CD71-binding fibronectin type III domain

Abstract

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

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Application No. 62 / 914,643 (filed October 14, 2019) and U.S. Provisional Application No. 62 / 949,020 (filed December 17, 2019), which are incorporated herein by reference in their entirety.

[0002] field This embodiment relates to a fibronectin type III domain (FN3) that specifically binds to differentiation antigen group 71 (CD71), and to a method for producing and using the molecule. [Background technology]

[0003] background CD71, also known as transferrin receptor 1, is a transmembrane receptor essential for iron transport into cells. It is highly expressed in many tumor types and at the blood-brain barrier, making it a crucial target for drug delivery. After binding to iron-loading transferrin, CD71 rapidly invaginates into the plasma membrane and efficiently recirculates back to the cell surface. Studies using CD71 antibody-drug conjugates suggest that targeting CD71 can improve the specificity and selectivity of drug delivery and expand the therapeutic index. Furthermore, studies using anti-CD71 monoclonal antibodies indicate that binding affinity may play a significant role in enabling transcellular transport across the blood-brain barrier. Antibodies with high affinity for CD71 rapidly translocate internally and alter normal receptor transport, resulting in the receptor being targeted to lysosomes for degradation instead of recirculation. In contrast, antibodies with low affinity for CD71 allow for receptor recirculation and higher brain exposure.

[0004] When high affinity and specificity for a target molecule are desired, antibodies or antibody fragments are the most widely used class of therapeutic proteins; however, non-antibody proteins can also be engineered to bind to such targets. These “alternative scaffold” proteins have advantages over traditional antibodies due to their small size, lack of disulfide bonds, high stability, ability to be expressed in prokaryotic hosts, and ease of purification. They readily bind to drugs / toxins, efficiently penetrate tissues, and are readily converted into multiselective binders. [Overview of the project] [Problems that the invention aims to solve]

[0005] One such alternative scaffold is the immunoglobulin (Ig) fold. This fold is found in the variable region of antibodies, and even in thousands of non-antibody proteins. One such Ig protein, the tenth fibronectin type III (FN3) repeat derived from human fibronectin, has been shown to be able to retain the overall Ig fold structure while allowing numerous mutations in the surface-exposed loop. Therefore, what is needed is an FN3 domain that can specifically bind to CD71, and a method for using such a molecule for cancer treatment. [Means for solving the problem]

[0006] Abstract In some embodiments, an FN3 domain (e.g., polypeptide) that specifically binds to the 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 does not exist naturally.

[0007] In some embodiments, an FN3 domain is provided that includes the amino acid sequence of SEQ ID NOs: 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, or 62. In some embodiments, FN3 domains are provided that include the amino acid sequences of SEQ ID NOs: 146, 214, 104, 259, 134, 92, 302, 235, 237, 152, 238, 136, 197, 212, 296, 226, 261, 307, 115, 112, 278, 297, 96, 222, 95, 233, 217, 252, 194, 164, 168, 174, 190, 257, 303, 284, 85, or 149. In some embodiments, FN3 domains are provided that include the amino acid sequences of SEQ ID NOs: 81-309. In some embodiments, the FN3 domain binds to CD71. In some embodiments, the FN3 domain binds to human CD71 at a site on CD71 that does not compete with transferrin binding to CD71. In some embodiments, FN3 domains are provided that include the amino acid sequences of SEQ ID NOs: 146, 214, 104, 259, 134, 92, 302, 235, 237, 152, 238, 136, 197, 212, 296, 226, 261, 307, 115, 112, 278, 297, 96, 222, 95, 233, 217, 252, 194, 164, 168, 174, 190, 257, 303, 284, 85, or 149. In some embodiments, the FN3 domains specifically bind to CD71. In some embodiments, polypeptides are provided that include one or more FN3 domains linked by linkers such as mobile linkers. In some embodiments, the polypeptide includes two, three, or four FN3 domains linked to each other by one or more linkers between the domains.

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

[0009] In some embodiments, vectors comprising the polynucleotides described herein are provided.

[0010] In some embodiments, host cells containing the vectors described herein are provided.

[0011] In some embodiments, methods for producing an FN3 domain are provided. In some embodiments, the method comprises culturing host cells containing a vector encoding or expressing an 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, pharmaceutical compositions are provided that include an FN3 domain bound to CD71 and a pharmaceutically acceptable carrier.

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

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

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

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

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

[0018] In some embodiments, a method is provided for treating cancer in subjects requiring treatment. In some embodiments, the method comprises administering a polypeptide that binds to CD71. In some embodiments, the polypeptide is an FN3 domain that binds to CD71. In some embodiments, the polypeptide is sequence numbers 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, The polypeptides provided herein include sequences such as 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 polypeptides linked or conjugated to a therapeutic agent.

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

[0020] In some embodiments, methods are provided for delivering a target agent to CD71-positive cells. In some embodiments, the method involves contacting cells with a target agent coupled to a CD71-binding FN3 domain, such as a polypeptide provided herein. In some embodiments, the target agent is a chemotherapeutic agent, drug, growth inhibitor, toxin, radioisotope, antitubulin agent, polynucleotide, siRNA molecule, antisense molecule, RNA molecule, DNA molecule, DNA minor groove binder, DNA replication inhibitor, alkylating agent, antibiotic, folate antimetabolites, antimetabolites, chemotherapy These are sensitizing agents, topoisomerase inhibitors, or vinca alkaloids.

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

[0022] In some embodiments, a method is provided for identifying FN3 proteins that bind to CD71 at a site that does not compete with or inhibit transferrin binding to CD71. [Modes for carrying out the invention]

[0023] Detailed explanation of disclosure As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include multiple referents unless the content explicitly indicates otherwise. Thus, for example, a reference to “a cell” includes combinations of two or more cells, etc.

[0024] The "fibronectin type III (FN3) domain" (FN3 domain) refers to a frequently occurring domain 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). Examples of FN3 domains include 15 different FN3 domains present in human tenascin C, 15 different FN3 domains present in human fibronectin (FN), and non-naturally synthesized FN3 domains, such as those described in U.S. Patent No. 8,278,419. Each FN3 domain is referred to by its domain number and protein name, for example, the third FN3 domain of tenascin (TN3), or the tenth FN3 domain of fibronectin (FN10).

[0025] The term "capture agent" refers to a substance that binds to a specific type of cell, enabling its isolation from other cells. Examples of capture agents include magnetic beads, magnetic fluids, encapsulation reagents, and molecules that bind to specific cell types.

[0026] "Sample" refers to a collection of similar fluids, cells, or tissues isolated from the subject, as well as fluids, cells, or tissues present within the subject. Examples of samples include tissue biopsies, fine-needle aspirations, surgically excised tissues, organ cultures, cell cultures, and biological fluids such as blood, serum and serosal fluids, plasma, lymph, urine, saliva, cystic fluid, tears, feces, sputum, mucosal secretions from secretory tissues and organs, vaginal secretions, ascites fluid, fluids from the pleura, pericoronium, peritoneum, abdominal cavity and other body cavities, fluids collected by bronchial lavage, synovial fluid, and liquid solutions that have come into contact with the subject or biological source, such as cell and organ culture media and lavage solutions containing cell or organ conditioned media.

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

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

[0029] "Specific binding" or "specific binding" means that it binds to its target, such as CD71, by approximately 1 x 10⁶ times. -6 M or less, for example, about 1x10 -7 M or smaller, approximately 1x10 -8 M or smaller, approximately 1x10 -9 M or smaller, approximately 1x10 -10 M or smaller, approximately 1x10 -11 M or smaller, approximately 1x10 -12 M or less, or approximately 1x10-13 The ability of an FN3 domain to bind with a dissociation constant (K D ) of M or less. 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 to the same given antigen (homolog) from other species such as Macaca Fascicularis (cynomolgus monkey, cyno) or Pan troglodytes (chimpanzee).

[0030] "Library" refers to a collection of variants. A library can be composed of polypeptide or polynucleotide variants.

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

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

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

[0034] "Cancer cells" or "tumor cells" refer to cancerous, precancerous, or transformed cells in vivo, ex vivo, or in tissue culture that exhibit spontaneous or induced phenotypic changes, not necessarily involving the uptake of new genetic material. Transformation can result from infection with transforming viruses and the incorporation of new genomic nucleic acids or the uptake of exogenous nucleic acids, but can also occur spontaneously or after exposure to carcinogens, thereby mutating endogenous genes. Transformation / cancer is exemplified, for example, by morphological changes, cell immortalization, abnormal growth control, lesion formation, proliferation, malignant lesions, tumor-specific marker levels, invasiveness, and tumor growth or suppression in appropriate animals such as nude mice, in vitro, in vivo, and ex vivo (Freshney, Culture of Animals). Cells: A Manual of Basic Technique (3rd edition, 1994)).

[0035] A “vector” refers to a polynucleotide that has the ability to replicate within a biological system or to be transported between such systems. Vector polynucleotides typically contain elements such as replication origins, polyadenylation signals, or selection markers that function to facilitate the replication or maintenance of these polynucleotides in a biological system. Examples of such biological systems include cells, viruses, animals, plants, and reconstituted biological systems that utilize biological components capable of replicating vectors. The polynucleotide containing the vector may be a DNA or RNA molecule or a hybrid thereof.

[0036] An "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.

[0037] A "polynucleotide" refers to a synthetic molecule containing a chain of nucleotides covalently linked by a sugar-phosphate backbone or other equivalent covalent chemistry. cDNA is a typical example of a polynucleotide.

[0038] A "polypeptide" or "protein" refers to a molecule containing at least two amino acid residues linked by peptide bonds to form a polypeptide. Small polypeptides with fewer than approximately 50 amino acids may be called "peptides."

[0039] "Valency" refers to the presence of a specific number of antigen-specific binding sites within a molecule. Thus, the terms "monovalent," "divalent," "tetravalent," and "hexavalent" refer to the presence of one, two, four, and six antigen-specific binding sites within a molecule.

[0040] "Subject" includes any human or non-human animal. "Non-human animal" includes all vertebrates, e.g., mammals and non-mammals, e.g., non-human primates, sheep, dogs, cats, horses, cattle, chickens, amphibians, reptiles, etc. Unless otherwise stated, the terms "patient" and "subject" are used interchangeably.

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

[0042] composition of a substance In some embodiments, a protein is provided comprising a polypeptide containing the amino acid sequence of SEQ ID NOs: 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, or 81-309. In some embodiments, a polypeptide comprising the sequence of SEQ ID NOs: 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, or 81-309 is provided, and at least 62%, 63%, 64%, 65%, 66%, 67%, 68% Proteins containing polypeptides that have amino acid sequences that are 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. 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 sequences 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 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to sequences 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46 The sequences 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, or 81-309 are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical. In some embodiments, the proteins are at least 95%, 96%, 97%, 98%, or 99% identical to sequence numbers 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, or 81-309.

