretions of secretory tissues and organs, vaginal secretions, ascites, pleural, pericardial, peritoneal, covering membranes and other body cavity fluids, fluids collected by bronchial lavage, synovial fluid, liquid solutions in contact with a subject or biological source, for example, cell and organ culture media, including cell or organ conditioned medium and lavage fluids, etc.

[0029] "Substitution" or "substitutional" or "mutation" or "mutant" refers to changing, deleting or inserting one or more amino acids or nucleotides in a polypeptide or polynucleotide sequence to produce a variant of the sequence.

[0030] "Variant" refers to a polypeptide or polynucleotide that differs from a reference polypeptide or reference polynucleotide by one or more modifications, such as substitutions, insertions, or deletions.

[0031] "Specifically binds" or "specific binding" refers to the FN3 domain being present at about 1x10 -6 M or smaller, such as about 1x10 -7 M or smaller, about 1x10 -8 M or smaller, about 1x10 -9 M or smaller, about 1x10 -10 M or smaller, about 1x10 -11 M or smaller, about 1x10 -12 M or smaller, or about 1x10 -13 M or less dissociation constant (K D ) to its target, such as CD71. Alternatively, "specific binding" refers to the ability of an FN3 domain to bind to 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 with other related antigens, such as the same predetermined antigen from other species (homologs) such as Macaca Fascicularis (crab-eating monkey, cyno) or Pan troglodytes (chimpanzee).

[0032] A "library" refers to a collection of variants. A library can be composed of polypeptide or polynucleotide variants.

[0033] "Stability" refers to the maintenance of a molecule's folded state under physiological conditions such that it retains at least one of its normal functional activities, such as the ability to bind to a predetermined antigen such as CD71.

[0034] "CD71" refers to a human CD71 protein having an amino acid sequence of SEQ ID NO: 32 or 80. In some embodiments, SEQ ID NO: 32 is a full-length human CD71 protein. In some embodiments, SEQ ID NO: 80 is the extracellular domain of human CD71.

[0035] "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.

[0036] "Cancer cell" or "tumor cell" refers to cancer cells, precancerous cells or transformed cells in vivo, in vitro and in tissue culture, which have spontaneous or induced phenotypic changes, not necessarily involving the uptake of new genetic material. Although transformation can be caused by infection with transforming viruses and the incorporation of new genomic nucleic acids or the uptake of exogenous nucleic acids, it can also occur spontaneously or after exposure to carcinogens, thereby mutating endogenous genes. Transformation / cancer is illustrated by, for example, morphological changes, cell immortalization, abnormal growth control, lesion formation, proliferation, malignancy, tumor-specific marker levels, invasiveness, tumor growth or inhibition in suitable animal hosts such as nude mice, in vitro, in vivo and in vitro (Freshney, Culture of Animal Cells: A Manual of Basic Technique (3rd ed. 1994)).

[0037] "Vector" refers to a polynucleotide that can replicate within a biological system or can be moved between such systems. Vector polynucleotides typically contain elements, such as an origin of replication, a polyadenylation signal, or a selectable marker, which functions to promote the replication or maintenance of these polynucleotides in a biological system. Examples of such biological systems can include cells, viruses, animals, plants, and reconstructed biological systems that utilize biological components that can replicate vectors. The polynucleotides comprising the vector can be DNA or RNA molecules or hybrids thereof.

[0038] "Expression vector" refers to a vector that can be used in a biological system or a reconstituted biological system to direct the translation of a polypeptide encoded by a polynucleotide sequence present in the expression vector.

[0039] "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.

[0040] "Polypeptide" or "protein" refers to a molecule comprising at least two amino acid residues linked by a peptide bond to form a polypeptide. Small polypeptides of less than about 50 amino acids may be referred to as "peptides."

[0041] "Valency" refers to the presence of a specific number of binding sites in a molecule that are specific for an antigen. Thus, the terms "monovalent," "divalent," "tetravalent," and "hexavalent" refer to the presence of one, two, four, and six binding sites in a molecule that are specific for an antigen in the molecule, respectively.

[0042] "Subject" includes any human or non-human animal. "Non-human animals" include all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc. Unless otherwise indicated, the terms "patient" or "subject" can be used interchangeably.

[0043] "Isolated" refers to a homogenous population of molecules (such as synthetic polynucleotides or polypeptides, such as FN3 domains) that have been substantially separated and / or purified from other components of a system (such as a recombinant cell) in which the molecule is produced, as well as proteins that have been subjected to at least one purification or isolation step. "Isolated FN3 domain" refers to a FN3 domain that is substantially free of other cellular material and / or chemicals, and includes FN3 domains isolated to a higher degree of purity, such as 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% purity.

[0044] Material composition

[0045] In some embodiments, proteins are provided, including polypeptides, 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, proteins include polypeptides, including 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. 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 of at least 62%, 63%, 64%, 65%, 66%, 67%, 68%, 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequence. 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 the 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, the protein is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the 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, the protein is at least 95%, 96%, 97%, 98% or 99% identical to the 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.

[0046] The polypeptides provided herein can be part of a larger polypeptide and can be referred to as a domain. Homology or identity between two domains in different polypeptides is based on similar domains rather than entire polypeptides. For example, if a polypeptide includes a polypeptide comprising an FN3 domain comprising SEQ ID NO: 81 and the domain is conjugated to a scFV antibody, another protein having a domain similar but not identical to SEQ ID NO: 81 can be at least 90% identical, even if the scFV has no homology. Thus, % identity can be based on a domain or the full length of a polypeptide. Methods for determining % identity are provided herein or are known to those of skill in the art.

[0047] In some embodiments, a fibronectin type III (FN3) domain that binds or specifically binds to a human CD71 protein (SEQ ID NO: 32 or 80) is provided. As provided herein, the FN3 domain can bind to the CD71 protein. It is also provided that, even if not explicitly stated, these domains can also specifically bind to the CD71 protein. Thus, for example, an FN3 domain that binds to CD71 will also include an FN3 domain protein that specifically binds to CD71. These molecules can be used, for example, in therapeutic and diagnostic applications and imaging. In some embodiments, polynucleotides encoding the FN3 domain disclosed herein or complementary nucleic acids thereof, vectors, host cells, and methods of making and using them are provided.

[0048] In some embodiments, isolated FN3 domains that bind or specifically bind to CD71 are provided.

[0049] 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,

[0050] The joint can be a G / S joint or the like.

[0051] In some embodiments, the FN3 domain can be present in an amount less than about 1×10 -7 M, for example, less than about 1x10 -8 M, less than about 1x10 -9 M, less than about 1x10 -10 M, less than about 1x10 -11 M, less than about 1x10 -12 M, or less than about 1x10 -13 The dissociation constant (K D) binding to CD71, as determined by surface plasmon resonance or Kinexa methods by one skilled in the art. The measured affinity of a particular FN3 domain-antigen interaction may vary if measured under different conditions (e.g., osmolarity, pH). Therefore, affinity and other antigen binding parameters (e.g., K D , K on , K off ) are measured using standardized solutions of protein scaffold and antigen and a standardized buffer (such as the buffer described herein).

[0052] In some embodiments, the FN3 domain can bind CD71 in a standard ELISA assay with a signal at least 5-fold higher than that obtained with a negative control.

[0053] In some embodiments, the FN3 domain that binds or specifically binds to CD71 comprises an initial methionine (Met) attached to the N-terminus of the molecule. In some embodiments, the FN3 domain that binds or specifically binds to CD71 comprises a cysteine ​​(Cys) attached to the C-terminus of the FN3 domain. The addition of an N-terminal Met and / or a C-terminal Cys can facilitate expression and / or conjugation of half-life extending molecules.

[0054] The FN3 domains may also include cysteine ​​substitutions, such as those described in U.S. Pat. No. 10,196,446, the entire contents of which are incorporated herein by reference. Briefly, in some embodiments, the polypeptides provided herein may include 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 of SEQ ID NO: 6 or SEQ ID NO: 1 of U.S. Pat. No. 10,196,446, and equivalent positions in related FN3 domains. In some embodiments, the substitution is at residue

[0055] 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.

[0056] Cysteine ​​substitution at a position in a domain or protein includes replacing an existing amino acid residue with a cysteine ​​residue. Other examples of cysteine ​​modifications can be found in, for example, U.S. Patent Application Publication No. 20170362301, the entire contents of which are incorporated herein by reference. Sequence alignment can be performed using, for example, BlastP from the NCBI website using default parameters.

[0057] In some embodiments, the FN3 domain that binds CD71 is internalized into a cell. In some embodiments, internalization of the FN3 domain can facilitate the delivery of a detectable label or therapeutic agent into a cell. In some embodiments, internalization of the FN3 domain can facilitate the delivery of a cytotoxic agent into a cell. A cytotoxic agent can be used as a therapeutic agent. In some embodiments, internalization of the FN3 domain can facilitate the delivery of any detectable label, therapeutic agent, and / or cytotoxic agent disclosed herein into a cell. In some embodiments, the cell is a tumor cell. In some embodiments, the cell is a hepatocyte.

[0058] In some embodiments, the CD71 binding FN3 domain is based on the Tencon sequence of SEQ ID NO: 1 or the Tencon 27 sequence of SEQ ID NO: 4, optionally with a substitution at residue position 11, 14, 17, 37, 46, 73, or 86 (corresponding to the residue numbering of SEQ ID NO: 4).

[0059] In some embodiments, the isolated FN3 domain that binds 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.

[0060] In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, or 61.

[0061] In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:33.

[0062] In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:34.

[0063] In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:35.

[0064] In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:36.

[0065] In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:37.

[0066] In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:38.

[0067] In some embodiments, the isolated FN3 domain that binds CD71 comprises SEQ ID NO:

[0068] 39 amino acid sequence.

[0069] In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:40.

[0070] In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:41.

[0071] In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:42.

[0072] In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:43.

[0073] In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:44.

[0074] In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:45.

[0075] In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:46.

[0076] In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:47.

[0077] In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:48.

[0078] In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:49.

[0079] In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:50.

[0080] In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:51.

[0081] In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:52.

[0082] In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:53.

[0083] In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:54.

[0084] In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:55.

[0085] In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:56.

[0086] In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:57.

[0087] In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:58.

[0088] In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:59.

[0089] In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:60.

[0090] In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:61.

[0091] In some embodiments, the isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:62.

[0092] In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 81. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 82. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 83. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 84. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 85. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 86. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 87. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 88. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 89. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 90. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 91. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 92. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 93. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 94. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 95. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 96. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 97. In some embodiments, the isolated FN3 domain that binds to 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 to CD71 comprises the amino acid sequence of SEQ ID NO: 102. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 103. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 104. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 105. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 106. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 107. In some embodiments, the isolated FN3 domain that binds to 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 to CD71 comprises the amino acid sequence of SEQ ID NO: 111. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 112. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 113. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 114. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 115. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 116. In some embodiments, the isolated FN3 domain that binds to 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 to CD71 comprises the amino acid sequence of SEQ ID NO: 123. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 124. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 125. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 126. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 127. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 128. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 129. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 130. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 131. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 132. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 133. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 134. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 135. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 136. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 137. In some embodiments, the isolated FN3 domain that binds to 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 to CD71 comprises the amino acid sequence of SEQ ID NO: 144. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 145. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 146. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 147. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 148. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 149. In some embodiments, the isolated FN3 domain that binds to 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: 152. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 153. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 154. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 155. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 156. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 157. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 158. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 159. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 160. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 161. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 162. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 163. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 164.In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 165. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 166. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 167. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 168. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 169. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 170. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 171. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 172. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 173. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 174. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 175. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 176. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 177. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 178. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 179. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 180. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 181. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 182. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 183. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 184. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 185.In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 186. In some embodiments, the isolated FN3 domain that binds to 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 to CD71 comprises the amino acid sequence of SEQ ID NO: 189. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 190. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 191. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 192. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 193. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 194. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 195. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 196. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 197. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 198. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 199. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 200. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 201. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 202. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 203. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 204. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 205. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 206.In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 207. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 208. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 209. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 210. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 211. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 212. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 213. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 214. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 215. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 216. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 217. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 218. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 219. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 220. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 221. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 222. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 223. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 224. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 225. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 226. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 227.In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 228. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 229. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 230. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 231. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 232. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 233. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 234. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 235. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 236. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 237. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 238. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 239. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 240. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 241. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 242. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 243. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 244. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 245. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 246. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 247. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 248.In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 249. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 250. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 251. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 252. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 253. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 254. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 255. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 256. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 257. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 258. In some embodiments, the isolated FN3 domain that binds to 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 to CD71 comprises the amino acid sequence of SEQ ID NO: 261. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 262. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 263. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 264. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 265. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 266. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 267. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 268. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 269.In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 270. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 271. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 272. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 273. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 274. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 275. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 276. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 277. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 278. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 279. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 280. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 281. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 282. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 283. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 284. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 285. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 286. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 287. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 288. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 289. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 290.In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 291. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 292. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 293. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 294. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 295. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 296. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 297. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 298. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 299. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 300. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 301. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 302. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 303. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 304. In some embodiments, the isolated FN3 domain that binds to CD71 comprises the amino acid sequence of SEQ ID NO: 305. In some embodiments, the isolated FN3 domain that binds to 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.

[0093] In some embodiments, the FN3 domain binds to human CD71 at a site on CD71 that does not compete with transferrin for 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.

[0094] In some embodiments, the isolated FN3 domain that binds CD71 includes an initial methionine (Met) attached to the N-terminus of the molecule.

[0095] 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 NO: 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 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. One of the amino acid sequences of 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 94%, 95%, 96%, 97%, 98% or 99% identical amino acid sequences.

[0096] Percent identity can be determined by aligning the two sequences using BlastP available through the NCBI website using default parameters.

[0097] Conjugates of the CD71-binding FN3 domain of the present disclosure

[0098] In some embodiments, an isolated FN3 domain that binds CD71 conjugated to a heterologous molecule is provided.

[0099] In some embodiments, the FN3 domain is conjugated to an oligonucleotide. For example, the oligonucleotide can be used to inhibit the expression of a gene or mRNA transcript. The oligonucleotide can be siRNA, miRNA, antisense oligonucleotide, etc.

[0100] In some embodiments, the peptides are conjugated to lipid nanoparticles, which can be used, for example, for cell-specific targeting.

[0101] 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 moiety for E3 ubiquitin ligases) to deliver the protein to the E3 ligase. These can be connected by a linker (such as a glycine-serine linker, etc.).

[0102] The FN3 domain that binds to CD71 can also be conjugated or linked to another FN3 domain that binds to a different target other than CD71. This would render the peptide multispecific (e.g., bispecific, trispecific, etc.), thereby allowing it to bind 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).

[0103] 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. The G / S repeat unit is four glycines followed by serine, for example GGGGS.

[0104] In some embodiments, the heterologous molecule is a detectable label or a therapeutic agent, such as but not limited to a cytotoxic agent.

[0105] In some embodiments, a CD71 binding FN3 domain conjugated to a detectable label is provided. Non-limiting examples of detectable labels are provided herein.

[0106] In some embodiments, a CD71-binding FN3 domain conjugated to a therapeutic agent is provided. Non-limiting examples of therapeutic agents are provided herein, such as, but not limited to, cytotoxic agents.

[0107] The CD71-binding FN3 domain conjugated to a detectable label can be used to assess CD71 expression on in vivo or in vitro samples, such as tumor tissue.

[0108] Detectable labels include compositions that, when conjugated to a CD71 -binding FN3 domain, render CD71 detectable by spectroscopic, photochemical, biochemical, immunochemical, or other chemical means.

[0109] 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, scintillators, avidin, streptavidin, protein A, protein G, antibodies or fragments thereof, polyhistidine, Ni 2+ , Flag tag, myc tag, heavy metals, enzymes, alkaline phosphatase, peroxidase, luciferase, electron donors / acceptors, acridinium esters, and colorimetric substrates.

[0110] The detectable label may emit a signal spontaneously, such as when the detectable label is a radioisotope. In some embodiments, the detectable label emits a signal due to stimulation by an external stimulus, such as a magnetic field or an electric field or an electromagnetic field.

[0111] Exemplary radioisotopes may be gamma-emitting, helical-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.

[0112] Exemplary metal atoms are metals with an atomic number greater than 20, such as calcium atoms, scandium atoms, titanium atoms, vanadium atoms, chromium atoms, manganese atoms, iron atoms, cobalt atoms, nickel atoms, copper atoms, zinc atoms, gallium atoms, germanium atoms, arsenic atoms, selenium atoms, bromine atoms, krypton atoms, rubidium atoms, strontium atoms, yttrium atoms, zirconium atoms, niobium atoms, molybdenum atoms, technetium atoms, ruthenium atoms, rhodium atoms, palladium atoms, silver atoms, cadmium atoms, indium atoms, tin atoms, antimony atoms, tellurium atoms, iodine atoms, xenon atoms, cesium atoms, barium atoms, lanthanum atoms , hafnium, tantalum, tungsten, rhenium, osmium, iridium, platinum, gold, mercury, thallium, lead, bismuth, francium, radium, actinium, cerium, praseodymium, neodymium, promethium, samarium, europium, gadolinium, terbium, dysprosium, holmium, erbium, thulium, ytterbium, lutetium, thorium, protactinium, uranium, neptunium, plutonium, americium, curium, berkelium, californium, einsteinium, fermium, mendelevium, nobelium, or lawrencium.

[0113] In some embodiments, the metal atom may be an alkaline earth metal having an atomic number greater than twenty.

[0114] In some embodiments, the metal atom may be a lanthanide element.

[0115] In some embodiments, the metal atom may be an actinide.

[0116] In some embodiments, the metal atom may be a transition metal.

[0117] In some embodiments, the metal atom may be metal-poor.

[0118] In some embodiments, the metal atoms may be gold atoms, bismuth atoms, tantalum atoms, and gadolinium atoms.

[0119] In some embodiments, the metal atom may be a metal having an atomic number of 53 (ie, iodine) to 83 (ie, bismuth).

[0120] In some embodiments, the metal atoms may be atoms suitable for magnetic resonance imaging.

[0121] The metal atom may be a metal ion in the +1, +2 or +3 oxidation state, such as Ba 2+ 、Bi 3+ , Cs + , Ca 2+ Cr 2+ Cr 3+ Cr 6+ 、Co 2+ 、Co 3+ , Cu + , Cu 2+ , Cu 3+ , Ga 3+ , Gd 3+ 、Au + 、Au 3+ , Fe 2+ , Fe 3+ 、F 3+ , Pb 2+ , Mn 2+ , Mn 3+ , Mn 4+ , Mn 7+ , Hg 2+ 、Ni 2+ 、Ni 3+ 、Ag + , Sr 2+ Sn 2+ Sn 4+ and Zn 2+ The metal atom may include a metal oxide, such as iron oxide, manganese oxide, or gadolinium oxide.

[0122] Suitable dyes include any commercially available dyes such as, for example, 5(6)-carboxyfluorescein, IRDye 680RD maleimide or IRDye 800CW, polypyridine ruthenium dyes, and the like.

[0123] Suitable fluorophores are fluorescein isothiocyanate (FITC), fluorescein thiosemicarbazide, rhodamine, Texas Red, CyDye (e.g., Cy3, Cy5, Cy5.5), Alexa Fluors (e.g., Alexa488, Alexa555, Alexa594; Alexa647), near-infrared (NIR) (700-900 nm) fluorescent dyes, as well as carbocyanine and aminostyryl dyes.

[0124] The FN3 domain that specifically binds CD71 conjugated to a detectable label can be used as an imaging agent, for example, to assess tumor distribution, diagnose the presence of tumor cells and / or tumor recurrence.

[0125] In some embodiments, the FN3 domain that specifically binds CD71 is conjugated to a therapeutic agent, such as but not limited to a cytotoxic agent.

[0126] In some embodiments, the therapeutic agent is a chemotherapeutic agent, a drug, a growth inhibitory agent, a toxin (eg, an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or a fragment thereof), or a radioactive isotope (ie, a radioconjugate).

[0127] The CD71-binding FN3 domains conjugated to therapeutic agents disclosed herein can be used to target the delivery of therapeutic agents to cells expressing CD71 (e.g., tumor cells) and for intracellular accumulation therein. While not being bound by any particular theory, this type of delivery may be beneficial in situations where systemic administration of these unconjugated agents may result in unacceptable levels of toxicity to normal cells.

[0128] In some embodiments, therapeutic agents may elicit their cytotoxic and / or cytostatic effects through mechanisms such as, but not limited to, tubulin binding, DNA binding, topoisomerase inhibition, DNA cross-linking, chelation, spliceosome inhibition, NAMPT inhibition, and HDAC inhibition.

[0129] 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, for example, a TLR7, 8, 9, RIG-I (dsRNA), and a STING (CpG) agonist. In some embodiments, the agent is daunomycin, doxorubicin, methotrexate, vindesine, a bacterial toxin such as diphtheria toxin, ricin, geldanamycin, maytansine, or calicheamicin.

[0130] 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), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, or tricothecenes.

[0131] In some embodiments, the therapeutic agent is a radionuclide such as 212 Bi, 131 I. 131 In, 90 Y or 186 Re.

[0132] In some embodiments, the therapeutic agent is a dolastatin or dolostatin peptide analog and derivative, auristatin or monomethyl auristatin phenylalanine. Exemplary molecules are disclosed in U.S. Patent Nos. 5,635,483 and 5,780,588. Dolastatin and auristatin have been shown to 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 anticancer and antifungal activity. The dolastatin or auristatin drug moiety can be linked 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.

[0133] In some embodiments, the therapeutic agent can be, for example, auristatins, camptothecins, duocarmycins, etoposide, maytansine and maytansines, taxanes, benzodiazepines, Benzodiazepine Drugs (such as pyrrolo[1,41-benzodiazepine PBD, indolebenzodiazepine Oxazolidine benzodiazepines class) or vinca alkaloids.

[0134] The FN3 domain that specifically binds CD71 can be conjugated to a detectable label using known methods.

[0135] In some embodiments, the detectable label is complexed to a chelator.

[0136] In some embodiments, a detectable label is conjugated to the CD71-binding FN3 domain via a linker.

[0137] Detectable labels, therapeutic compounds, or cytotoxic compounds can be directly or indirectly linked 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; dodecaborates), chelating moieties of 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 (TET A), N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), derivatives of iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipate HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl)hexanediamine), bis-diazo derivatives (such as bis(p-diazobenzoyl)ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate) and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene) and other chelating moieties. Suitable peptide linkers are well known.

[0138] In some embodiments, the CD71 binding FN3 domain is removed from the blood by renal clearance.

[0139] Isolation of CD71-binding FN3 domains from a Tencon sequence-based library

[0140] 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 seven beta strands as characterized for the FN3 domain, the beta strands are referred to as A, B, C, D, E, F and G, and the loops are referred to as AB, BC, CD, DE, EF and FG loops (Bork and Doolittle, Proc Natl Acad Sci USA 89: 8990-8992, 1992; U.S. Patent No. 6,673,901). These loops or selected residues within each loop can be randomized to construct a fibronectin type III (FN3) domain library that can be used to select new molecules that bind to CD71. Table 1 shows the position and sequence of each loop and beta strand in Tencon (SEQ ID NO: 1).

[0141] Therefore, the library designed based on the Tencon sequence may have a randomized FG loop, or a randomized BC and FG loop, such as the library TCL1 or TCL2 described below. The length of the Tencon BC loop is 7 amino acids, so 1, 2, 3, 4, 5, 6 or 7 amino acids can be randomized in the library designed based on the Tencon sequence and the BC loop is diversified. The length of the Tencon FG loop is 7 amino acids, so 1, 2, 3, 4, 5, 6 or 7 amino acids can be randomized in the library designed based on the Tencon sequence and the FG loop is diversified. The further diversity of the ring in the Tencon library can be achieved by the insertion and / or deletion of the residue at the ring. For example, the FG and / or BC loop can be extended by 1-22 amino acids, or reduced by 1-3 amino acids. The length of the FG loop in Tencon is 7 amino acids, and the corresponding loop range in the antibody heavy chain is 4-28 residues. In order to provide maximum diversity, the FG loop can be diversified in sequence and length to correspond to the antibody CDR3 length range of 4-28 residues. For example, the length of the FG loop can be further diversified by extending the loop by an additional 1, 2, 3, 4 or 5 amino acids.