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

[0044] In some embodiments, fibronectin type III (FN3) domains that bind to or specifically bind to the human CD71 protein (SEQ ID NO: 32 or 80) are provided. As provided herein, FN3 domains 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. Therefore, for example, an FN3 domain that binds to CD71 would also include an FN3 domain protein that specifically binds to CD71. These molecules can be used, for example, in therapeutic and diagnostic applications, as well as in imaging. In some embodiments, polynucleotides or complementary nucleic acids thereof encoding the FN3 domains disclosed herein, vectors, host cells, and methods for producing and using them are provided.

[0045] In some embodiments, isolated FN3 domains that bind to or specifically bind to CD71 are provided.

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

[0047] In some embodiments, the FN3 domain is determined by surface plasmon resonance or Kinexa method, as can be done by those skilled in the art, to be approximately 1 x 10⁻⁶ -7 Less than M, for example, about 1 x 10 -8 Less than M, approximately 1x10 -9 Less than M, approximately 1x10 -10 Less than M, approximately 1x10 -11 Less than M, approximately 1x10 -12 Less than M, or approximately 1x10 -13 Dissociation constant less than M (K D It can bind to CD71. The measured affinity of a particular FN3 domain-antigen interaction may vary when measured under different conditions (e.g., volume osmolality, pH). Therefore, affinity and other antigen-binding parameters (e.g., K D , K on , K off The measurement of ) is performed using standardized solutions of protein scaffolds and antigens, as well as standardized buffers such as those described herein.

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

[0049] In some embodiments, the FN3 domain that binds to or specifically binds to CD71 contains an initiator methionine (Met) ligated to the N-terminus of the molecule. In some embodiments, the FN3 domain that binds to or specifically binds to CD71 contains cysteine ​​(Cys) ligated to the C-terminus of the FN3 domain. The addition of N-terminal Met and / or C-terminal Cys may facilitate the expression and / or binding of half-life extension molecules.

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

[0051] Cysteine ​​substitution at a domain or protein site involves replacing an existing amino acid residue with a cysteine ​​residue. Other examples of cysteine ​​modification can be found, for example, in U.S. Patent Application Publication 20170362301 (which is incorporated herein by reference in its entirety). Sequence alignment can be performed, for example, using BlastP with default parameters on the NCBI website.

[0052] In some embodiments, the FN3 domain that binds to CD71 is internally translocated into the cell. In some embodiments, internal translocation of the FN3 domain can facilitate the delivery of detectable labels or therapeutic agents into the cell. In some embodiments, internal translocation of the FN3 domain can facilitate the delivery of cytotoxic agents into the cell. Cytotoxic agents can act as therapeutic agents. In some embodiments, internal translocation of the FN3 domain can facilitate the delivery of any detectable labels, therapeutic agents and / or cytotoxic agents disclosed herein into the cell. In some embodiments, the cells are tumor cells. In some embodiments, the cells are liver cells.

[0053] In some embodiments, the FN3 domain that binds to CD71 may be located at residue positions 11, 14, 17, 37, 46, 73, or 86 (residue number corresponding to SEQ ID NO: 4). Tencon array of sequence number 1 or Tencon array of sequence number 4 having substitutions in the ring) Based on a 27-array sequence.

[0054] In some embodiments, the isolated FN3 domain that binds to CD71 contains the amino acid sequence of SEQ ID NOs: 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

[0055] In some embodiments, the isolated FN3 domain that binds to CD71 contains the amino acid sequence of SEQ ID NOs. 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, or 61.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0087] In some embodiments, the FN3 domain binds to human CD71 at a site on CD71 that does not compete with transferrin binding to CD71. In some embodiments, the FN3 domain includes the sequence of SEQ ID NOs: 146, 214, 104, 259, 134, 92, 302, 235, 237, 152, 238, 136, 197, 212, 296, 226, 261, 307, 115, 112, 278, 297, 96, 222, 95, 233, 217, 252, 194, 164, 168, 174, 190, 257, 303, 284, 85, or 149.

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

[0089] In some embodiments, the isolated FN3 domain that binds to CD71 contains 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 to CD71 contains 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. In some embodiments, the isolated FN3 domain that binds to CD71 contains 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, isolated FN3 domains that bind to CD71 are sequence numbers 146, 214, 104, 259, 134, 92, 302, 235, 237, 152, 238, 136, 197, 212, 296, 226, 261, One of the amino acid sequences 307, 115, 112, 278, 297, 96, 222, 95, 233, 217, 252, 194, 164, 168, 174, 190, 257, 303, 284, 85, or 149, and 62%, 63%, 64% , containing amino acid sequences that are identical by 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%.

[0090] The identity percentage can be determined by aligning the two sequences using default parameters with BlastP, which is available via the NCBI website.

[0091] Conjugate of FN3 domains bound to CD71 of this disclosure In some embodiments, isolated FN3 domains that bind to CD71 conjugated to a heterologous molecule are provided.

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

[0093] In some embodiments, the peptide is conjugated to lipid nanoparticles, which can be used, for example, for cell-specific targeting.

[0094] In some embodiments, the protein is bound to a binding site that targets CD71 or another protein for proteolysis. For example, the protein may be bound to PROTACS (a binding site for E3 ubiquitin ligase), and as a result, the protein is delivered to the E3 ligase. These may be linked via linkers such as glycine-serine linkers.

[0095] The FN3 domain that binds to CD71 may also bind to or ligate to another FN3 domain that binds to a different target other than CD71. This allows the peptide to be multiselective (e.g., bispecific, triplicate, etc.), and as a result, it binds to CD71 and another, for example, a protein. In some embodiments, the CD71 FN3 binding domain is ligated to another FN3 domain that binds to an antigen expressed by tumor cells (tumor antigen).

[0096] In some embodiments, FN3 domains may be linked together by a linker to form a divalent FN3 domain. The linker may be a mobile linker. In some embodiments, the linker is a G / S linker. In some embodiments, the linker has one, two, three, or four G / S repeats. The G / S repeat unit consists of four glycine molecules followed by serine, e.g., GGGGS.

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

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

[0099] In some embodiments, an FN3 domain that binds to CD71 conjugated to a therapeutic agent is provided. The therapeutic agent, for example, a non-limited example of a cytotoxic agent, is provided. This will be provided in the details.

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

[0101] Examples of detectable labels include compositions that, when bound to the FN3 domain that binds to CD71, enable detection of CD71 by spectroscopic, photochemical, biochemical, immunochemical, or other chemical methods.

[0102] Examples of detectable labels include, but are not limited to, radioisotopes, magnetic beads, metal beads, colloidal particles, fluorescent dyes, high electron density reagents, enzymes (e.g., those commonly used in ELISA), biotin, digoxigenin, haptens, luminescent molecules, chemiluminescent molecules, fluorescent dyes, fluorophores, fluorescent quenchers, colored molecules, radioisotopes, scintillants, avidin, streptavidin, protein A, protein G, antibodies or their fragments, polyhistidine, and Ni. 2+ Examples include Flag tags, myc tags, heavy metals, enzymes, alkaline phosphatase peroxidase, luciferase, electron donors / electron acceptors, acridinium esters, and colorimetric analysis substrates.

[0103] A detectable label may spontaneously emit a signal, for example, if the detectable label is a radioactive isotope. In some embodiments, the detectable label emits a signal as a result of being stimulated by an external stimulus such as a magnetic field, electric field, or electromagnetic field.

[0104] Examples of radioactive isotopes may be those that emit gamma, Auger, beta, alpha, or positron. Examples of radioactive isotopes 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 American and 227 Ac is one example.

[0105] Examples of metal atoms include metals with atomic numbers greater than 20, such as calcium, scandium, titanium, vanadium, chromium, manganese, iron, cobalt, nickel, copper, zinc, gallium, germanium, arsenic, selenium, bromine, krypton, rubidium, strontium, yttrium, zirconium, niobium, molybdenum, technetium, ruthenium, rhodium, palladium, silver, cadmium, indium, tin, antimony, tellurium, iodine, xenon, cesium, barium, lanthanum, hafnium, tantalum, tungsten, and rhenium. The atoms are osmium atoms, iridium atoms, platinum atoms, gold atoms, mercury atoms, thallium atoms, lead atoms, bismuth atoms, francium atoms, radium atoms, actinium atoms, cerium atoms, praseodymium atoms, neodymium atoms, promethium atoms, samarium atoms, europium atoms, gadolinium atoms, terbium atoms, dysprosium atoms, holmium atoms, erbium atoms, thulium atoms, ytterbium atoms, lutetium atoms, thorium atoms, protactinium atoms, uranium atoms, neptunium atoms, plutonium atoms, americium atoms, curium atoms, bercklium atoms, californium atoms, einsteinium atoms, fermium atoms, mendelevium atoms, nobelium atoms, or lawrencium atoms.

[0106] In some embodiments, the metal atom is an alkaline earth atom with an atomic number greater than 20. It could be a metal-like substance.

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

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

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

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

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

[0112] In some embodiments, the metal atoms may be metals having atomic numbers ranging from 53 (i.e., iodine) to 83 (i.e., bismuth).

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

[0114] Metal atoms are metal ions in the form of +1, +2, or +3 oxidation states, for example, 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+ This is possible. The metal atoms may include metal oxides, such as iron oxide, manganese oxide, or gadolinium oxide.

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

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

[0117] The FN3 domain, which specifically binds to CD71 bound to a detectable label, can be used, for example, as a contrast agent for diagnosing tumor distribution, the presence of tumor cells, and / or evaluating tumor recurrence.

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

[0119] In some embodiments, the therapeutic agent is a chemotherapeutic agent, a drug, a growth inhibitor, a toxin (e.g., an enzyme-active toxin of bacterial, fungal, plant, or animal origin, or a fragment thereof), or a radioisotope (i.e., a radioconjugate).

[0120] The FN3 domain bound to CD71 in the therapeutic agent disclosed herein is CD It can be used for targeted delivery of therapeutic agents to cells expressing 71 (e.g., tumor cells) and for intracellular accumulation there. While not bound by any particular theory, this type of delivery may be useful when systemic administration of these unbound agents may produce unacceptable levels of toxicity to normal cells.

[0121] In some embodiments, therapeutic agents can induce their cytotoxic and / or cell division arrest effects through mechanisms such as tubulin binding, DNA binding, topoisomerase inhibition, DNA cross-linking, chelation, spliceosome inhibition, NAMPT inhibition, and HDAC inhibition.

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

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

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

[0125] In some embodiments, the therapeutic agents are dolostatin or dolostatin peptide analogs and derivatives, auristatin or monomethyl auristatin phenylalanine. Example molecules are disclosed in U.S. Patents 5,635,483 and 5,780,588. Dolostatin 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 to possess anticancer and antifungal activity. The dolostatin or auristatin drug moiety may be bound to the FN3 domain via the N (amino) or C (carboxyl) terminus of the peptide drug moiety (WO 02 / 088172), or via any cysteine ​​manipulated within the FN3 domain.