[0142] Libraries designed based on the Tencon sequence can also have random alternative surfaces formed on one side of the FN3 domain and include two or more β-strands and at least one loop. One such alternative surface is formed by amino acids in the C and F β-strands and CD and FG loops (C-CD-F-FG surface). A library design based on Tencon alternative C-CD-F-FG surface is described in U.S. Patent Publication No. 2013 / 0226834. Libraries designed based on the Tencon sequence also include libraries designed based on Tencon variants, such as Tencon variants with substitutions at residue positions 11, 14, 17, 37, 46, 73 or 86 (residue numbers corresponding to SEQ ID NO: 1), and which variants are shown to improve thermal stability. Exemplary Tencon variants are described in US Patent Publication No. 2011 / 0274623, and include Tencon27 (SEQ ID NO:4) having substitutions E11R, L17A, N46V, and E86I compared to the Tencon of SEQ ID NO:1.

[0143] Table 1. Tencon topology

[0144]

[0145]

[0146] Libraries based on Tencon and other FN3 sequences can be randomized at selected residue positions using random or defined sets of amino acids. For example, variants with random substitutions in the library can be generated using NNK codons encoding all 20 naturally occurring amino acids. In other diversification schemes, DVK codons can be used to encode 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 while reducing the frequency of stop codons. This can be achieved, for example, using The technology (http: / / www.sloning.com) synthesizes a library of FN3 domains with biased amino acid distribution at the positions to be diversified. The technology uses a pre-made double-stranded triplet library, which serves as a universal building block, sufficient for thousands of gene synthesis processes. The triplet library represents all possible sequence combinations required to build any desired DNA molecule. The codon names are based on the well-known IUB code.

[0147] FN3 domains that specifically bind to CD71 can be isolated by generating a FN3 library (e.g., a Tencon library) using cis display to link a DNA fragment encoding a scaffold protein to a DNA fragment encoding RepA to generate a pool of protein-DNA complexes formed after in vitro translation, wherein each protein is stably associated with the DNA encoding it (U.S. Pat. No. 7,842,476; Odegrip et al., Proc Natl Acad Sci USA 101, 2806-2810, 2004), 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 immunoassays, and competitive and non-competitive assays (see, e.g., Ausubel et al., eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York). The identified FN3 domains that specifically bind to CD71 were further characterized for their binding to CD71, modulation of CD71 activity, internalization, stability, and other desired features.

[0148] Any FN3 domain can be used as a template to generate an FN3 domain that specifically binds to CD71 to generate a library and screen the library for molecules that specifically bind to CD71 using the methods provided therein. Exemplary FN3 domains that can be used are the 3rd FN3 domain of tenascin C (TN3), Fibcon, and the 10th FN3 domain of fibronectin (FN10). Therefore, PCT applications WO 2010 / 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 synthesize a double-stranded cDNA cassette of the library 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. Pat. No. 5,643,768) can be used. The library of FN3 domain variants can be expressed as a fusion protein displayed on the surface of any suitable phage, for example. Methods for displaying fusion polypeptides on the surface of phage are well known (U.S. Pat. App. Pub. No. 2011 / 0118144; International Patent Publication No. WO2009 / 085462; U.S. Pat. No. 6,969,108; U.S. Pat. No. 6,172,197; U.S. Pat. No. 5,223,409; U.S. Pat. No. 6,582,915; U.S. Pat. No. 6,472,147).

[0149] In some embodiments, the FN3 domain that binds CD71 is based on the Tencon sequence of SEQ ID NO: 1 or the Tencon27 sequence of SEQ ID NO: 4, SEQ ID NO: 1 or SEQ ID NO: 4, optionally with substitutions at residue positions 11, 14, 17, 37, 46, 73 and / or 86.

[0150] In some embodiments, the FN3 protein or polypeptide is a protein or polypeptide that binds to human CD71 at a site on CD71 that does not compete with transferrin for binding to CD71. As used herein, a site on CD71 that does not compete with transferrin for binding to CD71 refers to an epitope or portion of CD71 where the binding of the FN3 protein does not compete or inhibit the binding of transferrin to CD71. Competition or lack of competition can be complete or partial. In some embodiments, the binding also does not inhibit transferrin from being internalized into cells through its interaction with CD71.

[0151] In some embodiments, a method for identifying an FN3 protein that binds to CD71 at a site that does not compete or inhibit transferrin binding to CD71 is provided. In some embodiments, the method comprises contacting CD71 with a test FN3 protein in the presence of transferrin or an agent that binds to a transferrin binding site of CD71; and identifying a test FN3 protein that binds to CD71 in the presence of transferrin or an agent that binds to a transferrin binding site of CD71. 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 a transferrin binding site of CD71. 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 a transferrin binding site of CD71. In some embodiments, the method comprises preparing 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 a transferrin binding site of CD71. 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 a transferrin binding site of CD71 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 a transferrin binding site of CD71. 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.

[0152] In some embodiments, a FN3 protein is provided, wherein the FN3 protein is identified according to any of the methods provided herein.

[0153] The FN3 domain that specifically binds to CD71 can be modified to improve its properties, such as improving thermal stability and the 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 thermal stable sequences, design of stable disulfide bonds, mutations that increase α-helical propensity, engineering of salt bridges, changes in protein surface charge, directed evolution, and composition of 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 chains during manufacturing. Residues that can be replaced to improve the thermal stability of Tencon (SEQ ID NO: 1) are 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 introduced into the FN3 domain containing molecules disclosed herein.

[0154] The measurement of protein stability and protein instability can be viewed as the same or different aspects of protein integrity. Proteins can be sensitive or "unstable" to denaturation caused by heat, ultraviolet or ionizing radiation, changes in ambient osmolarity and pH if in liquid solution, mechanical shear forces applied by small pore filtration, ultraviolet radiation, ionizing radiation (such as gamma radiation), chemical or thermal dehydration, or any other action or force that may cause protein structural disruption. The stability of a molecule can be determined using standard methods. For example, the stability of a molecule can be determined by measuring thermal melting ("T m ”) temperature (i.e., the temperature in degrees Celsius (°C) at which half of the molecules unfold). Typically, T m The higher it is, the more stable the molecule. In addition to heat, the chemical environment can also change the protein's ability to maintain a specific three-dimensional structure.

[0155] In some embodiments, a CD71-binding FN3 domain can exhibit increased stability compared to the same domain prior to engineering, as determined by T m An increase of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% or more of a measurement.

[0156] Chemical denaturation can also be measured by a variety of methods. Chemical denaturants include guanidine hydrochloride, guanidine thiocyanate, urea, acetone, organic solvents (DMF, benzene, acetonitrile), salts (ammonium sulfate, lithium bromide, lithium chloride, sodium bromide, calcium chloride, sodium chloride); reducing agents (e.g., dithiothreitol, β-mercaptoethanol, dinitrobenzene, and hydrides such as sodium borohydride), nonionic and ionic detergents, acids (e.g., hydrochloric acid (HCl), acetic acid (CH3COOH), haloacetic acids), hydrophobic molecules (e.g., phospholipids), and targeted denaturants. Quantification of the degree of denaturation can rely on the loss of functional properties (such as the ability to bind to a target molecule), or on physicochemical properties such as aggregation tendency, exposure of residues that were previously inaccessible to solvents, or destruction or formation of disulfide bonds.

[0157] FN3 domains that bind to CD71 can be produced in the form of monomers, dimers, or multimers, for example, as a means of increasing the valency of target molecule binding and thus increasing affinity, or as a means of producing bispecific or multispecific scaffolds that bind two or more different target molecules simultaneously. Dimers and multimers can be produced by linking monospecific, bispecific, or multispecific protein scaffolds, for example, by including amino acid linkers, such as linkers comprising 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)5AAA (SEQ ID NO: 70). Dimers and multimers can be linked to each other in the N- to C-direction. The use of natural and artificial peptide linkers to link polypeptides into new 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; and U.S. Pat. No. 5,856,456).

[0158] Half-life extension

[0159] The FN3 domain that specifically binds to CD71 can introduce other subunits, for example, through covalent interactions. In some embodiments, the FN3 domain that specifically binds to CD71 also includes a half-life extension portion. Exemplary half-life extension portions 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 Fc regions of IgG1, IgG2, IgG3, IgG4, IgM, IgA, and IgE. In some embodiments, the FN3 domain that specifically binds to CD71 can introduce 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 half-life extension portion 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.

[0160] All or part of the antibody constant region can be linked to the CD71-binding FN3 domain to confer antibody-like properties, particularly those associated with the Fc region, such as Fc effector functions, such as C1q binding, complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, downregulation of cell surface receptors (e.g., B cell receptor; BCR), and can be further modified by modifying the residues in Fc responsible for these activities (for review; see Strohl, Curr Opin Biotechnol. 20, 685-691, 2009).

[0161] Additional moieties may be introduced into the FN3 domain that specifically binds to CD71, such as polyethylene glycol (PEG) molecules, such as PEG5000 or PEG20,000, fatty acids and fatty acid esters of different chain lengths, such as laurate, myristate, stearate, eicosate, behenate, oleate, arachidonate, suberic acid, tetradecanedioic acid, octadecanedioic acid, docosandioic acid, etc., polylysine, octane, carbohydrates (glucan, cellulose, oligosaccharides or polysaccharides) to obtain the desired properties. These moieties may be fused directly to the protein scaffold coding sequence and may be produced by standard cloning and expression techniques. Alternatively, well-known chemical coupling methods may be used to link the moieties to the recombinantly produced molecules disclosed herein.

[0162] A polyethylene glycol moiety can be added to the CD71-binding FN3 domain, for example, by introducing a cysteine ​​residue at the C-terminus of the molecule, or engineering a cysteine ​​into a residue position facing away from the CD71-binding surface of the molecule and attaching a polyethylene glycol group to the cysteine ​​using well-known methods.

[0163] The functionality of the FN3 domains that specifically bind to CD71 into which additional moieties have been introduced can be compared by several well-known assays. For example, the altered properties due to the introduction of an Fc domain and / or Fc domain variant can be assessed by using soluble forms of receptors such as FcγRI, FcγRII, FcγRIII or FcRn receptors in Fc receptor binding assays, or using well-known cell-based assays measuring, for example, ADCC or CDC, or assays that assess the pharmacokinetic properties of the molecules disclosed herein in in vivo models.

[0164] Polynucleotides, vectors, host cells

[0165] In some embodiments, nucleic acids encoding FN3 domains that specifically bind to CD71 are provided as isolated polynucleotides, or as part of an expression vector or as part of a linear DNA sequence (including a linear DNA sequence for in vitro transcription / translation), a vector compatible with prokaryotic, eukaryotic or filamentous phage expression, secretion and / or display of the composition or its directed mutagenesis. Certain exemplary polynucleotides are disclosed herein, however, given 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 present disclosure.

[0166] In some embodiments, the isolated polynucleotide encodes an FN3 domain that specifically binds to CD71, the domain 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.

[0167] Polynucleotides disclosed herein can be produced and assembled into complete single-stranded or double-stranded molecules by solid phase polynucleotide synthesis on chemical synthesis such as automated polynucleotide synthesizer. Alternatively, polynucleotides disclosed herein can be produced by conventional cloning subsequently by other techniques such as PCR. The technology for producing or obtaining polynucleotides of given known sequences is well known in the art.

[0168] The polynucleotides disclosed herein may include at least one non-coding sequence, such as a promoter or enhancer sequence, an intron, a polyadenylation signal, a cis sequence that promotes RepA binding, etc. The polynucleotide sequence may also include additional sequences encoding additional amino acids, encoding, for example, a marker or tag sequence such as a histidine tag or HA tag to facilitate purification or detection of the protein, a signal sequence, a fusion protein partner such as RepA, Fc, or a phage coat protein such as pIX or pilll.

[0169] In some embodiments, a vector comprising at least one polynucleotide disclosed herein is provided. Such a vector can be a plasmid vector, a viral vector, a vector for baculovirus expression, a vector based on a transposon, or any other vector suitable for introducing a polynucleotide disclosed herein into a given organism or genetic background by any means. Such a vector can be an expression vector comprising a nucleic acid sequence element that can control, regulate, cause, or allow the expression of polypeptides encoded by such a vector. Such an element can include a transcription enhancer binding site, an RNA polymerase initiation site, a ribosome binding site, and other sites that promote the expression of coded polypeptides in a given expression system. Such an expression system can be a cell-based or cell-free system well known in the art.

[0170] In some embodiments, a host cell comprising the vector is provided. The FN3 domain that specifically binds to CD71 can optionally be produced by a cell line, a mixed cell line, an immortalized cell, or a clonal population of immortalized cells known in the art. See, for example, Ausubel, et al., ed., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NY, NY (1987-2001); Sambrook, et al., Molecular Cloning: A Laboratory Manual, 2 nd Edition, Cold Spring Harbor, NY (1989); Harlow and Lane, Antibodies, a Laboratory Manual, Cold Spring Harbor, NY (1989); Colligan, et al., eds., Current Protocols in Immunology, John Wiley & Sons, Inc., NY (1994-2001); Colligan et al., Current Protocols in Protein Science, John Wiley & Sons, NY, NY, (1997-2001).

[0171] The host cell selected for expression can be of mammalian origin or can 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 derived, immortalized or transformed cells thereof. Alternatively, the host cell can be selected from species or organisms that cannot glycosylate polypeptides, such as prokaryotic cells or organisms, such as BL21, BL21 (DE3), BL21-GOLD (DE3), XL1-Blue, JM109, HMS174, HMS174 (DE3), and any natural or engineered Escherichia coli, Klebsiella or Pseudomonas strains.

[0172] 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.

[0173] The CD71-binding FN3 domains can be purified from recombinant cell culture by well-known methods, such as by 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).

[0174] Anti-idiotypic antibodies

[0175] In some embodiments, the anti-idiotypic antibody binds to the FN3 domain.

[0176] In some embodiments, the anti-idiotypic antibody that binds to the FN3 domain comprises the amino acid sequence of one 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.

[0177] Reagent test kit

[0178] In some embodiments, kits are provided that include an FN3 domain that binds CD71.

[0179] The kits can be used for therapeutic purposes and as diagnostic kits.

[0180] In some embodiments, the kit comprises an FN3 domain that binds CD71 and a reagent for detecting the FN3 domain. In some embodiments, the kit comprises a bivalent FN3 domain.

[0181] The kits may include one or more other elements, including: instructions for use; other reagents, such as labels, reagents for chelation or other coupling, radioprotective compositions; devices or other materials for preparing the FN3 domain that binds CD71 for administration for imaging, diagnostic, or therapeutic purposes; a pharmaceutically acceptable carrier; and devices or other materials for administration to a subject.

[0182] In some embodiments, the kit includes a CD71-binding FN3 domain comprising an amino acid sequence of one of SEQ ID NOs: 33-50.

[0183] In some embodiments, the kit includes a CD71-binding FN3 domain comprising an amino acid sequence of one of SEQ ID NOs: 51-61.

[0184] In some embodiments, the kit includes a CD71-binding FN3 domain comprising an amino acid sequence of one of SEQ ID NOs: 81-309.

[0185] In some embodiments, the kit includes a CD71-binding FN3 domain comprising an amino acid sequence of one 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.

[0186] Use of the CD71 binding FN3 domain

[0187] The FN3 domain or its conjugate that specifically binds to CD71 can be used to diagnose, monitor, regulate, treat, alleviate, help prevent the occurrence or reduce (symptoms) of human diseases or specific pathologies in cells, tissues, organs, body fluids or generally in the host.

[0188] In some embodiments, the FN3 domain or its conjugate that specifically binds to CD71 can also be used to image CD71-positive tumor tissue in a subject. The methods disclosed herein can be used for animal patients belonging to any classification. Examples of such animals include mammals, such as humans, rodents, dogs, cats, and farm animals.

[0189] In some embodiments, methods of diagnosing a subject having or likely to develop tissue cancer based on CD71 expression by cancer tissue cells, methods of predicting the success of immunotherapy, methods of prognosis, and methods of treatment are provided.

[0190] In some embodiments, a method for detecting cancer cells expressing CD71 in a tumor tissue is provided, the method comprising: obtaining a sample of the tumor tissue from a subject; detecting whether CD71 is expressed in the tumor tissue by contacting the sample of the tumor tissue with a CD71-binding FN3 domain comprising one 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 detecting binding between CD71 and the FN3 domain.

[0191] In some embodiments, CD71 cells are cells involved in CNS diseases, inflammatory / immune diseases, such as MS and infectious diseases of the brain.

[0192] In some embodiments, the tissue can be tissue of any organ or anatomical system that expresses CD71.

[0193] In some embodiments, CD71 expression can be assessed using known methods such as immunohistochemistry or ELISA.

[0194] In some embodiments, a method of isolating cells expressing CD71 is provided, the method comprising: obtaining a sample from a subject; contacting the sample with a CD71-binding FN3 domain comprising an amino acid sequence of one of SEQ ID NOs: 33-50, and isolating cells bound to the FN3 domain.

[0195] In some embodiments, a method of isolating cells expressing CD71 is provided, the method comprising: obtaining a sample from a subject; contacting the sample with a CD71-binding FN3 domain comprising an amino acid sequence of one of SEQ ID NOs: 51-61, and isolating cells bound to the FN3 domain.

[0196] In some embodiments, a method for detecting cancer cells expressing CD71 in a tumor tissue is provided, the method comprising: conjugating a CD71-binding FN3 domain comprising an amino acid sequence of one 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 to which the conjugate binds.

[0197] In some embodiments, a method for detecting cancer cells expressing CD71 in a tumor tissue is provided, the method comprising: conjugating a CD71-binding FN3 domain comprising an amino acid sequence of one 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 binds.

[0198] In some embodiments, a method of treating a subject having cancer is provided, the method comprising administering to the subject an FN3 domain that binds 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 another target, etc.).

[0199] In some embodiments, the subject has a solid tumor.

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

[0201] 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.

[0202] In some embodiments, the solid tumor is renal cell carcinoma (RCC).

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

[0204] In some embodiments, the solid tumor is nasopharyngeal carcinoma (NPC).

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

[0206] In some embodiments, the solid tumor is prostate cancer. In some embodiments, the solid tumor is castration-resistant prostate cancer.

[0207] In some embodiments, the solid tumor is gastric cancer.

[0208] In some embodiments, the solid tumor is ovarian cancer.

[0209] In some embodiments, the solid tumor is gastric cancer.

[0210] In some embodiments, the solid tumor is liver cancer.

[0211] In some embodiments, the solid tumor is pancreatic cancer.

[0212] In some embodiments, the solid tumor is thyroid cancer.

[0213] In some embodiments, the solid tumor is head and neck squamous cell carcinoma.

[0214] In some embodiments, the solid tumor is esophageal cancer or gastrointestinal cancer.

[0215] In some embodiments, the solid tumor is breast cancer.

[0216] In some embodiments, the solid tumor is fallopian tube cancer.

[0217] In some embodiments, the solid tumor is brain cancer.

[0218] In some embodiments, the solid tumor is urethral cancer.

[0219] In some embodiments, the solid tumor is a genitourinary cancer.

[0220] In some embodiments, the solid tumor is endometriosis.

[0221] In some embodiments, the solid tumor is cervical cancer.

[0222] In some embodiments, the solid tumor is a metastatic lesion of cancer.

[0223] In some embodiments, the subject has a hematological malignancy.

[0224] In some embodiments, the hematological malignancy is a lymphoma, myeloma, or leukemia. In some embodiments, the hematological malignancy is a B-cell lymphoma. In some embodiments, the hematological malignancy is Burkitt's lymphoma. In some embodiments, the hematological malignancy is Hodgkin's lymphoma. In some embodiments, the hematological malignancy is non-Hodgkin's lymphoma.

[0225] In some embodiments, the hematological malignancy is myelodysplastic syndrome.

[0226] In some embodiments, the hematological malignancy is acute myeloid leukemia (AML). In some embodiments, the hematological malignancy is chronic myeloid leukemia (CML). In some embodiments, the hematological malignancy is chronic myeloid leukemia (CMML).

[0227] In some embodiments, the hematological malignancy is multiple myeloma (MM).

[0228] In some embodiments, the hematological malignancy is a plasmacytoma.

[0229] In some embodiments, the compositions or pharmaceutical compositions provided herein can be administered alone or in combination with other therapeutic agents, i.e., administered simultaneously or sequentially. In some embodiments, other or additional therapeutic agents are other anti-tumor agents or therapeutic agents. Different tumor types and tumor stages may require the use of various auxiliary compounds useful for treating cancer. For example, the compositions provided herein can be used in combination with various chemotherapeutic agents (such as Taxol, tyrosine kinase inhibitors, leucovorin, fluorouracil, irinotecan, phosphatase inhibitors, MEK inhibitors, etc.). The composition can also be used in combination with drugs (such as anti-PD-1 or anti-CTLA-4, etc.) that regulate the immune response to tumors. Other treatments can be drugs that regulate the immune system, such as antibodies targeting PD-1 or PD-L1.

[0230] In some embodiments, the FN3 domain or its conjugate that specifically binds to CD71 and can be used for diagnosis, monitoring, regulation, treatment, alleviation, helping to prevent the occurrence or alleviation of symptoms for human diseases or specific pathologies in cells, tissues, organs, body fluids or general hosts also exhibits the property of being able to cross the blood-brain barrier. The blood-brain barrier (BBB) ​​prevents most large molecules (such as 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, so the passage of small and large substances from the blood into the central nervous system is controlled by the BBB. This structure makes the treatment and management of patients with neurological diseases and disorders (such as brain cancer) difficult because many therapeutic agents cannot be delivered across the BBB with ideal efficiency. Other conditions involving BBB disruption include: stroke, diabetes, convulsions, hypertensive encephalopathy, acquired immunodeficiency syndrome, craniocerebral injury, multiple sclerosis, Parkinson's disease (PD) and Alzheimer's disease. This ability is particularly useful for treating brain cancers, including, for example, astrocytomas, medulloblastomas, gliomas, ependymomas, germ cell tumors (pinealomas), glioblastomas multiforme, oligodendrogliomas, schwannomas, retinoblastomas, and congenital tumors; or spinal cord cancers, such as neurofibromas, meningiomas, gliomas, and sarcomas. In certain embodiments, a FN3 domain that specifically binds to CD71 comprising an amino acid sequence of one of SEQ ID NOs: 33-50, or a conjugate thereof, can be used to deliver a therapeutic agent or a cytotoxic agent, for example, across the blood-brain barrier. In certain embodiments, a FN3 domain that specifically binds to CD71 comprising an amino acid sequence of one of SEQ ID NOs: 51-61, or a conjugate thereof, can be used to deliver a therapeutic agent or a cytotoxic agent, for example, across the blood-brain barrier. In some embodiments, the protein comprises the following sequence: 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, 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.

[0231] In some embodiments, the polypeptide that facilitates 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.

[0232] "Treat" or "treatment" refers to therapeutic treatment and preventive measures, wherein the purpose is to prevent or slow down (mitigate) 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, relief of symptoms, reduction in disease extent, stabilization of the disease state (i.e., no worsening), delay or slowing of disease progression, improvement or alleviation of the disease state, and relief (whether partial or complete), whether detectable or undetectable. "Treatment" can also mean prolonging survival compared to the expected survival without treatment. People in need of treatment include people who already have a disease or disorder and people who are prone to a disease or disorder or people who want to prevent a disease or disorder.

[0233] A "therapeutically effective amount" refers to an amount effective to achieve the desired therapeutic outcome, at dosages and for periods of time necessary. A therapeutically effective amount of an 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 FN3 domain that binds to CD71 are improved patient well-being, reduction or shrinkage of tumor size, stagnation or slowing of tumor growth, and / or the absence of metastasis of cancer cells to other parts of the body.

[0234] Administration / Pharmaceutical Composition

[0235] In some embodiments, a pharmaceutical composition of an FN3 domain that specifically binds to CD71, optionally conjugated to a detectable marker, therapeutic agent or cytotoxic agent disclosed herein, and a pharmaceutically acceptable carrier is provided. For therapeutic use, the FN3 domain that specifically binds to CD71 can be prepared into a pharmaceutical composition comprising an effective amount of the domain or molecule as an active ingredient in a pharmaceutically acceptable carrier. "Carrier" refers to a diluent, adjuvant, excipient or carrier that is administered with the active compound. Such a carrier can be a liquid, such as water and oil, including those of petroleum, animal, plant or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. 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 composition may contain pharmaceutically acceptable auxiliary substances as needed to approach physiological conditions, such as pH adjusters and buffers, stabilizers, thickeners, lubricants and colorants, etc. The concentration of the molecules disclosed herein in such pharmaceutical formulations can vary widely, i.e., from less than about 0.5%, typically at least about 1% to as much as 15 or 20% by weight, and will be selected according to the particular mode of administration chosen, primarily based on desired dose, fluid volume, viscosity, etc. Suitable carriers and formulations, including other human proteins, such as human serum albumin, are described, for example, in Remington: The Science and Practice of Pharmacy, 21 st Edition, Troy, DBed., Lipincott Williams and Wilkins, Philadelphia, PA 2006, Part 5, Pharmaceutical Manufacturing pp. 691-1092, see especially pp. 958-989.