[0126] In some embodiments, the therapeutic agent may be, for example, auristatins, camptothecin, duocalmycins, etoposides, mytansins and mytansinoids, taxanes, benzodiazepines or benzodiazepine-containing drugs (e.g., pyrrolo[1,41-benzodiazepines (PBD), indolinobenzodiazepines, and oxazolidinobenzodiazepines) or vinca alkaloids.

[0127] The FN3 domain, which specifically binds to CD71, can be bound to a detectable label using known methods.

[0128] In some embodiments, the detectable label is complexed with a chelating agent.

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

[0130] Detectable labels, therapeutic compounds, or cytotoxic compounds can be directly or indirectly linked to the FN3 domain bound to CD71 using known methods. Suitable linkers are known in the art and include, for example, prosthetic groups, non-phenolic linkers (derivatives of N-succinimidyl benzoate; dodecaborate), chelates of both macrocyclic compounds and acyclic chelating agents, for example, derivatives of 1,4,7,10-tetraazacyclododecane-1,4,7,10,tetraacetic acid (DOTA), derivatives of diethylenetriaminepentaacetic acid (DTPA), derivatives of S-2-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), and derivatives of 1,4,8,11-tetraazacyclododecane-1,4,8,11-tetraacetic acid (TETA), N-succinimidyl- Examples include 3-(2-pyridyldithiol)propionate (SPDP), iminothiolane (IT), difunctional derivatives of imide esters (e.g., dimethylHCl adipoimoidate), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bis-azide compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (e.g., bis(p-diazoniumbenzoyl)ethylenediamine), diisocyanates (e.g., toluene 2,6-diisocyanate), and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene), and other chelated moieties. Suitable peptide linkers are well known.

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

[0132] Isolation of CD71-binding FN3 domains from Tencon sequence-based libraries Tencon (SEQ ID NO: 1) is a naturally occurring fibronectin type III (FN3) domain designed from a consensus sequence of 15 FN3 domains derived 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-chains characteristic of the FN3 domain, with these beta-chains referred to as A, B, C, D, E, F, and G, and the loops referred to as AB, BC, CD, DE, EF, and FG loops (Bork and Doolittle, Proc Natl Acad Sci USA 89:8990-8992, 1992; U.S. Patent No. 6,673,901). These loops, or selected residues within each loop, can be randomized to construct a library of fibronectin type III (FN3) domains that can be used to select novel molecules that bind to CD71. Table 1 shows the location and sequence of each loop and beta chain in Tencon (SEQ ID NO: 1).

[0133] Therefore, a library designed based on the Tencon sequence may have randomized FG loops, or randomized BC and FG loops, for example, library TCL1 or TCL2 as described below. The Tencon BC loop is 7 amino acid long, and therefore 1, 2, 3, 4, 5, 6 or 7 amino acids are diversified in the BC loop and randomized in a library designed based on the Tencon sequence. The Tencon FG loop is 7 amino acid long, and therefore 1, 2, 3, 4, 5, 6, or 7 amino acids can be diversified in the FG loop and randomized in libraries designed based on the Tencon sequence. Further diversity in the loop in the Tencon library can be achieved by insertion and / or deletion of residues in the loop. For example, the FG and / or BC loops can be extended by only 1 to 22 amino acids, or reduced by only 1 to 3 amino acids. While the FG loop in Tencon is 7 amino acid long, the corresponding loop in the antibody heavy chain ranges from 4 to 28 residues. To obtain maximum diversity, the FG loop can be diversified in length, corresponding to the sequence and even the 4 to 28 residue length range of the antibody CDR3. 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.

[0134] Libraries designed based on the Tencon sequence may also have randomized alternative surfaces that form on the side of the FN3 domain and may include two or more beta chains and at least one loop. One such alternative surface is formed by amino acids in the C and F beta chains, as well as the CD and FG loop (C-CD-F-FG surface). A library design based on the Tencon alternative C-CD-F-FG surface is described in U.S. Patent Publication 2013 / 0226834. Libraries designed based on the Tencon sequence also include libraries designed based on Tencon variants, e.g., Tencon variants having substitutions at residue positions 11, 14, 17, 37, 46, 73, or 86 (residue numbering corresponding to SEQ ID NO: 1), and these variants exhibit improved thermal stability. An example Tencon variant is described in U.S. Patent Publication 2011 / 0274623 and includes Tencon27 (SEQ ID NO: 4), which has substitutions E11R, L17A, N46V, and E86I when compared to Tencon SEQ ID NO: 1.

[0135] [Table 1]

[0136] Libraries based on Tencon and other FN3 sequences can be randomized at selected residue positions using random or predetermined sets of amino acids. For example, variants in a library with random substitutions can be generated using NNK codons that encode 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 simultaneously reducing the frequency of stop codons. Libraries of FN3 domains with an amino acid distribution biased towards the positions to be diversified can be synthesized, for example, using Slonomics® technology (http: / / www.sloning.com). This technology uses a library of pre-constructed double-stranded triplets that act as versatile building blocks sufficient for thousands of gene synthesis processes. The triplet library represents all possible sequence combinations necessary to construct any desired DNA molecule. Codon designations follow the well-known IUB code.

[0137] The FN3 domain, which specifically binds to CD71, can be isolated by producing an FN3 library, such as the Tencon library, using cis display to ligate a DNA fragment encoding a scaffold protein to a DNA fragment encoding RepA, thereby generating a pool of protein-DNA complexes formed in vitro after translation, in which each protein is stably bound to the DNA encoding it (U.S. Patent No. 7,842,476; Odegrip et al., Proc Natl Acad Sci USA 101, 2806-2810, 2004), and then assaying the library for specific binding to PSMA by any of the methods known in the art and described in the examples. Well-known methods that can be used include ELISA, Sandwich In. These include thymunoassays, as well as competitive and non-competitive assays (e.g., Ausubel et al., 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York). Identified FN3 domains that specifically bind to CD71 are further characterized for their binding to CD71, regulation of CD71 activity, internal translocation, stability, and other desirable features.

[0138] FN3 domains that specifically bind to CD71 can be generated by using any FN3 domain as a template to generate a library and then screening the library for molecules that specifically bind to CD71 using the method shown. Examples of FN3 domains that may be used are the third FN3 domain of tenascin C (TN3), Fibcon, and the tenth FN3 domain of fibronectin (FN10). Accordingly, PCT applications WO2010 / 051274, WO 2011 / 137319, and WO 2013 / 049275 are incorporated herein by reference in their entirety. Standard cloning and expression techniques are used to clone the library into a vector, to synthesize a double-stranded cDNA cassette of the library, and to express or translate the library in vitro. For example, ribosome displays (Hanes and Pluckthun, Proc Natl Acad Sci USA, 94, 4937-4942, 1997), mRNA displays (Roberts and Szostak, Proc Natl Acad Sci USA, 94, 12297-12302, 1997), or other cell-free systems (U.S. Patent No. 5,643,768) may be used. Libraries of FN3 domain variants may be expressed, for example, as fusion proteins presented on the surface of any suitable bacteriophage. Methods for presenting fusion polypeptides on the surface of bacteriophages are well known (U.S. Patent Publication 2011 / 0118144; International Patent Publication WO2009 / 085462; U.S. Patents 6,969,108; U.S. Patents 6,172,197; U.S. Patents 5,223,409; U.S. Patents 6,582,915; U.S. Patents 6,472,147).

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

[0140] In some embodiments, the FN3 protein or polypeptide binds to human CD71 at a site on CD71 that does not compete with transferrin binding to CD71. As used herein, a site on CD71 that does not compete with transferrin binding to CD71 refers to an epitope or portion of CD71 at which the binding of the FN3 protein does not compete with or inhibit the binding of transferrin to CD71. Competition or non-competition may be complete or partial. In some embodiments, the binding also does not inhibit the internal translocation of transferrin into the cell via its interaction with CD71.

[0141] In some embodiments, methods are provided for identifying FN3 proteins that bind to CD71 at a site that neither competes with nor inhibits transferrin binding to CD71. In some embodiments, the method includes contacting CD71 with a test FN3 protein in the presence of transferrin or a drug that binds to the CD71 transferrin binding site; and identifying a test FN3 protein that binds to CD71 in the presence of transferrin or a drug that binds to the CD71 transferrin binding site. In some embodiments, the method includes isolating a test FN3 protein that binds to CD71 in the presence of transferrin or a drug that binds to the CD71 transferrin binding site. In some embodiments, the method includes sequencing a test FN3 protein that binds to CD71 in the presence of transferrin or a drug that binds to the CD71 transferrin binding site. In some embodiments, the method includes producing or obtaining a nucleic acid sequence encoding a test FN3 protein that binds to CD71 in the presence of transferrin or a drug that binds to the CD71 transferrin binding site. In some embodiments, the method includes expressing a test FN3 protein that binds to CD71 in the presence of transferrin or a drug that binds to the CD71 transferrin binding site from a nucleic acid sequence encoding a test FN3 protein that binds to CD71 in the presence of transferrin or a drug that binds to the CD71 transferrin binding site. In some embodiments, the test FN3 protein is expressed in cells. In some embodiments, the method includes isolating and / or purifying the expressed test FN3 protein.

[0142] In some embodiments, FN3 proteins identified according to any of the methods provided herein are provided.

[0143] The FN3 domain, which specifically binds to CD71, can be modified to improve its properties, such as thermal stability and reversibility of thermal folding and unfolding. Several methods have been applied to increase the apparent thermal stability of proteins and enzymes, including rational design based on comparison with highly similar thermally stable sequences, design to stabilize disulfide crosslinks, mutations that increase alpha-helix tendency, manipulation of salt crosslinks, modification of protein surface charge, directional evolution, and construction 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 a cold chain in manufacturing. Residues that can be substituted to improve the thermal stability of Tencon (SEQ ID NO: 1) are at residue positions 11, 14, 17, 37, 46, 73, or 86, as described in U.S. Patent Publication 2011 / 0274623. Substitutions corresponding to these residues may be incorporated into the FN3 domain-containing molecules disclosed herein.

[0144] Measures of protein stability and protein instability can be assessed as the same or different aspects of protein integrity. Proteins are susceptible to or "unstable" to denaturation caused by heat, ultraviolet or ionizing radiation, ambient osmotic pressure and pH in liquid solutions, mechanical shear forces applied by small-pore filtration, ultraviolet radiation, ionizing radiation, e.g., gamma ray irradiation, chemical or thermal dehydration, or any other action or force that can cause protein structural breakdown. Molecular stability can be determined by standard methods. For example, molecular stability can be assessed by thermal melting ("T"). m The temperature, the Celsius temperature (°C) at which half of the molecule is unfolded, can be determined by measuring it using standard methods. Typically, T m The higher the temperature, the more stable the molecule. In addition to heat, the chemical environment also alters a protein's ability to maintain a specific three-dimensional structure.

[0145] In some embodiments, the FN3 domain that binds to CD71 is T m The increase may indicate an increase in stability 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, compared to the same domain before the operation.

[0146] Chemical denaturation can also be measured by various methods. Examples of chemical denaturation include guanidine hydrochloride, guanidine thiocyanate, urea, acetone, organic solvents (DMF, benzene, acetonitrile), and salts (ammonium sulfate, lithium bromide, lithium chloride, sodium bromide). Examples include solvents, calcium chloride, sodium chloride; reducing agents (e.g., dithiothreitol, beta-mercaptoethanol, dinitrothiobenzene, and hydrides, e.g., sodium boride); nonionic and ionic surfactants; acids (e.g., hydrochloric acid (HCl), acetic acid (CH3COOH), halogenated acetic acid); hydrophobic molecules (e.g., phospholipids); and targeted denaturants. The degree of denaturation may depend on functional properties, such as loss of ability to bind to target molecules, or physiological and chemical properties, such as tendency to aggregate, exposure of residues previously inaccessible to the solvent, or disruption or formation of disulfide bonds.

[0147] The FN3 domain that binds to CD71 may be generated as a monomer, dimer, or polymer, for example, as a means of increasing valence and, consequently, increasing the avidity of target molecule binding, or as a means of generating a bispecific or multiselective scaffold that simultaneously binds to two or more different target molecules. Dimers and polymers can be generated by linking single-characteristic, bispecific, or multiselective protein scaffolds, for example, by including an amino acid linker, such as a linker containing polyglycine, glycine and serine, or alanine and proline. Examples of 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), and (AP) 10 (Sequence ID 68), (AP) 20 Examples include (SEQ ID NO: 69) and A(EAAAK)5AAA (SEQ ID NO: 70). Dimers and polymers can be linked together in the N-to-C direction. The use of artificial peptide linkers, in addition to naturally occurring peptide linkers, to link polypeptides into novel linked fusion polypeptides is also well known in the literature (Hallewell et al., J Biol Chem 264, 5260-5268, 1989; Alfthan et al., Protein Eng. 8, 725-731, 1995; Robinson & Sauer, Biochemistry 35, 109-116, 1996; U.S. Patent No. 5,856,456).

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

[0149] All or part of the antibody constant region can be bound to the CD71-binding FN3 domain to confer antibody-like properties, particularly those related to the Fc region, such as Fc effector functions including C1q binding, complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, and downregulation of cell surface receptors (e.g., B cell receptors; BCRs), and can be further modified by altering the residues in Fc that are responsible for these activities (see Strohl, Curr Opin Biotechnol. 20, 685-691, 2009 for an overview).

[0150] For the desired properties, polyethylene glycol (PEG) molecules, such as PEG5000 or PEG20,000, fatty acids and fatty acid esters of various chain lengths, such as lauric acid, are used. Further moieties such as polylysine, octane, and carbohydrates (dextran, cellulose, oligosaccharides, or polysaccharides), including esters, myristic acid esters, stearate esters, arachidic acid esters, behenate esters, oleate esters, arachidonic acid esters, octanedioic acid, tetradecanedioic acid, octadecanedioic acid, and docosanedioic acid, can be incorporated into the FN3 domain that specifically binds to CD71. These moieties can be directed to fusion with sequences encoding protein scaffolds and can be generated by standard cloning and expression techniques. Alternatively, these moieties can be conjugated to recombinant-produced molecules disclosed herein using well-known chemical coupling methods.

[0151] The pegylated portion can be added to the CD71-binding FN3 domain, for example, by incorporating a cysteine ​​residue into the C-terminus of the molecule, or by manipulating the cysteine ​​to a residue position oriented away from the CD71-binding plane of the molecule, and then binding the pegylated group to the cysteine ​​using a known method.

[0152] The FN3 domain, which specifically binds to CD71 with additional components incorporated, can be compared for functionality using several well-known assays. For example, the altered properties resulting from the incorporation of the Fc domain and / or Fc domain variants can be assayed 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 that measure ADCC or CDC, for example, or by evaluating the pharmacokinetic properties of the molecules disclosed herein in an in vivo model.

[0153] Polynucleotides, vectors, host cells In some embodiments, nucleic acids encoding the FN3 domain that specifically binds to CD71 are provided, either as isolated polynucleotides, as part of an expression vector, or as part of a linear DNA sequence, such as linear DNA sequences, compositions, or vectors compatible with prokaryotic, eukaryotic, or filamentous phage expression, secretion, and / or display, used for in vitro transcription / translation. However, specific example polynucleotides are disclosed herein, and other polynucleotides encoding the FN3 domain disclosed herein are also within the scope of this disclosure, taking into account the degeneracy of the genetic code or codon priority in a given expression system.

[0154] In some embodiments, the isolated polynucleotides encode an FN3 domain that specifically binds to CD71, comprising the amino acid sequence of SEQ ID NOs: 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, or 81-309.

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

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

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

[0158] In some embodiments, host cells containing the vector are provided. The FN3 domain, which specifically binds to CD71, may be produced by cell lines, mixed cell lines, immortalized cells, or clonal populations of immortalized cells, as is known in the art. See, for example, Ausubel et al., *Current Protocols in Molecular Biology*, John Wiley & Sons, Inc., NY, NY (1987-2001); Sambrook et al., *Molecular Cloning: A Laboratory Manual*, 2nd edition, Cold Spring Harbor, NY (1989); Harlow and Lane, *Antibodies*, a Laboratory Manual, Cold Spring Harbor, NY (1989); Colligan et al., *Current Protocols in Immunology*, John Wiley & Sons, Inc., NY (1994-2001); and Colligan et al., *Current Protocols in Protein Science*, John Wiley & Sons, NY, NY (1997-2001).

[0159] The host cells selected for expression may be of mammalian origin, or may be selected from COS-1, COS-7, HEK293, BHK21, CHO, BSC-1, He G2, SP2 / 0, HeLa, myeloma, lymphoma, yeast, insect or plant cells, or any derivative thereof, immortalized or transformed cells. Alternatively, the host cells may be selected from species or organisms incapable of glycosylation of polypeptides, such as prokaryotic cells or prokaryotes like BL21, BL21(DE3), BL21-GOLD(DE3), XL1-Blue, JM109, HMS174, HMS174(DE3), and from any natural or engineered Escherichia coli (E. coli) species, Klebsiella species, or Pseudomonas species strains.

[0160] A method for producing an isolated FN3 domain that binds to CD71, comprising culturing isolated host cells under conditions such that an isolated FN3 domain that binds to CD71 is expressed in some embodiments, and purifying the FN3 domain.

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

[0162] Anti-idiotype antibodies In some embodiments, anti-idiotype antibodies bind to the FN3 domain.

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

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

[0165] The kit can be used for therapeutic purposes and as a diagnostic kit.

[0166] In some embodiments, the kit includes a CD71-binding FN3 domain and a reagent for detecting the FN3 domain. In some embodiments, the kit includes a divalent FN3 domain. The kit may include one or more other elements, including: instructions for use; other reagents, e.g., agents useful for labeling, chelation or other coupling methods, radioprotective compositions; devices or other materials for preparing the CD71-binding FN3 domain for administration for imaging, diagnostic or therapeutic purposes; a pharmaceutically acceptable carrier; and other devices or materials for administration to a subject.

[0167] In some embodiments, the kit includes an FN3 domain that binds to CD71, comprising one of the amino acid sequences from SEQ ID NOs. 33 to 50.

[0168] In some embodiments, the kit includes an FN3 domain that binds to CD71, comprising one of the amino acid sequences from SEQ ID NOs. 51 to 61.

[0169] In some embodiments, the kit includes a CD71-binding FN3 domain containing one amino acid sequence from SEQ ID NOs. 81-309.

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

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

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

[0173] In some embodiments, methods are provided for diagnosing subjects with tissue cancer or those at risk of developing tissue cancer based on the expression of CD71 by cancer tissue cells, for predicting the success of immunotherapy, for determining prognosis, and for treatment.

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

[0175] In some embodiments, CD71 cells are cells involved in CNS diseases, inflammatory / immune diseases, such as cerebral stenosis (MS) and infectious diseases.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0197] In some embodiments, the solid tumor is esophageal or gastrointestinal cancer.

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

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

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

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

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

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

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

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

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

[0207] In some embodiments, the hematological malignancy is lymphoma, myeloma, or leukemia. In some embodiments, the hematological malignancy is B-cell lymphoma. In some embodiments, the hematological malignancy is Burkitt lymphoma. In some embodiments, the hematological malignancy is Hodgkin lymphoma. In some embodiments, the hematological malignancy is non-Hodgkin lymphoma.

[0208] In some embodiments, hematological malignancies are myelodysplastic syndromes.

[0209] 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 myelomonocytic leukemia (CMML).

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

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

[0212] In some embodiments, the compositions or pharmaceutical compositions provided herein may be administered alone or in combination with other therapeutic agents, i.e., simultaneously or sequentially. In some embodiments, other or further therapeutic agents are other antitumor agents or therapeutic agents. Different tumor types and tumor stages may require the use of various adjunct compounds useful for treating cancer. For example, the compositions provided herein may be used in combination with various chemotherapeutic agents such as taxol, tyrosine kinase inhibitors, leucovorin, fluorouracil, irinotecan, phosphatase inhibitors, and MEK inhibitors, among others. The compositions may also be used in combination with drugs that modulate the immune response against tumors, such as anti-PD-1 or anti-CTLA-4, among others. Further treatments may be immune-modulating agents, such as antibodies targeting PD-1 or PD-L1.

[0213] In some embodiments, FN3 domains or their conjugates that specifically bind to CD71, which can be used to diagnose, monitor, regulate, treat, alleviate, prevent or reduce the onset of symptoms or specific pathological conditions of human diseases in cells, tissues, organs, fluids, or generally in the host, also exhibit properties that allow them to cross the blood-brain barrier. The blood-brain barrier (BBB) ​​prevents most macromolecules (e.g., DNA, RNA, and polypeptides) and many small molecules from entering the brain. The BBB is mainly composed of specialized endothelial cells with highly restrictive, tight junctions, and as a result, the passage of substances of all sizes 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 (e.g., brain cancer) difficult because many therapeutic agents cannot be delivered across the BBB with the desired efficacy. Further conditions involving BBB disruption include: stroke, diabetes, epileptic seizures, hypertensive encephalopathy, acquired immunodeficiency syndrome, traumatic brain injury, multiple sclerosis, Parkinson's disease (PD), and Alzheimer's disease. This ability is particularly useful for treating brain cancers, which include, for example: astrocytoma, medulloblastoma, glioma, ependymoma, germ cell tumor (pineal glandoma), glioblastoma multiforme, oligodendroglioma, schwannoma, retinoblastoma, and congenital tumors; or cancers of the spinal cord, such as neurofibroma, meningioma, glioma, and sarcoma. In certain embodiments, it is specific to CD71 containing one amino acid sequence from SEQ ID NOs: 33-50. FN3 domains or their conjugates that bind to CD71 are useful, for example, for delivering therapeutic agents or cytotoxic drugs across the blood-brain barrier. In certain embodiments, FN3 domains or their conjugates that specifically bind to CD71 containing one of the amino acid sequences from SEQ ID NOs. 51-61 are useful, for example, for delivering therapeutic agents or cytotoxic drugs across the blood-brain barrier. In some embodiments, this protein is used for SEQ ID NOs. 146, 214, 104, 259, 134, 92, 302, 235, 237, 152, 238, 136, 197, 212, 296, 226, 261, 307, 115, 112, 278, 297, 96, 222, 95, 233, 217, 252, 194, Includes sequences of 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.