[0236] The administration mode for the therapeutic use of the FN3 domains disclosed herein can be any suitable route for delivering the agent to the host, such as parenteral administration, for example, intradermal, intramuscular, intraperitoneal, intravenous or subcutaneous, pulmonary; transmucosal (oral, intranasal, vaginal, rectal), using tablets, capsules, solutions, powders, gels, formulations in granules; and contained in syringes, implants, osmotic pumps, cartridges, mini-pumps; or other means known in the art and understood by those skilled in the art. Site-specific administration can be achieved by, for example, intraarticular, intrabronchial, intraperitoneal, intracapsular, intracartilaginous, intracavitary, intracavitary, intracerebellar, intraventricular, intracolonic, intracervical, intragastric, intrahepatic, intracardiac, intraosseous, intrapelvic, intrapericardial, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravascular, intravesical, intralesional, vaginal, rectal, oral, sublingual, intranasal or transdermal delivery.

[0237] The pharmaceutical composition can be provided as a kit comprising a container comprising the pharmaceutical composition as described herein. The pharmaceutical composition can be provided, for example, in the form of an injectable solution for single or multiple doses, or as a sterile powder to be reconstituted before injection. Alternatively, such a kit can include a dry powder disperser, a liquid aerosol generator or a nebulizer for administration of the pharmaceutical composition. Such a kit can further include written information about the indications and uses of the pharmaceutical composition.

[0238] Example

[0239] The following examples are used to illustrate the embodiments disclosed herein. These examples are provided for illustrative purposes only, and the embodiments should not be construed as being limited to these examples, but rather should be construed as covering any and all variations that become apparent due to the teachings provided herein. Those skilled in the art will readily recognize various non-critical parameters that can be changed or modified to produce substantially similar results.

[0240] Example 1. Construction of Tencon library with randomized loops

[0241] 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 connecting seven beta strands. These loops or selected residues within each loop can be randomized to construct a fibronectin type III (FN3) domain library, which can be used to select new molecules that bind to specific targets.

[0242] Tencon:

[0243] LPAPKNLVVSEVTEDSLRLSWTAPDAAFDSFLIQYQESEKVGEAINL TVPGSERSYDLTGLKPGTEYTVSIYGVKGGHRSNPLSAEFTT(SEQ ID NO 1)

[0244] Various libraries were generated using the Tencon scaffold and various design strategies. In general, libraries TCL1 and TCL2 produced good binders. The generation of TCL1 and TCL2 libraries is described in detail in International Patent Publication No. WO / 2014081944A2.

[0245] Construction of TCL1 library

[0246] A library TCL1 designed to randomize only the FG loop of Tencon (SEQ ID NO: 1) was constructed for use in a cis display system (Jacobs et al., Protein Engineering, Design, and Selection, 25: 107-117, 2012). In this system, single-stranded DNA incorporating sequences of Tac promoter, Tencon library coding sequence, RepA coding sequence, cis element and ori element was produced. After expression in an in vitro transcription / translation system, a complex of Tencon-RepA fusion protein and DNA encoding it was produced in cis. The complex bound to the target molecule was then isolated and amplified by polymerase chain reaction (PCR), as described below.

[0247] Construction of the TCL1 library for cis display was achieved by consecutive rounds of PCR to produce two halves of the final linear double-stranded DNA molecule; the 5' fragment contained the promoter and Tencon sequence, while the 3' fragment contained the repA gene and the cis- and ori elements. The two halves were combined by restriction digestion to produce the entire construct. The TCL1 library was designed to introduce random amino acids only in the FG loop of Tencon. NNS codons were used for the construction of this library, making it possible to introduce all 20 amino acids and a stop codon into the FG loop. The TCL1 library includes six independent sub-libraries, each with a different random FG loop length (7 to 12 residues) to further increase diversity.

[0248] TCL1 library (SEQ ID NO: 2)

[0249] LPAPKNLVVSEVTEDSLRLSWTAPDAAFDSFLIQYQESEKVGEAINLTVPGSERSYDLTGLKPGTEYTVSIYGVX 7-12 PLSAEFTT;

[0250] in

[0251] X1, X2, X3, X4, X5, X6, X7 are any amino acids; and

[0252] X8, X9, X 10 , X 11 and X 12 Any amino acid or missing

[0253] Construction of TCL2 library

[0254] Construct TCL2 library, wherein both BC and FG loops of Tencon are randomized, and the amino acid distribution of each position is strictly controlled. Table 2 shows the amino acid distribution of the desired loop position in the TCL2 library. The designed amino acid distribution has two purposes. First, based on the analysis of the Tencon crystal structure and / or homology modeling, the library is biased towards residues that are structurally important for Tencon folding and stability. For example, position 29 is fixed to a subset of only hydrophobic amino acids because the residue is buried in the hydrophobic core of Tencon folding. The second layer of design includes biasing the amino acid distribution towards residues preferentially found in the heavy chain HCDR3 of antibodies to effectively produce high affinity binders (Birtalan et al., J Mol Biol 377:1518-28, 2008; Olson et al., Protein Sci 16:476-84, 2007). To achieve this goal, the "designed distribution" in Table 2 refers to the following distribution: 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 has no methionine, cysteine ​​and stop codons.

[0255] TCL2 library (SEQ ID NO: 3)

[0256] LPAPKNLVVSEVTEDSLRLSWX1X2X3X4X5X6X7X8SFLIQYQESEKVG EAINLTVPGSERSYDLTGLKPGTEYTVSIYGVX9X 10 X 11 X 12 X 13 SX 14 X 15 LSA EFTT;

[0257] X1 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val;

[0258] X2 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val;

[0259] X3Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val;

[0260] X4 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val;

[0261] X5 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val;

[0262] X6 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val;

[0263] X7 is Phe, Ile, Leu, Val or Tyr;

[0264] X8 is Asp, Glu or Thr;

[0265] X9 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val;

[0266] X 10 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val;

[0267] X 11 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val;

[0268] X 12 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val;

[0269] X 13is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val;

[0270] X 14 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr, or Val; and

[0271] X 15 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, He, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val.

[0272] Table 2. Residue distribution in the TCL2 library

[0273] Residue Position* WT residues Distribution of TCL2 in the library 22 T Design distribution 23 A Design distribution 24 P Distribution of 50% P+ Design 25 D Design distribution 26 A 20% A +20% G+ distribution of designs 27 A Design distribution 28 F 20% F, 20% I, 20% L, 20% V, 20% Y 29 D 33% D, 33% E, 33% T 75 K Design distribution 76 G Design distribution 77 G Design distribution 78 H Design distribution 79 R Design distribution 80 S 100％S 81 N Design distribution 82 P Distribution of 50% P+ Design

[0274] *Residue numbering is based on the Tencon sequence of SEQ ID NO: 1

[0275] Subsequently, these libraries were improved in various ways, including the construction of libraries on a stable Tencon framework (U.S. Patent No. 8,569,227) that introduced replacement E11R / L17A / N46V / E86I (Tencon27; SEQ ID NO: 4) compared to wild-type Tencon, and changes in randomized positions in BC and FG loops. Tencon27 is described in International Patent Application No. WO2013049275. Thus, a new library designed to randomize only the FG loop of Tencon (library TCL9) or the combination of BC and FG loops (library TCL7) was generated. These library constructions are used for cis display systems (Odegrip et al., Proc. Natl. Acad. Sci. USA 101: 2806-2810, 2004). The details of the design are as follows:

[0276] Stable Tencon (Tencon27) (SEQ ID NO: 4)

[0277] LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFLIQYQESEKVGEAIVL TVPGSERSYDLTGLKPGTEYTVSIYGVKGGHRSNPLSAIFTT

[0278] TCL7 (randomized FG and BC loops) (SEQ ID NO: 5)

[0279] LPAPKNLVVSRVTEDSARLSWX1X2X3X4X5X6X7X8X9FDSFLIQYQESE KVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVX 10 X 11 X 12 X 13 X 14 X 15 X 16 X 17 X 18 X 19 SNPLSAIFTT;

[0280] in

[0281] X1, X2, X3, X4, X5, X6, 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

[0282] X7, X8, X9, 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.

[0283] TCL9 (randomized FG loop) (SEQ ID NO: 6)

[0284] LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFLIQYQESEKVGEAIVL TVPGSERSYDLTGLKPGTEYTVSIYGVX1X2X3X4X5X6X7X8X9X 10 X 11 X 12 SN PLSAIFTT;

[0285] X1, X2, X3, X4, X5, X6, and X7 are A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, or Y; and

[0286] X 8、 X 9、 X 10、 X 11 and X 12is A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, Y, or missing.

[0287] For library construction, DNA fragments encoding random 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 eliminate stop codons. Two different sets of DNA molecules that randomized the BC loop or FG loop were synthesized independently and then combined using PCR to generate a complete library product.

[0288] Construction of FG loop library (TCL9)

[0289] A set of synthetic DNA molecules was generated, which consisted of the 5'Tac promoter followed by the complete gene sequence of Tencon (except for the random codons in the FG loop) (SEQ ID NO: 26-31). For the FG loop randomization, all amino acids except cysteine ​​and methionine were encoded in equal percentages. The length of the diversified parts was such that they encoded 7, 8, 9, 10, 11 or 12 amino acids in the FG loop. Each sublibrary of length variation was synthesized separately on a 2ug scale and then amplified by PCR using oligonucleotides Sloning-FOR (SEQ ID NO: 9) and Sloning-Rev (SEQ ID NO: 10).

[0290] The 3' fragment of the library is a constant DNA sequence, comprising elements for display, including PspOMI restriction site, the coding region of repA gene and cis- and ori elements. Using plasmid (pCR4Blunt) (Invitrogen) as a template, PCR reaction is carried out using M13 forward and M13 reverse primers to amplify the fragment. The resulting PCR product is digested overnight with PspOMI and gel purified. In order to connect the 5' part of the library DNA to the 3' DNA comprising the repA gene, 2pmol (~540ng to 560ng) of 5' DNA is connected to equimolar (~1.25μg) 3' repA DNA at 37°C overnight in the presence of NotI and PspOMI enzymes and T4 ligase. The library product connected is amplified by 12 PCR cycles using oligonucleotides POP2250 (SEQ ID NO:11) and DigLigRev (SEQ ID NO:12). For each sublibrary, the resulting DNA from 12 PCR reactions was pooled and purified by Qiagen spin columns. The yield of each sublibrary for TCL9 ranged from 32-34 μg.

[0291] Construction of FG / BC loop library (TCL7)

[0292] The TCL7 library provides a library with randomized Tencon BC and FG rings. In this library, a length of 6-9 amino acid whose BC ring is mixed with a length of 7-12 amino acid whose randomized FG ring is combined. Synthetic Tencon fragments BC6, BC7, BC8 and BC9 (respectively SEQ ID NO:13-16) have been produced to include the Tencon gene, the N-terminal portion of the gene encoding protein until and including residue VX, so that the BC ring is replaced with 6,7,8 or 9 random amino acids, which are represented by " N " strings in the sequence provided herein. These fragments were synthesized before finding L17A, N46V and E83I mutations (CEN5243), but these mutations were introduced in the molecular biology step described below. In order to combine the fragment with the fragment of the randomized FG ring of the encoding, the following steps were taken.

[0293] First, the 5' sequence of encoding Tac promoter and Tencon is used to generate DNA fragments (130mer-L17A, SEQ ID NO:17) of nucleotides encoding amino acid A17 by PCR using oligonucleotides POP2222ext (SEQ ID NO:18) and LS1114 (SEQ ID NO:19). This is done to include L17A mutation (CEN5243) in the library. Next, using BC6, BC7, BC8 or BC9 as template and oligonucleotides LS1115 (SEQ ID NO:20) and LS1117 (SEQ ID NO:21), the DNA fragments of Tencon residues R18-V75 including randomized BC ring are amplified by PCR. This PCR step introduces BsaI site at 3' end. Subsequently, oligonucleotides POP2222ext and LS1117 are used as primers to connect these DNA fragments by overlapping PCR. The PCR product of gained 240bp is collected and purified by Qiagen PCR purification kit. The purified DNA was digested with BsaI-HF and gel purified.