[0214] In some embodiments, polypeptides that can facilitate the transport of therapeutic agents across the blood-barrel barrier are proteins containing the sequences of SEQ ID NOs: 146, 214, 104, 259, 134, 92, 302, 235, 237, 152, 238, 136, 197, 212, 296, 226, 261, 307, 115, 112, 278, 297, 96, 222, 95, 233, 217, 252, 194, 164, 168, 174, 190, 257, 303, 284, 85, or 149.

[0215] "To treat" or "treatment" refers to therapeutic and preventive measures, the purpose of which is to prevent or slow (reduce) undesirable physiological changes or impairments, such as the development or spread of cancer. In some embodiments, beneficial or desirable clinical outcomes include, but are not limited to, symptom relief, reduction in disease severity, stabilization (i.e., non-worsening) of the disease, delay or slowing of disease progression, recovery or mitigation of the disease state, and remission (partial or complete), whether detectable or undetectable. "Treatment" may also mean extending survival compared to the survival predicted without treatment. Those requiring treatment include those who already have a condition or impairment, those who are prone to developing a condition or impairment, or those seeking to prevent a condition or impairment.

[0216] The "therapeutic effective dose" refers to the amount of medication that is effective in achieving the desired therapeutic outcome in the required dosage and duration. The therapeutic effective dose of FN3 domains that specifically bind to CD71 can vary depending on factors such as the individual's disease state, age, sex, and weight. Examples of indicators of effective CD71-binding FN3 domains include improved patient health, reduction or shrinkage of tumor size, cessation or slowed tumor growth, and / or absence of metastasis of cancer cells to other locations in the body.

[0217] Administration / Pharmaceutical Composition In some embodiments, pharmaceutical compositions are provided comprising a CD71-specifically binding FN3 domain, optionally conjugated to a detectable label, therapeutic agent, or cytotoxic agent, and a pharmaceutically acceptable carrier, as disclosed herein. For therapeutic use, the CD71-specifically binding FN3 domain may be prepared as a pharmaceutical composition containing an effective amount of the domain or molecule as the active ingredient in a pharmaceutically acceptable carrier. "Carrier" refers to a diluent, adjuvant, excipient, or vehicle administered together with the active compound. Such vehicles may be liquids such as water and oil, which may be of petroleum, animal, plant, or synthetic origin, such as peanut oil, soybean oil, mineral oil, or sesame oil. For example, 0.4% saline and 0.3% glycine may 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 is necessary to approximate a physiological state. Depending on the circumstances, pharmaceutically acceptable auxiliary substances may be included, such as pH adjusters and buffers, stabilizers, thickeners, lubricants, and colorants. 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 mass, and is selected according to the specific dosage form chosen, mainly based on the required dose, fluid volume, viscosity, etc. Suitable vehicles and formulations include other human proteins, such as human serum albumin, e.g., Remington: See The Science and Practice of Pharmacy, 21st edition, edited by Troy, DB, Lipincott Williams and Wilkins, Philadelphia, PA 2006, Part 5, Pharmaceutical Manufacturing, pp. 691–1092, in particular pp. 958–989.

[0218] The modes of administration for the therapeutic use of the FN3 domain disclosed herein may be any suitable route for delivering the drug to the host, as is well known in the art, e.g., parenteral administration, e.g., intradermal, intramuscular, intraperitoneal, intravenous or subcutaneous, transpulmonary; transmucosal administration (oral, intranasal, intravaginal, rectal) using formulations in the form of tablets, capsules, liquids, powders, gels, or particles; and contained in syringes, implantable devices, osmotic pumps, cartridges, micropumps; or other means as understood by those skilled in the art. Site-specific administration can be achieved, for example, by intra-articular, intra-bronchial, intra-abdominal, intra-sacral, intra-cartilage, intracavitary, intra-intracelial, intra-cerebellar, intraventricular, intra-colon, intra-cervical, intra-cervical, intra-cervical, intra-cervical, intra-cervical, intra-pulmonary, intra-rectal, intra-renal, intra-retinal, intra-spinal cord, intra-synovial sac, intra-thoracic, intra-uterine, intravascular, intra-bladder, intra-focal, intra-vaginal, rectal, buccal, sublingual, intra-nasal, or percutaneous delivery.

[0219] Pharmaceutical compositions may be supplied as kits comprising a container for the pharmaceutical compositions described herein. Pharmaceutical compositions may be provided, for example, in the form of an injectable liquid for single or multiple doses, or as a sterile powder to be reconstituted before injection. Alternatively, such kits may include a dry powder disperser, a liquid aerosol generator, or a sprayer for administering the pharmaceutical composition. Such kits may further include written information regarding the indications and uses of the pharmaceutical composition. [Examples]

[0220] The following examples are useful for illustrating the embodiments disclosed herein. These examples are provided for illustrative purposes only, and the embodiments should not be construed in any way as being limited to these examples, but rather as encompassing any and all variations that are evident as a result of the teachings provided herein. Those skilled in the art will readily understand the various non-essential parameters that may be changed or modified to obtain essentially similar results.

[0221] Example 1. Construction of a Tencon library containing a randomization loop Tencon (SEQ ID NO: 1) is a fibronectin type III (FN3) domain of an immunoglobulin-like scaffold designed from the consensus sequences of 15 FN3 domains derived 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 loops exposed on six surfaces connecting seven beta strands. These loops, or selected residues within each loop, can be randomized to construct a library of fibronectin type III (FN3) domains that can be used to select novel molecules that bind to specific targets.

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

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

[0224] Construction of the TCL1 library for use with cis display was achieved by performing PCR in multiple successive rounds to generate two halves of the final linear double-stranded DNA molecule; the 5' fragment contains the promoter and Tencon sequence, while the 3' fragment contains the repA gene as well as the cis and ori elements. These two halves were combined by restriction digestion to generate the whole construct. The TCL1 library was designed to incorporate random amino acids only into the FG loop of Tencon. The NNS codon was used in the construction of this library, resulting in the possible incorporation of all 20 amino acids and one stop codon into the FG loop. The TCL1 library contains six separate sublibraries, each having a different randomized FG loop length of 7-12 residues to further increase the diversity.

[0225] TCL1 library (SEQ ID NO: 2) LPAPKNLVVSEVTEDSLRLSWTAPDAAFDSFLIQYQESEKVGEAINLTVPGSERSYDLTGLKPGTEYTVSIYGVX 7-12 PLSAEFTT; Here X1, X2, X3, X4, X5, X6, X7 are any amino acids; and X8, X9, X 10 , X 11 and X 12 It is either any amino acid or it is deleted.

[0226] Building a TCL2 Library We constructed a TCL2 library in which both the BC and FG loops of Tencon were randomized and the amino acid distribution at each position was strictly controlled. Table 2 shows the amino acid distribution at the desired loop positions in the TCL2 library. The designed amino acid distribution had two objectives. First, based on analysis of the Tencon crystal structure and / or homology modeling, we biased the library to residues predicted to be structurally important for Tencon folding and stability. For example, since the residue at position 29 is embedded in the hydrophobic core of the Tencon fold, we fixed position 29 to only a subset of hydrophobic amino acids. The second layer of the design involved biasing the amino acid distribution to the amino acid distribution of residues preferentially found in the heavy chain HCDR3 of the antibody to efficiently generate a high-affinity binder (Birtalan et al., J Mol Biol 377:1518-). 28, 2008; Olson et al., Protein Sci 16:476-84, 2007). For this purpose, 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 lacks methionine, cysteine, and stop codons.

[0227] TCL2 Library (SEQ ID NO: 3) LPAPKNLVVSEVTEDSLRLSWX1X2X3X4X5X6X7X8SFLIQYQESEKVGEAINLTVPGSERSYDLTGLKPGTEYTVSIYGVX9X 10 X 11 X 12 X 13 SX 14 X 15 LSAEFTT; Here, X1 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr, or Val; X2 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr, or Val; X3 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr, or Val; X4 is Ala, Arg, As X5 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr, or Val; X6 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr, or Val; X7 is Phe, Ile, Leu, Val, or Tyr; X8 is Asp, Glu, or Thr; X9 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr, or Val; X 10 These are Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr, or Val; X 11These are Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr, or Val; X 12 These are Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr, or Val; X 13 These are Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr, or Val; X 14 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr, or Val; and X 15 These are Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.

[0228] [Table 2]

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

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

[0231] TCL7 (Randomized FG and BC Loop) (SEQ ID NO: 5) LPAPKNLVVSRVTEDSARLSWX1X2X3X4X5X6X7X8X9FDSFLIQYQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVX 10 X 11 X 12 X 13 X 14 X 15 X 16 X 17 X 18 X 19 SNPLSAIFTT; Here, 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 X7, X8, X9, X 17 , X 18 and X 19 is either A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, Y, or is missing.

[0232] TCL9 (Randomized FG Loop) (SEQ ID NO: 6) LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFLIQYQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIYGV X1X2X3X4X5X6X7X8X9X 10 X 11 X 12 SNPLSAIFTT; 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 X 8、 X 9、 X 10、 X 11 and X 12 is either A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, Y, or is missing.

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

[0234] Building an FG Loop Library (TCL9) A set of synthetic DNA molecules was prepared consisting of the 5'Tac promoter and the complete Tencon gene sequence with randomized codons removed from the subsequent FG loop (SEQ ID NOs. 26-31). For FG loop randomization, all amino acids except cysteine ​​and methionine were coded in equal percentages. The lengths of the diversified portions were varied so that they coded 7, 8, 9, 10, 11, or 12 amino acids in the FG loop. Sublibraries of each length variation were synthesized individually on a 2ug scale and then amplified by PCR using oligonucleotides Sloning-FOR (SEQ ID NO: 9) and Sloning-Rev (SEQ ID NO: 10).

[0235] The 3' fragment of the library is a constant DNA sequence containing elements for display, including the PspOMI restriction site, the coding region of the repA gene, and cis and ori elements. This fragment was amplified by PCR using plasmid (pCR4Blunt) (Invitrogen) as a template with M13 forward and M13 reverse primers. The resulting PCR product was digested overnight with PspOMI and then gel-purified. To ligate the 5' portion of the library DNA to the 3' DNA containing the repA gene, 2 pmol (approximately 540 ng-560 ng) of 5' DNA was ligated overnight at 37°C in the presence of NotI and PspOMI enzymes and T4 ligase to equimolar concentration (approximately 1.25 μg) of 3' repA DNA. The ligated library product was amplified by 12 cycles of PCR using oligo-POP2250 (SEQ ID NO: 11) and DigLigRev (SEQ ID NO: 12). For each sublibrary, the DNA obtained as a result of 12 PCR reactions was combined and purified using a Qiagen spin column. The yield of each TCL9 sublibrary ranged from 32 to 34 μg.

[0236] Building the FG / BC Loop Library (TCL7) The TCL7 library contains randomized Tencon BC and FG loops. The library comprises BC loops of 6–9 amino acid length combinatorially mixed with randomized FG loops of 7–12 amino acid length. Synthetic Tencon fragments BC6, BC7, BC8, and BC9 (sequences 13–16, respectively) were prepared to contain the Tencon gene encoding up to residue VX, including residue VX, in the N-terminal portion of the protein, so that the BC loop is replaced with one of 6, 7, 8, or 9 randomized amino acids, which is represented by the string "N" in the sequences provided herein. These fragments were synthesized prior to the discovery of the L17A, N46V, and E83I mutations (CEN5243), which were introduced in the molecular biology steps described below. The following steps were taken to combine these fragments with the fragments encoding the randomized FG loops.

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

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

[0239] The digested BC and FG fragments were ligated together in a single step using a three-way ligation method. Four ligation reactions were set up with 16 possible combinations, and each ligation reaction combined two BC loop lengths and two FG loop lengths. Each ligation contained approximately 300 ng of total BC fragment and 300 ng of FG fragment. These four ligation pools were then amplified by PCR using oligoPOP2222 (SEQ ID NO: 24) and SDG28 (SEQ ID NO: 25). Next, 7.5 μg of each reaction product was digested with Not1 and purified using a Qiagen PCR purification column. 5.2 μg of this DNA was ligated to an equimolar amount of RepA DNA fragment (approximately 14 μg), digested with PspOMI, and the product was amplified by PCR using oligoPOP2222.

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

[0241] Tencon and other FN3 domains contain two sets of CDR-like loops on opposite sides of the molecule; the first set is formed by the BC, DE, and FG loops, and the second set is formed by the AB, CD, and EF loops. The two sets of loops are separated by a beta chain that forms the center of the FN3 structure. If the image of Tencon is rotated 90 degrees, another surface may be visualized. This slightly recessed surface is formed by the CD and FG loops, as well as two antiparallel beta chains, the C and F beta chains, and is referred to herein as the C-CD-F-FG surface. The C-CD-F-FG surface can be used as a template for designing a library of protein scaffold interaction surfaces by randomizing a subset of residues that form the surface. The beta chain has a repeating structure in which the side chains of all other residues are exposed on the surface of the protein. Thus, a library can be constructed by randomizing some or all of the surface-exposed residues in the beta chain. By selecting appropriate residues in the beta chain, the compromise on the intrinsic stability of the Tencon scaffold should be minimized while providing a unique scaffold surface for interaction with other proteins.

[0242] Library TCL14 (sequence number 7) was designed as Tencon27 scaffolding (sequence number 4).

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

[0244] TCL14 library (sequence number 7): LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFX1IX2YX3EX4X5X6X7GEAIVLTVPGSERSYDLTGLKPGTEYX8VX9IX 10 GVKGGX 11 X 12 SX 13 PLSAIFTT; Here, X1, X2, X3, X4, X5, X6, X7, X8, X9, X 10 , X11 , X 12 and X 13 These are A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, Y, C, or M.

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

[0246] Thirteen positions in Tencon were selected for randomization: L32, Q34, and Q36 in the C chain; S38, E39, K40, and V41 in the CD loop; T68, S70, and Y72 in the F chain; and H78, R79, and N81 in the FG loop. In the C and F chains, S30 and E66 are located just beyond the CD and FG loops, and Since they do not appear to be part of the C-CD-F-FG surface, they were not randomized. For the CD loop, glycine, which provides mobility, may be beneficial in the loop region, and since E43 is in the surface junction, G42 and E43 were not randomized. For the FG loop, K75, G76, G77, and S80 were excluded. Although glycine was excluded for the reasons mentioned above, careful examination of the crystal structure revealed that S80 helps to form a stable FG loop by making key contact with the core. K75 is oriented away from the surface of the C-CD-F-FG surface and was not a very attractive candidate for randomization. The above residues were not randomized in the original TCL14 design, but they may be included in subsequent library designs for novel selection or to generate further diversity, for example, for affinity-mature libraries for specific hits of selected TCL14 targets.

[0247] Following the production of TCL14, three further Tencon libraries of a similar design were produced. These two libraries, TCL19, TCL21, and TCL23, were randomized at the same positions as TCL14 (see above), but with altered amino acid distributions at these positions (Table 3). TCL19 and TCL21 were designed to contain an equal distribution of 18 native amino acids (5.55% each) at all positions, excluding only cysteine ​​and methionine. TCL23 was designed so that each randomization position approximated the amino acid distribution found in the HCDR3 loop of a functional antibody, as shown in Table 3 (Birtalan et al., J.Mol.Biol.377: 1518-1528, 2008). Similar to the TCL21 library, cysteine ​​and methionine were excluded.

[0248] A third additional library was constructed to expand the potential target binding surface of the other libraries. In this library, TCL24, four additional Tencon positions were randomized compared to libraries TCL14, TCL19, TCL21, and TCL23. These positions include N46 and T48 from the D chain, and S84 and I86 from the G chain. The side chains of residues at positions 46, 48, 84, and 86 are surface-exposed from the β-chains D and G, and are structurally adjacent to the randomized positions on the C and F chains, thus increasing the accessible surface area for binding to target proteins; these residues were therefore specifically selected. The amino acid distribution used at each position for TCL24 was the same as that described for TCL19 and TCL21 in Table 3.

[0249] TCL24 Library (SEQ ID NO: 8) LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFX1IX2YX3EX4X5X6X7GEAIX8LX9VPGSERSYDLTGLKPGTEYX 10 VX 11 IX 12 GVKGGX 13 X 14 SX 15 PLX 16 AX 17 FTT; Here, X1, X2, X3, X4, X5, X6, X 10 , X 11 , X 12 , X 13、 X 14 , X 15 , X 16 and X 17 These are A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, Y, or W.

[0250] [Table 3]

[0251] Generation of TCL21, TCL23, and TCL24 libraries The TCL21 library was generated using Colibra library technology (Isogenica) to control the amino acid distribution. The TCL19, TCL23, and TCL24 gene fragments were generated using Slonomics technology (Morphosys) to control the amino acid distribution. Each library was amplified using PCR after the 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 loop library.

[0252] Example 3: Selection of fibronectin type III (FN3) domains that bind to CD71 Panning and biochemical screening Human CD71-specific FN3 domains were selected by CIS display (Odegrip et al., 2004) using recombinant biotinylated CD71 extracellular domains with an N-terminal 6His tag (Sino Biologics). For in vitro transcription and translation (ITT), 3 μg of DNA from FN3 domain libraries TCL18, TCL19, TCL21, TCL23, and TCL24 was 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 a series of rounds of further panning. Between each round, the concentration of target CD71 was successively increased from 400 nM to 100 nM. High-affinity binders were isolated by reducing the concentration and increasing the washing stringency. The output from the fifth round of panning was subjected to four further off-rate selections. The biotinylated target antigen concentration was reduced from 25 nM in rounds 6 and 7 to 2.5 nM in rounds 8 and 9.

[0253] Following panning, the genes encoding the selected FN3 domains were amplified by PCR, subcloned into a modified pET vector containing a ligase-independent cloning site, and transformed into BL21(DE3)(Stratagene) cells for soluble expression in Escherichia coli (E. coli) using standard molecular biology techniques. Gene sequences encoding the C-terminal polyhistidine tag were added to each FN3 domain to enable purification and detection.

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

[0255] [Table 4]

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

[0257] [Table 5]

[0258] High-throughput expression and conjugation The identified clones were grown in 24-well deep block plates in a double 5 mL culture. Briefly, they were cultured in 5 mL of TB medium supplemented with 50 μg / mL kanamycin. 150 μL of culture was seeded overnight into each well and grown at 37°C for approximately 3 hours with shaking at 220 rpm (OD600 approx. 1). The culture was induced using IPTG and further incubated at 37°C for 4 hours at 220 rpm to a final concentration of 1 mM. The bacterial pellet was collected by centrifugation at 2250xg for 15 minutes. 600 μL / well BugBuster HT (Novagen) with 0.2 mg / mL lysozyme (Sigma) was added to each well; the pellet was separated by pipette and then vigorously shaken on a shaking platform for approximately 30 minutes until the pellet dissolved. The plate was rotated at 2250xg for 15 minutes to clear the lysate, and two 600 μL aliquots were combined for each sample. His-tagged FN3 domains were purified using a HisTrap plate (GE) according to the manufacturer's instructions, followed by replacement of the buffer with TBS using a Zeba Spin 7K desalting plate (Thermo Scientific). Protein concentration was assessed by Nanodrop. For binding to GlyGly-VC-MMAF, the FN3 domain (30 μM) was mixed with 150 μM GlyGlyVC-MMAF (Concortis) and 1 μM Sortase A to a total volume of 200 μL. Binding was allowed to proceed at room temperature for 1.5 hours, followed by a GE Healthcare 96-well His Multitrap. The solution was repurified using an HP plate according to the manufacturer's instructions. Buffer exchange to PBS was performed using a Zeba desalting plate, followed by sterile filtration using a Multiscreen HTS GV plate (Durapore) with centrifugation at 3000xg for 2 minutes. The concentration was assessed using Nanodrop.

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

[0260] Combination of selected clones by dose-response ELISA Selected clones were analyzed by ELISA to determine the EC50 value for binding. Briefly, Maxisorb plates were coated with streptavidin at 5 μg / ml overnight at 4C. The plates were then blocked at room temperature for 1 hour using a Starting Block (ThermoFisher) and washed with a TBS-Tween. Biotinylated CD71 (2 μg / ml) was captured onto streptavidin plates, and serially diluted centyrins were added to appropriate wells at room temperature for 1 hour. After washing, bound centyrins were detected using HRP and anti-V5 tagged antibodies conjugated to POD substrates and a luminescent plate reader. The luminescence values ​​were plotted as a function of concentration and fitted to the dose-response using PRISM to determine the EC50 value for binding.