[0294] Using FG7, FG8, FG9, FG10, FG11 and FG12 as templates, the fragment encoding the FG loop was amplified by PCR using oligonucleotides SDG10 (SEQ ID NO: 22) and SDG24 (SEQ ID NO: 23) to introduce a BsaI restriction site and the N46V and E86I variants (CEN5243).

[0295] The digested BC fragment and FG fragment are connected together in one step using 3-way connection. Four ligation reactions in 16 possible combinations are set, and each ligation reaction combines two BC loop lengths and 2 FG loop lengths. Each connection contains a total BC fragment of ~300ng and a FG fragment of 300ng. These 4 connection pools are then amplified by PCR using oligonucleotides POP2222 (SEQ ID NO:24) and SDG28 SEQ ID NO:25). Then 7.5μg of each reaction product is digested with Not1 and purified with Qiagen PCR purification columns. 5.2μg of this DNA is connected to an equimolar amount of RepA DNA fragment (~14μg) digested with PspOMI, and the product is amplified by PCR using oligonucleotides POP2222.

[0296] Example 2: Generation of Tencon Libraries with Alternative Binding Surfaces

[0297] The choice of residues randomized in a particular library design determines the overall shape of the resulting interaction surface. X-ray crystallographic analysis of FN3 domains selected from libraries in which the BC, DE, and FG loops were randomized, which comprise scaffold proteins that bind maltose binding protein (MBP), revealed a largely curved interface with an active site suitable for MBP (Koide et al., Proc. Natl. Acad. Sci. USA 104: 6632-6637, 2007). In contrast, the ankyrin repeat scaffold proteins selected to bind to MBP were found to have a flatter interaction surface and bind to the outer surface of MBP away from the active substance (Binz et al., Nat. Biotechnol. 22: 575-582, 2004). These results suggest that the shape (curved vs. flat) of the binding surface of the scaffold molecule may determine which target proteins or specific epitopes on the target proteins can be effectively bound by the scaffold. Published efforts around engineering protein scaffolds containing FN3 domains for protein binding rely on engineering adjacent loops for target binding, creating a curved binding surface. This approach may limit the number of targets and epitopes accessible through such scaffolds.

[0298] Tencon and other FN3 domains contain two sets of CDR-like loops located 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 β-strand that forms the center of the FN3 structure. If the image of Tencon is rotated 90 degrees, an alternative surface can be seen. This slightly concave surface is formed by the CD and FG loops and two antiparallel β-strands (C and F β-strands), referred to herein as the C-CD-F-FG surface. The C-CD-F-FG surface can be used as a template to design a library of protein scaffold interaction surfaces by randomizing a subset of residues that form the surface. The β-strand has a repeating structure, and the side chains of each other residue are exposed on the protein surface. Therefore, a library can be constructed by randomizing some or all of the surface-exposed residues in the β-strand. By selecting appropriate residues in the β-strand, the inherent stability of the Tencon scaffold should be minimally compromised while providing a unique scaffold surface for interactions with other proteins.

[0299] Library TCL14 (SEQ ID NO:7) was designed into the Tencon27 scaffold (SEQ ID NO:4).

[0300] A full description of the methods used to construct this library is described in U.S. Patent Publication No. 2013 / 0226834.

[0301] TCL14 library (SEQ ID NO:7):

[0302] LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFX1IX2YX3EX4X5X6X7GEAIVLTVPGSERSYDLTGLKPGTEYX8VX9IX 10 GVKGGX 11 X 12 SX 13 PLSAIFTT;

[0303] in

[0304] X1, X2, X3, X4, X5, X6, X7, X8, X9, 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.

[0305] The two β-strands that form the C-CD-F-FG surface in Tencon27 have a total of 9 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 6 potential residues: S38, E39, K40, V41, G42, and E43, and the FG loop has 7 potential residues: K75, G76, G77, H78, R79, S80, and N81. The selected residues were chosen for inclusion in the TCL14 design because the theoretical size of the library is larger if all 22 residues are randomized.

[0306] Thirteen positions in Tencon were selected 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 were not randomized because they are just outside the CD and FG loops and do not appear to be part of the C-CD-F-FG surface. For the CD loop, G42 and E43 were not randomized to glycines, providing flexibility that may be valuable in the loop region, and E43 is located at the junction of the surface. K75, G76, G77, and S80 were excluded from the FG loop. Glycine was excluded for the reasons mentioned above, while close inspection of the crystal structure showed that S80 makes critical contacts with the core to help form a stable FG loop. K75 is far from the surface facing the C-CD-F-FG surface and is a less attractive candidate for randomization. Although the above residues were not randomized in the initial TCL14 design, they can be included in subsequent library designs to provide additional diversity for de novo selection or for affinity maturation libraries against, for example, selected TCL14 target-specific hits.

[0307] After the generation of TCL14, three additional Tencon libraries of similar design were generated. These two libraries, TCL19, TCL21 and TCL23, were randomized at the same positions as TCL14 (see above), but the amino acid distribution occurring at these positions was changed (Table 3). TCL19 and TCL21 were designed to include an equal distribution of 18 natural amino acids at each position (about 5.55% each), excluding only cysteine ​​and methionine. TCL23 was designed so that each randomized position was close to 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.

[0308] A third additional library was constructed to expand the potential target binding surface of the other library libraries. In this library, TCL24, 4 additional Tencon positions were randomized compared to libraries TCL14, TCL19, TCL21, and TCL23. These positions include N46 and T48 from the D chain and S84 and I86 from the G chain. Positions 46, 48, 84, and 86 were specifically selected because the side chains of these residues are surfaces exposed from beta chains D and G and are structurally adjacent to the randomized portions of the C and F chains, thereby increasing the surface area that can bind to the target protein. The amino acid distribution used by TCL24 at each position is the same as described in Table 3 for TCL19 and TCL21.

[0309] TCL24 library (SEQ ID NO: 8)

[0310] LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFX1IX2YX3EX4X5X6X7GE AIX8LX9VPGSERSYDLTGLKPGTEYX 10 VX 11 IX 12 GVKGGX 13 X 14 SX 15 PLX 16 AX 17 FTT;

[0311] in

[0312] X1, X2, X3, X4, X5, X6, 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.

[0313] Table 3. Amino acid frequency (%) at each randomized position in TCL21, TCL23 and TCL24.

[0314] Amino Acids TCL19 TCL21 TCL23 TCL24 Ala 5.6 5.6 6.0 5.6 Arg 5.6 5.6 6.0 5.6 Asn 5.6 5.6 3.9 5.6 Asp 5.6 5.6 7.5 5.6 Cys 0.0 0.0 0.0 0.0 Gln 5.6 5.6 1.5 5.6 Glu 5.6 5.6 2.5 5.6 Gly 5.6 5.6 15.0 5.6 His 5.6 5.6 2.3 5.6 Ile 5.6 5.6 2.5 5.6 Leu 5.6 5.6 5.0 5.6 Lys 5.6 5.6 1.5 5.6 Met 0.0 0.0 0.0 0.0 Phe 5.6 5.6 2.5 5.6 Pro 5.6 5.6 4.0 5.6 Ser 5.6 5.6 10.0 5.6 Thr 5.6 5.6 4.5 5.6 Trp 5.6 5.6 4.0 5.6 Tyr 5.6 5.6 17.3 5.6 Val 5.6 5.6 4.0 5.6

[0315] Generation of TCL21, TCL23 and TCL24 libraries

[0316] The TCL21 library was generated using Colibra library technology (Isogenica) to control the amino acid distribution. TCL19, TCL23 and TCL24 gene fragments were generated using Slonomics technology (Morphosys) to control the amino acid distribution. PCR was used to amplify each library after initial synthesis and then ligated to the gene for RepA for use in selection using the CIS display system (Odegrip et al., Proc. Natl. Acad. Sci. USA 101: 2806-2810, 2004), as described above for the libraries.

[0317] Example 3: Selection of fibronectin type III (FN3) domains that bind CD71

[0318] Panning and biochemical screening

[0319] FN3 domains specific for human CD71 were selected by CIS-Display (Odegrip et al 2004) using recombinant biotinylated CD71 extracellular domains (Sino Biologics) with an N-terminal 6H tag. For in vitro transcription and translation (ITT), 3 μg of DNA from the FN3 domain libraries TCL18, TCL19, TCL21, TCL23, and TCL24 were used, and unbound library members were removed by washing. The DNA was eluted from the target protein by heating and amplified by PCR using KOD polymerase for further panning rounds. High affinity binders were isolated by successively reducing the concentration of target CD71 from 400 nM to 100 nM in each round and increasing the stringency of washing. Four additional rounds of off-rate selection were performed on the output from the fifth round of panning. The concentration of biotinylated target antigen was reduced from 25 nM in rounds 6 and 7 to 2.5 nM in rounds 8 and 9.

[0320] After 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 enable purification and detection.

[0321] To screen for FN3 domains that specifically bind to CD71, streptavidin-coated Maxisorp plates (Nunc catalog 436110) were blocked in Start 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. The plates were rinsed with TBST and the diluted lysates were applied to the plates for 1 hour. After additional rinses, the 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 signals at least 10-fold higher than the ELISA signal of the streptavidin control was sequenced, resulting in 23 unique, readable FN3 domain sequences isolated from round 9 of screening (Table 4).

[0322] Table 4. Summary of screening hits

[0323]

[0324]

[0325] Size Exclusion Chromatography Analysis

[0326] Size exclusion chromatography was used to determine the aggregation state of the anti-CD71 FN3 domains. An aliquot (10 μL) of each purified FN3 domain was injected onto a Superdex 75 5 / 150 column (GE Healthcare) at a flow rate of 0.3 mL / min in a mobile phase of PBS pH 7.4. The elution from the column was monitored by absorbance at 280 nm. Tencon protein was included as a control in each run. Agilent ChemStation software was used to analyze the elution profiles. Table 5 lists selected SEC parameters for the 18 identified FN3 domains.

[0327] Table 5. Summary of size exclusion chromatography analysis

[0328]

[0329]

[0330] High-throughput expression and conjugation

[0331] The identified clones were grown in 5 mL replicate cultures in 24-well deep block plates. In brief, 150 μL of overnight culture was inoculated in 5 mL / well of TB medium supplemented with 50 μg / mL kanamycin and grown at 37°C with 220 rpm (OD600~1) shaking for about 3 hours. The culture was induced to a final concentration of 1 mM with IPTG for another 4 hours at 37°C, 220 rpm. The bacterial precipitate was recovered by centrifugation at 2250×g for 15 minutes. 600 μL / well of BugBuster HT (Novagen) supplemented with 0.2 mg / mL lysozyme (Sigma) was added to each well; the precipitate was dissociated with a pipette and then vigorously shaken on a shaking platform for about 30 minutes until the precipitate dissolved. The plate was rotated at 2250×g for 15 minutes to clarify the lysate, and then 2600 μL aliquots of each sample were combined. The His-tagged FN3 domain was purified on His Trap plates (GE) according to the manufacturer's instructions, and then buffer exchanged into TBS using Zeba Spin 7K Desalting Plates (Thermo Scientific). Protein concentration was assessed by Nanodrop. For conjugation with GlyGly-VC-MMAF, FN3 domain (30 μM) was mixed with 150 μM GlyGlyVC-MMAF (Concortis) and 1 μM SortaseA in a total volume of 200 μL. Conjugation was allowed to proceed for 1.5 hours at room temperature according to the manufacturer's instructions and purified again using 96-well His Multitrap HP plates from GE Healthcare. Buffer exchange into PBS was achieved using Zeba Desalting Plates, followed by sterile filtration using Multiscreen HTS GV Plates (Durapore) and centrifugation at 3000×g for 2 minutes. Concentration was assessed by Nanodrop.

[0332] Identification of the SK-BR3-binding FN3 domain

[0333] 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 FACS buffer containing HiLyte-tagged FN3 domains. The cells were incubated for 2 hours at 37°C, 5% CO2. The cells were washed 3 times with FACS buffer and finally resuspended in 100 μL FACS buffer. Fluorescence was detected by Intellictye. Cell populations were identified by FSC-SSC dot plots and FL4 MFI was then recorded. Data were normalized to the mean of 8 unstained cells and dose response curves were fitted using GraphPad.