[0261] Identification of the internally translocated FN3 domain via toxin conjugates. The FN3 domain was conjugated to the cytotoxic tubulin inhibitor monomethyl auristatin F (MMAF) via an enzymatically cleavable Val-Cit linker or an incleavable PEG4 linker (VC-MMAF) using the method described for NEM conjugation. Cell toxicity was assessed by measuring the viability of SKBR-3 cells after exposure to the cysteine ​​variant-cytotoxic conjugate. Cells were plated in 50 μL / well of phenol red RPMI medium (Gibco, 11875093) containing 10% fetal bovine serum (Gibco) at a rate of 3000 cells / well in a white well, opaque bottom, tissue culture treated plate (Fisher, PI15042). The cells were attached overnight at 37°C under a humidified 5% CO2 atmosphere. The cells were treated with 25 μL of fresh medium and 25 μL of 4× inhibitor prepared in fresh medium. Cell viability was determined at 72 hours by an endpoint assay using Cell TiterGlo (Promega). IC50 values ​​were determined by fitting the data to a sigmoid dose-response equation using a variable gradient with GraphPad Prism (GraphPad Software). The results are shown in Table 6, demonstrating that the FN3 domain binding to CD71 underwent internal translocation and was cytotoxic.

[0262] [Table 6]

[0263] Bivalent FN3 protein The bivalent FN3 protein is synthesized using two FN3 domains linked by a 4-repeat G / S linker. The bivalent FN3 protein was bound to VC-MMAF as described and evaluated for cytotoxicity in SK-BR3 cells. The IC50 value for the bivalent molecule was found to be superior.

[0264] Competition for transferrin binding and internal migration The FN3 domain VCMMAF conjugate was screened for competition with human transferrin using the cytotoxic assay described above. The FN3 domain was screened in the absence or presence of 0.6 μM holo-human transferrin (T0665-100MG). Table 7 shows the IC50 values ​​for the FN3 domain toxin conjugates screened in SK-BR3 cells in the absence or presence of the competitor.

[0265] [Table 7]

[0266] Array 1 = Original Tencon array LPAPKNLVVSEVTEDSLRLSWTAPDAAFDSFLIQYQESEKVGEAINLTVPGSERSYDLTGLKPGTEYTVSIYGVKGGHRSNPLSAEFTT

[0267] Sequence ID 2 = TCL1 Library LPAPKNLVVSEVTEDSLRLSWTAPDAAFDSFLIQYQESEKVGEAINLTVPGSERSYDLTGLKPGTEYTVSIYGV(X) 7-12 PLSAEFTT; Here, X1, X2, X3, X4, X5, X6, X7 are any amino acids; and X8, X9, X 10 , X 11 and X 12 It is either any amino acid or a missing one.

[0268] Sequence ID 3 = TCL2 Library LPAPKNLVVSEVTEDSLRLSWX1X2X3X4X5X6X7X8SFLIQYQESEKVGEAINLTVPGSERSYDLTGLKPGTEYTVSIYGVX9X 10 X 11 X 12 X 13 SX 14 X 15LSAEFTT; Here, X1 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr, or Val; X2 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr, or Val; X3 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr, or Val; X4 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr, or Val; X5 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr, or Val; X6 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr, or Val; X7 is Phe, Ile, Leu, Val, or Tyr; X8 is Asp, Glu, or Thr; X9 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr, or Val; X 10 These are Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr, or Val; X 11 These are Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr, or Val; X 12 These are Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr, or Val; X13 These are Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr, or Val; X 14 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr, or Val; and X 15 These are Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.

[0269] Sequence ID 4 = Stabilized Tencon LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFLIQYQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVKGGHRSNPLSAIFTT

[0270] Sequence ID 5 = TCL7 (FG and BC loops) LPAPKNLVVSRVTEDSARLSWX1X2X3X4X5X6X7X8X9FDSFLIQYQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVX 10 X 11 X 12 X 13 X 14 X 15 X 16 X 17 X 18 X 19 SNPLSAIFTT; Here, 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 X7, X8, X9, X 17, X 18 and X 19 is either A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, Y, or is missing.

[0271] Sequence ID 6 = TCL9 (FG loop) LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFLIQYQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIYGV X1X2X3X4X5X6X7X8X9X 10 X 11 X 12 SNPLSAIFTT; Here, 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 X 8、 X 9、 X 10、 X 11 and X 12 is either A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, Y, or is missing.

[0272] TCL14 Library (SEQ ID NO: 7) LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFX1IX2YX3EX4X5X6X7GEAIVLTVPGSERSYDLTGLKPGTEYX8VX9IX 10 GVKGGX 11 X 12 SX 13 PLSAIFTT; Here, X1, X2, X3, X4, X5, X6, X7, X8, X9, X 10 , X 11 , X 12 and X 13 These are A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, Y, C, or M.

[0273] TCL24 Library (SEQ ID NO: 8) LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFX1IX2YX3EX4X5X6X7GEAIX8LX9VPGSERSYDLTGLKPGTEYX 10 VX 11 IX 12 GVKGGX 13 X 14 SX 15 PLX 16 AX 17 FTT; Here, X1, X2, X3, X4, X5, X6, X 10 , X 11 , X 12 , X 13、 X 14 , X 15 , X 16 and X 17 These are A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, Y, or W.

[0274] Sequence ID 9 = Sloning-FOR GTGACACGGCGGTTAGAAC

[0275] Sequence ID 10 = Sloning-REV GCCTTTGGGAAGCTTCTAAG

[0276] Sequence ID 11 = POP2250 CGGCGGTTAGAACGCGGCTACAATTAATAC

[0277] Sequence ID 12 = DigLigRev CATGATTACGCCAAGCTCAGAA

[0278] Sequence ID 13 = BC9 GTGACACGGCGGTTAGAACGCGGCTACAATTAATACATAACCCCATCCCCCTGTTGACAATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGGATCTACCATGCTGCCGGCGCCGAAAAACCTGGTTGTTTCTGAAGTTACCGAAGACTCTCTGCGTCTGTCTTGGNNNNNNNNNNNNNNNNNNNNNNNNNNNTTYGACTCTTTCCTGATCCAGTACCAGGAATCTGAAAAAGTTGGTGAAGCGATCAACCTGACCGTTCCGGGTTCTGAACGTTCTTACGACCTGACCGGTCTGAAACCGGGTACCGAATACACCGTTTCTATCTACGGTGTTCTTAGAAGCTTCCCAAAGGC (where N is any base)

[0279] Sequence ID 14 = BC8 GTGACACGGCGGTTAGAACGCGGCTACAATTAATACATAACCCCATCCCCCTGTTGACAATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGGATCTACCATGCTGCCGGCGCCGAAAAACCTGGTTGTTTCTGAAGTTACCGAAGACTCTCTGCGTCTGTCTTGGNNNNNNNNNNNNNNNNNNNNNNNNTTYGACTCTTTCCTGATCCAGTACCAGGAATCTGAAAAAGTTGGTGAAGCGATCAACCTGACCGTTCCGGGTTCTGAACGTTCTTACGACCTGACCGGTCTGAAACCGGGTACCGAATACACCGTTTCTATCTACGGTGTTCTTAGAAGCTTCCCAAAGGC (where N is any base)

[0280] Sequence ID 15 = BC7 GTGACACGGCGGTTAGAACGCGGCTACAATTAATACATAACCCCATCCCCCTGTTGACAATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGGATCTACCATGCTGCCGGCGCCGAAAAACCTGGTTGTTTCTGAAGTTACCGAAGACTCTCTGCGTCTGTCTTGGNNNNNNNNNNNNNNNNNNNNNTTYGACTCTTTCCTGATCCAGTACCAGGAATCTGAAAAAGTTGGTGAAGCGATCAACCTGACCGTTCCGGGTTCTGAACGTTCTTACGACCTGACCGGTCTGAAACCGGGTACCGAATACACCGTTTCTATCTACGGTGTTCTTAGAAGCTTCCCAAAGGC (where N is any base)

[0281] Sequence ID 16 = BC6 GTGACACGGCGGTTAGAACGCGGCTACAATTAATACATAACCCCATCCCCCTGTTGACAATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGGATCTACCATGCTGCCGGCGCCGAAAAACCTGGTTGTTTCTGAAGTTACCGAAGACTCTCTGCGTCTGTCTTGGNNNNNNNNNNNNNNNNNNNNTTYGACTCTTTCCTGATCCAGTACC AGGAATCTGAAAAAGTTGGTGAAGCGATCAACCTGACCGTTCCGGGTTCTGAACGTTCTTACGACCTGACCGGTCTGAAACCGGGTACCGAATACACCGTTTCTATCTACGGTGTTCTTAGAAGCTTCCCAAAGGC (where N is any base)

[0282] Sequence ID 17 = 130mer - L17A CGGCGGTTAGAACGCGGCTACAATTAATACATAACCCCATCCCCCTGTTGACAATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGGATCTACCATGCTG

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

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

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

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

[0287] Dimensions 22=SDG10 CATGCGGTCTCTTCCGAAAAAGTTGGTGAAGCGATCGTCCTGACCGTTCCGGGT

[0288] range 23=SDG24 GGTGGTGAAGATCGCAGACAGCGGGTTAG

[0289] Enclosure 24=POP2222 CGGCGGTTAGAACGCGGCTAC

[0290] Dimensions 25=SDG28 AAGATCAGTTGCGGCCGCTAGACTAGAACCGCTGCCACCGCCGGTGGTGAAGATCGCAGAC

[0291] Sequence ID 26 = FG12 GTGACACGGCGGTTAGAACGCGGCTACAATTAATACATAACCCCATCCCCCTGTTGACAATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGGATCTACCATGCTGCCGGCGCCGAAAAACCTGGTTGTTTCTCGCGTTACCGAAGACT CTGCGCGTCTTGTCTTGGACCGCGCCGGACGCGGCGTTCGACTCTTTCTGATCCAGTACCAGGAATCTGAAAAAGTTGGTGAAGCGATCGTGCTGACCGTTCCGGGTTCTGAACGTTCTTACGACCTGACCGGTCTGAAACCGGGTACCGAATACACCGTTTCTATCTACGGTGTTNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNTCTAACCCGCTGTCTGCGATCTTCACCACCGGCGGTCACCATCACCATCACCATGGCAGCGGTTCTAGTCTAGCGGCCGCAACTGATCTTGGC (where N is any base)