[0334] Binding of selected clones by dose response ELISA

[0335] Selected clones were analyzed by ELISA to determine the EC50 value of binding. Briefly, the Maxisorb plates were coated with 5 μg / ml of streptavidin at 4°C overnight. The plates were then blocked with StartingBlock (ThermoFisher) for 1 hour at room temperature and then washed with TBS-Tween. Biotinylated CD71 (2 μg / ml) was captured onto the streptavidin plate, and serially diluted Centyrins were added to the appropriate wells for 1 hour at room temperature. After washing, bound Centyrins were detected with an anti-V5 tag antibody conjugated to HRP and POD substrate, and luminescence was read by a plate reader. Luminescence values ​​were plotted as a function of concentration, and dose responses were fitted using PRISM to determine the EC50 value of binding.

[0336] Internalizing FN3 domains were identified by toxin conjugates. FN3 domains were conjugated to the cytotoxic tubulin inhibitor monomethyl auristatin F (MMAF) via an enzymatically cleavable Val-Cit linker or a non-cleavable PEG4 linker (VC-MMAF) using the 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 seeded 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) with 10% fetal bovine serum (Gibco). Cells were allowed to attach overnight at 37°C in a humidified 5% CO2 atmosphere. Cells were treated with 25uL fresh medium and 25uL of 4x inhibitors formulated in fresh medium. Cell viability was determined by endpoint assay at 72 hours using Cell TiterGlo (Promega). IC50 values ​​were determined by fitting the data to a sigmoidal dose response equation with a variable slope using GraphPad Prism (GraphPad Software). The results are shown in Table 6 and indicate that the FN3 domain bound to CD71 is internalized and cytotoxic.

[0337] Table 6: IC50 of CD71 FN3-MMAF conjugate molecules in SKBR-3 cells

[0338]

[0339]

[0340] Bivalent FN3 protein

[0341] A bivalent FN3 protein was generated using two FN3 domains connected by a 4-repeat G / S linker. The bivalent FN3 protein was conjugated to VC-MMAF as described and the cytotoxicity in SK-BR3 cells was evaluated. The IC50 value was found to be better for the bivalent molecule.

[0342] Competition for transferrin binding and internalization

[0343] 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 saturated human transferrin (T0665-100MG). IC50 values ​​for FN3 domain toxin conjugates on SK-BR3 cells screened in the absence or presence of competitor are shown in Table 7.

[0344] Table 7: IC50 of CD71 FN3 domain-MMAF conjugate molecules on SKBR-3 cells (+ / - human transferrin)

[0345]

[0346]

[0347] SEQ ID NO: 1 = Original Tencon sequence

[0348] LPAPKNLVVSEVTEDSLRLSWTAPDAAFDSFLIQYQESEKVGEAINL TVPGSERSYDLTGLKPGTEYTVSIYGVKGGHRSNPLSAEFTT

[0349] SEQ ID NO:2 = TCL1 library

[0350] LPAPKNLVVSEVTEDSLRLSWTAPDAAFDSFLIQYQESEKVGEAINLTVPGSERSYDLTGLKPGTEYTVSIYGV(X) 7-12 PLSAEFTT;

[0351] in

[0352] X1, X2, X3, X4, X5, X6, X7 are any amino acids; and

[0353] X8, X9, X 10 , X 11 and X 12 Any amino acid or missing

[0354] SEQ ID NO:3 = TCL2 library

[0355] LPAPKNLVVSEVTEDSLRLSWX1X2X3X4X5X6X7X8SFLIQYQESEKVG EAINLTVPGSERSYDLTGLKPGTEYTVSIYGVX9X 10 X 11 X 12 X 13 SX 14 X 15 LSA EFTT;

[0356] in

[0357] X1 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val;

[0358] X2 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val;

[0359] X3Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val;

[0360] X4 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val;

[0361] X5 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val;

[0362] X6 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val;

[0363] X7 is Phe, Ile, Leu, Val or Tyr;

[0364] X8 is Asp, Glu or Thr;

[0365] X9 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val;

[0366] X 10 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val;

[0367] X 11 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val;

[0368] X 12 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val;

[0369] X 13 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val;

[0370] X 14 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr, or Val; and

[0371] X 15 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, He, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val.

[0372] SEQ ID NO:4=Stable Tencon

[0373] LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFLIQYQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVKGGHRSNPLSAIFTT

[0374] SEQ ID NO:5 = TCL7 (FG and BC loop)

[0375] LPAPKNLVVSRVTEDSARLSWX1X2X3X4X5X6X7X8X9FDSFLIQYQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVX 10 X 11 X 12 X 13 X 14 X 15 X 16 X 17 X 18 X 19 SNPLSAIFTT;

[0376] in

[0377] X1, X2, X3, X4, X5, X6, 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

[0378] X7, X8, X9, 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

[0379] SEQ ID NO:6 = TCL9 (FG loop)

[0380] LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFLIQYQESEKVGEAIVL TVPGSERSYDLTGLKPGTEYTVSIYGVX1X2X3X4X5X6X7X8X9X 10 X 11 X 12 SN PLSAIFTT;

[0381] in

[0382] X1, X2, X3, X4, X5, X6, and X7 are A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, or Y; and

[0383] X 8、 X 9、 X 10、 X 11 and X 12is A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, Y, or missing.

[0384] TCL14 library (SEQ ID NO:7)

[0385] LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFX1IX2YX3EX4X5X6X7GE AIVLTVPGSERSYDLTGLKPGTEYX8VX9IX 10 GVKGGX 11 X 12 SX 13 PLSAIFTT;

[0386] in

[0387] X1, X2, X3, X4, X5, X6, X7, X8, X9, 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.

[0388] TCL24 library (SEQ ID NO: 8)

[0389] LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFX1IX2YX3EX4X5X6X7GE AIX8LX9VPGSERSYDLTGLKPGTEYX 10 VX 11 IX 12 GVKGGX 13 X 14 SX 15 PLX 16 AX 17 FTT;

[0390] in

[0391] X1, X2, X3, X4, X5, X6, 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.

[0392] SEQ ID NO:9=Sloning-FOR

[0393] GTGACACGGCGGTTAGAAC

[0394] SEQ ID NO:10 = Sloning-REV

[0395] GCCTTTGGGAAGCTTCTAAG

[0396] SEQ ID NO:11 = POP2250

[0397] CGGCGGTTAGAACGCGGCTACAATTAATAC

[0398] SEQ ID NO:12 = DigLigRev

[0399] CATGATTACGCCAAGCTCAGAA

[0400] SEQ ID NO:13 = BC9

[0401] GTGACACGGCGGTTAGAACGCGGCTACAATTAATACATAACCCCATCCCCCTGTTGACAATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGGATCTACCATGCTGCCGGCGCCGAAAAACCTGGTTGTTTCTGAAGTTACCGAAGACTCTCTGCGTCTGTCTTGGNNNNNNNNNNNNNNNNNNNNNNNNNNNTTYGACTCTTTCCTGATCCAGTACCAGGAATCTGAAAAAGTTGGTGAAGCGATCAACCTGACCGTTCCGGGTTCTGAACGTTCTTACGACCTGACCGGTCTGAAACCGGGTACCGAATACACCGTTTCTATCTACGGTGTTCTTAGAAGCTTCCCAAAGGC (where N is any base)

[0402] SEQ ID NO:14 = BC8

[0403] GTGACACGGCGGTTAGAACGCGGCTACAATTAATACATAACCCCATCCCCCTGTTGACAATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGGATCTACCATGCTGCCGGCGCCGAAAAACCTGGTTGTTTCTGAAGTTACCGAAGACTCTCTGCGTCTGTCTTGGNNNNNNNNNNNNNNNNNNNNNNNNTTYGACTCTTTCCTGATCCAGTACCAGGAATCTGAAAAAGTTGGTGAAGCGATCAACCTGACCGTTCCGGGTTCTGAACGTTCTTACGACCTGACCGGTCTGAAACCGGGTACCGAATACACCGTTTCTATCTACGGTGTTCTTAGAAGCTTCCCAA AGGC(where N is any base)

[0404] SEQ ID NO:15 = BC7

[0405] GTGACACGGCGGTTAGAACGCGGCTACAATTAATACATAACCCCATCCCCCTGTTGACAATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGGATCTACCATGCTGCCGGCGCCGAAAAACCTGGTTGTTTCTGAAGTTACCGAAGACTCTCTGCGTCTGTCTTGGNNNNNNNNNNNNNNNNNNNNNTTYGACTCTTTCCTGATCCAGTACCAGGAATCTGAAAAAGTTGGTGAAGCGATCAACCTGACCGTTCCGGGTTCTGAACGTTCTTACGACCTGACCGGTCTGAAACCGGGTACCGAATACACCGTTTCTATCTACGGTGTTCTTAGAAGCTTCCCAAAGGC(where N is any base)

[0406] SEQ ID NO:16 = BC6

[0407] GTGACACGGCGGTTAGAACGCGGCTACAATTAATACATAACCCCATCCCCCTGTTGACAATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGGATCTACCATGCTGCCGGCGCCGAAAAACCTGGTTGTTTCTGAAGTTACCGAAGACTCTCTGCGTCTGTCTTGGNNNNNNNNNNNNNNNNNNTTYGACTCTTTCCTGATCCAGTACCAGGAATCTGAAAAAGTTGGTGAAGCGATCAACCTGACCGTTCCGGGTTCTGAACGTTCTTACGACCTGACCGGTCTGAAACCGGGTACCGAATACACCGTTTCTATCTACGGTGTTCTTAGAAGCTTCCCAAAGGC (where N is any base)

[0408] SEQ ID NO:17 = 130mer-L17ACGGCGGTTAGAACGCGGCTACAATTAATACATAACCCCATCCCCCTGTTGACAATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGGATCTACCATGCTG

[0409] SEQ ID NO:18 = POP222ext

[0410] CGG CGG TTA GAA CGC GGC TAC AAT TAA TAC

[0411] SEQ ID NO:19 = LS1114

[0412] CCA AGA CAG ACG GGC AGA GTC TTC GGT AAC GCG AGA AACAAC CAG GTT TTTCGG CGC CGG CAG CAT GGT AGA TCC TGT TTC

[0413] SEQ ID NO:20 = LS1115

[0414] CCG AAG ACT CTG CCC GTC TGT CTT GG

[0415] SEQ ID NO:21(LS1117).

[0416] CAG TGG TCT CAC GGA TTC CTG GTA CTG GAT CAG GAA AGAGTC GAA

[0417] SEQ ID NO:22(SDG10).

[0418] CATGCGGTCTCTTCCGAAAAAGTTGGTGAAGCGATCGTCCTGACCGTTCCGGGT

[0419] SEQ ID NO:23(SDG24).

[0420] GGTGGTGAAGATCGCAGACAGCGGGTTAG

[0421] SEQ ID NO:24(POP2222).

[0422] CGGCGGTTAGAACGCGGCTAC

[0423] SEQ ID NO:25;SDG28

[0424] AAGATCAGTTGCGGCCGCTAGACTAGAACCGCTGCCACCGCCGG TGGTGAAGATCGCAGAC

[0425] SEQ ID NO:26FG12

[0426] GTGACACGGCGGTTAGAACGCGGCTACAATTAATACATAACCCCATCCCCCTGTTGACAATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGGATCTACCATGCTGCCGGCGCCGAAAAACCTGGTTGTTTCTCGCGTTACCGAAGACTCTGCGCGTCTGTCTTGGACCGCGCCGGACGCGGCGTTCGACTCTTTCCTGATCCAGTACCAGGAATCTGAAAAAGTTGGTGAAGCGATCGTGCTGACCGTTCCGGGTTCTGAACGTTCTTACGACCTGACCGGTCTGAAACCGGGTACCGAATACACCGTTTCTATCTACGGTGTTNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNTCTAACCCGCTGTCTGCGATCTTCACCACCGGCGGTCACCATCACCATCACCATGGCAGCGGTTCTAGTCTAGCGGCCGCAACTGATCTTGGC (where N is any base)

[0427] SEQ ID NO:27 = FG11

[0428] GTGACACGGCGGTTAGAACGCGGCTACAATTAATACATAACCCCATCCCCCTGTTGACAATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGGATCTACCATGCTGCCGGCGCCGAAAAACCTGGTTGTTTCTCGCGTTACCGAAGACTCTGCGCGTCTGTCTTGGACCGCGCCGGACGCGGCGTTCGACTCTTTCCTGATCCAGTACCAGGAATCTGAAAAAGTTGGTGAAGCGATCGTGCTGACCGTTCCGGGTTCTGAACGTTCTTACGACCTGACCGGTCTGAAACCGGGTACCGAATACACCGTTTCTATCTACGGTGTTNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNTCTAACCCGCTGTCTGCGATCTTCACCACCGGCGGTCACCATCACCATCACCATGGCAGCGGTTCTAGTCTAGCGGCCGCAACTGATCTTGGC(where N is any base)

[0429] SEQ ID NO:28 = FG10

[0430] GTGACACGGCGGTTAGAACGCGGCTACAATTAATACATAACCCCATCCCCCTGTTGACAATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGGATCTACCATGCTGCCGGCGCCGAAAAACCTGGTTGTTTCTCGCGTTACCGAAGACTCTGCGCGTCTGTCTTGGACCGCGCCGGACGCGGCGTTCGACTCTTTCCTGATCCAGTACCAGGAATCTGAAAAAGTTGGTGAAGCGATCGTGCTGACCGTTCCGGGTTCTGAACGTTCTTACGACCTGACCGGTCTGAAACCGGGTACCGAATACACCGTTTCTATCTACGGTGTTNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNTCTAACCCGCTGTCTGCGATCTTCACCACCGGCGGTCACCATCACCATCACCATGGCAGCGGTTCTAGTCTAGCGGCCGCAACTGATCTTGGC (where N is any base)

[0431] SEQ ID NO:29 = FG9

[0432] GTGACACGGCGGTTAGAACGCGGCTACAATTAATACATAACCCCATCCCCCTGTTGACAATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGGATCTACCATGCTGCCGGCGCCGAAAAACCTGGTTGTTTCTCGCGTTACCGAAGACTCTGCGCGTCTGTCTTGGACCGCGCCGGACGCGGCGTTCGACTCTTTCCTGATCCAGTACCAGGAATCTGAAAAAGTTGGTGAAGCGATCGTGCTGACCGTTCCGGGTTCTGAACGTTCTTACGACCTGACCGGTCTGAAACCGGGTACCGAATACACCGTTTCTATCTACGGTGTTNNNNNNNNNNNNNNNNNNNNNNNNNNNTCTAACCCGCTGTCTGCGATCTTCACCACCGGCGGTCACCATCACCATCACCATGGCAGCGGTTCTAGTCTAGCGGCCGCAACTGATCTTGGC (where N is any base)

[0433] SEQ ID NO:30 = FG8

[0434] GTGACACGGCGGTTAGAACGCGGCTACAATTAATACATAACCCCATCCCCCTGTTGACAATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGGATCTACCATGCTGCCGGCGCCGAAAAACCTGGTTGTTTCTCGCGTTACCGAAGACTCTGCGCGTCTGTCTTGGACCGCGCCGGACGCGGCGTTCGACTCTTTCCTGATCCAGTACCAGGAATCTGAAAAAGTTGGTGAAGCGATCGTGCTGACCGTTCCGGGTTCTGAACGTTCTTACGACCTGACCGGTCTGAAACCGGGTACCGAATACACCGTTTCTATCTACGGTGTTNNNNNNNNNNNNNNNNNNNNNNNNTCTAACCCGCTGTCTGCGATCTTCACCACCGGCGGTCACCATCACCATCACCATGGCAGCGGTTCTAGTCTAGCGGCCGCAACTGATCTTGGC(where N is any base)

[0435] SEQ ID NO:31 = FG7

[0436] GTGACACGGCGGTTAGAACGCGGCTACAATTAATACATAACCCCATCCCCCTGTTGACAATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGGATCTACCATGCTGCCGGCGCCGAAAAACCTGGTTGTTTCTCGCGTTACCGAAGACTCTGCGCGTCTGTCTTGGACCGCGCCGGACGCGGCGTTCGACTCTTTCCTGATCCAGTACCAGGAATCTGAAAAAGTTGGTGAAGCGATCGTGCTGACCGTTCCGGGTTCTGAACGTTCTTACGACCTGACCGGTCTGAAACCGGGTACCGAATACACCGTTTCTATCTACGGTGTTNNNNNNNNNNNNNNNNNNNNNTCTAACCCGCTGTCTGCGATCTTCACCACCGGCGGTCACCATCACCATCACCATGGCAGCGGTTCTAGTCTAGCGGCCGCAACTGATCTTGGC(where N is any base)

[0437] SEQ ID NO:32 = Human mature CD71

[0438] MTKEYQDLQHLDNEESDHHQLRKGPPPPQPLLQRLCSGPRLLLLSLGLSLLLLVVVCVIGSQNSQLQEELRGLRETFSNFTASTEAQVKGLSTQGGNVGRKMKSLESQLEKQQKDLSEDHSSLLLHVKQFVSDLRSLSCQMAALQGNGSERTCCPVNWVEHERSCYWFSRSGKAWADADNYCRLEDAHLVVVTSWEEQKFVQHHIGPVNTWMGLHDQNGPWKWVDGTDYETGFKNWRPEQPDDWYGHGLGGGEDCAHFTDDGRWNDDVCQRPYRWVCETELDKASQEPPLL

[0439] SEQ ID NO:80 = Human mature CD71 extracellular domain

[0440] QNSQLQEELRGLRETFSNFTASTEAQVKGLSTQGGNVGRKMKSLESQLE

[0441] KQQKDLSEDHSSLLLHVKQFVSDLRSLSCQMAALQGNGSERTCCPVNW

[0442] VEHERSCYWFSRSGKAWADADNYCRLEDAHLVVVTSWEEQKFVQHHI

[0443] GPVNTWMGLHDQNGPWKWVDGTDYETGFKNWRPEQPDDWYGHGLG

[0444] GGEDCAHFTDDGRWNDDVCQRPYRWVCETELDKASQEPPLL

[0445]

[0446]

[0447]

[0448] Example 4: Selection of fibronectin type III (FN3) domains that bind CD71

[0449] Panning and biochemical screening methods for identifying FN3 domains that bind to CD71 but 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 the panning) or negative controls (irrelevant Fc-fusion recombinant proteins and human serum albumin) in the presence of transferrin or in the case of FN3 proteins bound to the CD71 transferrin binding site for 1 hour. The concentration of transferrin was up to 35 μM. Without being bound by any particular theory, including transferrin or FN3 proteins bound to the CD71 transferrin binding site pushed the selection of FN3 domains to those domains that did not compete or inhibit transferrin binding to CD71. The plates were rinsed with TBST and the diluted lysate was applied to the plates for 1 hour. After additional washing, the 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 signals at least 10-fold higher than the ELISA signal of the streptavidin control was sequenced to obtain the FN3 domain sequences isolated from the screen.

[0450] Example 5: Selection of fibronectin type III (FN3) domains that bind CD71 and do not compete with transferrin

[0451] To identify CD71 binding FN3 domains that do not compete or compete minimally with transferrin, a biased CIS display strategy was designed. Briefly, the output recovered after 5 rounds of selection on the ECD of human CD71 was used (Example 3). Additional rounds of off-rate selection were performed as described in Example 3, with the addition of 1) a wash step with human saturated transferrin to elute Centyrin bound to the same site as transferrin before the final elution step or 2) elution of FN3 domain binders with monoclonal antibody OKT9. FN3 domains recovered from the transferrin wash strategy and OKT9 elution strategy were PCR amplified and cloned into pET vectors as previously described (Example 3). Binding of 228 FN3 domains that specifically bind to huCD71 to the huCD71 ECD was confirmed by ELISA (Table 8). A subset of unique binders was analyzed by SEC (Table 9), conjugated to MMAF and internalization assessed by cell viability assay in SKBR-3 cells + / - saturated 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.

[0452] Table 8. Summary of screening hits

[0453]

[0454]

[0455]

[0456]

[0457]

[0458]

[0459]

[0460] Table 9. Summary of size exclusion chromatography analysis

[0461]

[0462]

[0463]

[0464]

[0465]

[0466]

[0467] Table 10: IC50 of CD71 FN3 domain-MMAF conjugate molecule 5 on SKBR-3 cells + / - human transferrin

[0468]

[0469]

[0470]

[0471]

[0472]

[0473]

[0474]

[0475]

[0476]

[0477]

[0478]

[0479]

[0480]

[0481]

[0482]

[0483]

[0484]

[0485]

[0486]

[0487] 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 maleimide chemistry through cysteine ​​uniquely engineered into the FN3 domain. Cysteine ​​replacements can be such as those provided herein and also as those provided in U.S. Patent Application 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 be able to knockdown the target mRNA in vitro. The FN3 domain-oligonucleotide conjugate was administered intravenously in mice at doses up to 10 mg / kg oligonucleotide load. Mice were sacrificed at various time points after dosing; skeletal muscle, cardiac muscle, and various other tissues were recovered and stored in RNAlater TM (Sigma Aldrich) until needed. Use standard qPCR ΔΔC T Methods and primers specific for target and control genes were used to assess target gene knockdown. The target gene was found to be knocked down in muscle, and this knockdown was enhanced by conjugating siRNA or ASO to the CD71 FN3 binding domain.

[0488] Example 7. General method

[0489] Standard methods in molecular biology are described in: Sambrook, Fritsch and Maniatis (1982 & 1989 2 nd Edition,2001 3 rd Edition) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Sambrook and Russell (2001) Molecular Cloning, 3 rded., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Wu (1993) Recombinant DNA, Vol. 217, Academic Press, San Diego, CA). Standard methods also appear in Ausbel, et al. (2001) Current Protocols in Molecular Biology, Vols. 1-4, John Wiley and Sons, Inc. New York, NY, which describes 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).

[0490] Methods for protein purification, 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 modifications, post-translational modifications, production of fusion proteins, 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) Bio Directory, 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 for characterizing ligand / receptor interactions can be used (see, e.g., Coligan, et al. (2001) Current Protocols in Immunology, Vol. 4, John Wiley, Inc., New York).

[0491] 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 as incorporated by reference. Applicants intend that this statement of incorporation by reference pursuant to 37 CFR § 1.57(b)(1) refers 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 pursuant to 37 CFR § 1.57(b)(2), even if such citations are not directly adjacent to the express statement of incorporation by reference. The inclusion of a specific statement of incorporation by reference in the specification, if any, does not in any way diminish this general statement of incorporation by reference. Citation of a reference herein does not constitute an admission that the reference is relevant prior art, nor does it constitute any admission of the contents or date of these publications or documents.

[0492] The scope of the present embodiment is not limited by the specific embodiments described herein. In fact, various modifications of the embodiments, except those described herein, will become apparent to those skilled in the art through the foregoing description. Such modifications are intended to fall within the scope of the appended claims.

[0493] The foregoing written description is considered 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. A polypeptide comprising an amino acid sequence that is at least 90% identical to the following amino acid sequence: 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, 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 the following amino acid sequence: SEQ ID NO:146, 214, 104, 259, 134, 92, 302, 235, 237, 152, 238, 136, 197, 212, 296, 2 26, 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.

2. The polypeptide of claim 1, wherein the polypeptide comprises two of the following: 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, 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.

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

4. The polypeptide of claim 3, wherein the polypeptide comprises an amino acid sequence that is at least 90% identical to a polypeptide having the following sequence: SEQ ID NO: 146, 214, 104, 259, 134, 92, 302, 235, 237, 152, 238, 136, 197, 212, 296, 226, 261, 307, {GOODW-24007-CNDIV / 01993881v1}1 115, 112, 278, 297, 96, 222, 95, 233, 217, 252, 194, 164, 168, 174, 190, 257, 303, 284, 85 or 149.

5. The polypeptide of claim 3, wherein the polypeptide 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.

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

7. The polypeptide of claim 6, wherein the detectable label is a radioisotope, magnetic beads, metal beads, colloidal particles, fluorescent dyes, electron-dense reagents, enzymes, biotin, digoxigenin, or a hapten.

8. The polypeptide of claim 6 or 7, wherein the detectable label is an auristatin, monomethyl auristatin phenylalanine, a dolastatin, a chemotherapeutic agent, a drug, a growth inhibitory agent, a toxin, or a radioisotope.

9. The polypeptide of claim 6, wherein the therapeutic agent is a chemotherapeutic agent, a drug, an antibody, a growth inhibitory agent, a toxin, a radioisotope, an anti-tubulin agent, a polynucleotide, an siRNA molecule or its sense or antisense strand, an antisense molecule or its strand, 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 chemosensitizer, a topoisomerase inhibitor, or a vinca alkaloid.

10. The polypeptide of claim 6, wherein the therapeutic agent can cause 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.

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