[0292] Sequence ID 27 = FG11 GTGACACGGCGGTTAGAACGCGGCTACAATTAATACATAACCCCATCCCCCTGTTGACAATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGGATCTACCATGCTGCCGGCGCCGAAAAACCTGGTTGTTTCTCGCGTTACCGAAGACTCTGCGCGTCTGTCTTGGACCGCGCCGGACGCGGCGTTCGACTCTTTCCTGATCCAGTACCAGGAATCTGAAAAAGTTGGTGAAGCGATCGTGCTGACCGTTCCGGGTTCTGAACGTTCTTACGACCTGACCGGTCTGAAACCGGGTACCGAATACACCGTTTCTATCTACGGTGTTNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNTCTAACCCGCTGTCTGCGATCTTCACCACCGGCGGTCACCATCACCATCACCATGGCAGCGGTTCTAGTCTAGCGGCCGCAACTGATCTTGGC (where N is any base)

[0293] Sequence ID 28 = FG10 GTGACACGGCGGTTAGAACGCGGCTACAATTAATACATAACCCCATCCCCCTGTTGACAATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGGATCTACCATGCTGCCGGCGCCGAAAAACCTGGTTGTTTCTCGCGTTACCGAAGACTCTGCGCGTCTGTCTTGGACCGCGCCGGACGCGGCGTTCGACTCTTTCCTGATCCAGTACCAGGAATCTGAAAAAGTTGGTGAAGCGATCGTGCTGACCGTTCCGGGTTCTGAACGTTCTTACGACCTGACCGGTCTGAAACCGGGTACCGAATACACCGTTTCTATCTACGGTGTTNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNTCTAACCCGCTGTCTGCGATCTTCACCACCGGCGGTCACCATCACCATCACCATGGCAGCGGTTCTAGTCTAGCGGCCGCAACTGATCTTGGC (where N is any base)

[0294] Sequence ID 29 = FG9 GTGACACGGCGGTTAGAACGCGGCTACAATTAATACATAACCCCATCCCCCTGTTGACAATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGGATCTACCATGCTGCCGGCGCCGAAAAACCTGGTTGTTTCTCGCGTTACCGAAGACTCTGCGCGTCTGTCTTGGACCGCGCCGGACGCGGCGTTCGACTCTTTCCTGATCCAGTACCAGGAATCTGAAAAAGTTGGTGAAGCGATCGTGCTGACCGTTCCGGGTTCTGAACGTTCTTACGACCTGACCGGTCTGAAACCGGGTACCGAATACACCGTTTCTATCTACGGTGTTNNNNNNNNNNNNNNNNNNNNNNNNNNNTCTAACCCGCTGTCTGCGATCTTCACCACCGGCGGTCACCATCACCATCACCATGGCAGCGGTTCTAGTCTAGCGGCCGCAACTGATCTTGGC (where N is any base)

[0295] Sequence ID 30 = FG8 GTGACACGGCGGTTAGAACGCGGCTACAATTAATACATAACCCCATCCCCCTGTTGACAATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGGATCTACCATGCTGCCGGCGCCGAAAAACCTGGTTGTTTCTCGCGTTACCGAAGACTCTGCGCGTCTGTCTTGGACCGCGCCGGACGCGGCGTTCGACTCTTTCCTGATCCAGTACCAGGAATCTGAAAAAGTTGGTGAAGCGATCGTGCTGACCGTTCCGGGTTCTGAACGTTCTTACGACCTGACCGGTCTGAAACCGGGTACCGAATACACCGTTTCTATCTACGGTGTTNNNNNNNNNNNNNNNNNNNNNNNNTCTAACCCGCTGTCTGCGATCTTCACCACCGGCGGTCACCATCACCATCACCATGGCAGCGGTTCTAGTCTAGCGGCCGCAACTGATCTTGGC (where N is any base)

[0296] Sequence ID 31 = FG7 GTGACACGGCGGTTAGAACGCGGCTACAATTAATACATAACCCCATCCCCCTGTTGACAATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGGATCTACCATGCTGCCGGCGCCGAAAAACCTGGTTGTTTCTCGCGTTACCGAAGACTCTGCGCGTCTGTCTTGGACCGCGCCGGACGCGGCGTTCGACTCTTTCCTGATCCAGTACCAGGAATCTGAAAAAGTTGGTGAAGCGATCGTGCTGACCGTTCCGGGTTCTGAACGTTCTTACGACCTGACCGGTCTGAAACCGGGTACCGAATACACCGTTTCTATCTACGGTGTTNNNNNNNNNNNNNNNNNNNNNTCTAACCCGCTGTCTGCGATCTTCACCACCGGCGGTCACCATCACCATCACCATGGCAGCGGTTCTAGTCTAGCGGCCGCAACTGATCTTGGC (where N is any base)

[0297] Sequence ID 32 = Human mature CD71 MTKEYQDLQHLDNEESDHHQLRKGPPPPQPLLQRLCSGPRLLLLSLGLSLLLLVVVCVIGSQNSQLQEELRGLRETFSNFTASTEAQVKGLSTQGGNVGRKMKSLESQLEKQQKDLSEDHSSLLLHVKQFVSDLRSLSCQMAALQ GNGSERTCCPVNWVEHERSCYWFSRSGKAWADADNYCRLEDAHLVVVTSWEEQKFVQHHIGPVNTWMGLHDQNGPWKWVDGTDYETGFKNWRPEQPDDWYGHGLGGGEDCAHFTDDGRWNDDVCQRPYRWVCETELDKASQEPPLL

[0298] Sequence ID 80 = Human mature CD71 extracellular domain QNSQLQEELRGLRETFSNFTASTEAQVKGLSTQGGNVGRKMKSLESQLEKQQKDLSEDHSSLLLHVKQFVSDLRSLSCQMAALQGNGSERTCCPVNWVEHERSCYWFSRSGKAWA DADNYCRLEDAHLVVTSWEEQKFVQHHIGPVNTWMGLHDQNGPWKWVDGTDYETGFKNWRPEQPDDWYGHGLGGGEDCAHFTDDGRWNDDVCQRPYRWVCETELDKASQEPPLL

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

[0300] Example 4: Selection of fibronectin type III (FN3) domains that bind to CD71 A panning and biochemical screening method for identifying FN3 domains that bind to CD71 and do not inhibit transferrin binding to CD71. To screen for FN3 domains that specifically bind to CD71 and do not inhibit transferrin binding to CD71, streptavidin-coated Maxisorp plates (Nunc catalog 436110) were blocked in a Starting Block T20 (Pierce) for 1 hour and then coated with biotinylated CD71 (using the same antigen as panning) or negative control (unrelated Fc fusion recombinant protein and serum albumin) in the presence of transferrin for 1 hour, or in the presence of FN3 protein that binds to the CD71 transferrin binding site. The transferrin concentration was up to 35 μM. Although not bound by any particular theory, including transferrin or FN3 protein that binds to the CD71 transferrin binding site leads to the selection of FN3 domains that do not compete with or inhibit transferrin binding to CD71. The plates were rinsed with TBST, and diluted lysates were applied to the plates for 1 hour. After further rinsing, the wells were treated with HRP-conjugated anti-V5 tag antibody (Abcam, ab1325) for 1 hour and then tested using POD (Roche, 11582950001). DNA from FN3 domain lysates exhibiting an ELISA signal at least 10 times higher than that of the streptavidin control was sequenced to obtain FN3 domain sequences isolated from the screening.

[0301] Example 5: Selection of a fibronectin type III (FN3) domain that binds to CD71 and does not compete with transferrin. A biased CIS display strategy was designed to identify CD71-binding FN3 domains that do not compete with transferrin or compete minimally with it. Briefly, the output recovered after five rounds of panning with human CD71 ECD was used (Example 3). Further rounds of off-rate selection were performed as described in Example 3, by adding either 1) a washing step to elute centyrins bound at the same site as transferrin using human holotransferrin before the final elution step, or 2) elution of the FN3 domain binder using the monoclonal antibody OKT9. The FN3 domains recovered from the transferrin washing strategy and the OKT9 elution strategy were PCR amplified and cloned into a pET vector as previously described (Example 3). 228 FN3 domains specifically bound to huCD71 were confirmed by ELISA for binding to huCD71 ECD (Table 8). A subset of the unique binder was analyzed by SEC (Table 9), conjugated to MMAF, and its internal translocation was evaluated by a cell viability assay in SKBR-3 cells + / - holohuman transferrin (Table 10). The polypeptide was found to have been internally translocated via the receptor. The hit data and sequences are identified in the table below.

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

[0303] Table 10-1 Table 10-2 Table 10-3 Table 10-4 Table 10-5 Table 10-6

[0304] Table 11-1 Table 11-2

[0305] Table 12-1 Table 12-2 Table 12-3 Table 12-4 Table 12-5 Table 12-6 Table 12-7 Table 12-8 [Table 12-9] [Table 12-10]

[0306] Example 6. Knockdown of mRNA in muscle cells using CD71 FN3 domain-oligonucleotide conjugate. Cysteine ​​specifically manipulated in the FN3 domain. Using maleimide chemistry, the muCD71-binding FN3 domain was conjugated to an siRNA oligonucleotide or antisense oligonucleotide (ASO). Cysteine ​​substitutions may be as shown herein and in U.S. Patent Application Publication No. 20150104808 (incorporated herein by reference as a whole). The siRNA or ASO was modified using standard chemical modifications and confirmed to allow for in vitro knockdown of targeted mRNA. The FN3 domain-oligonucleotide conjugate was administered intravenously to mice at doses up to 10 mg / kg oligonucleotide payload. Mice were sacrificed at various time points after administration; skeletal muscle, cardiac muscle and various other tissues were collected, and RNAlater TM (Sigma Aldrich) was stored until needed. Target gene knockdown was performed using standard qPCR ΔΔC T The method was evaluated using primers specific to the target gene and control gene. The target gene was found to be knocked down in muscle, and such knockdown was enhanced by binding siRNA or ASO to the CD71 FN3 binding domain.

[0307] Example 7. General Method Standard methods in molecular biology are described in Sambrook, Fritsch, and Maniatis (1982 and 1989, 2nd edition; 2001, 3rd edition) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Sambrook and Russell (2001) Molecular Cloning, 3rd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Wu (1993) Recombinant DNA, Vol. 217, Academic Press, San Diego, CA). Standard methods can also be found 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), complex carbohydrate and protein expression (Vol. 3), and bioinformatics (Vol. 4).

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

[0309] All references cited herein are invoked by reference to the same extent as each individual publication, database entry (e.g., Genbank sequence or GeneID entry), patent application, or patent is specifically and individually indicated as being invoked by reference. This statement of referencing by reference is intended by the applicants in accordance with 37 CFR §1.57(b)(1) to relate to each individual publication, database entry (e.g., Genbank sequence or GeneID entry), patent application, or patent (each of which is clearly identified in accordance with 37 CFR §1.57(b)(2)), even if such reference is not directly adjacent to a dedicated statement of referencing by reference. Including a dedicated statement of referencing by reference, where present, in the specification does not in any way weaken this general statement of referencing by reference. The citation of references herein is not intended to be an acknowledgment that the references relate to the prior art, nor does it constitute any acknowledgment of the content or date of these publications or documents.

[0310] This embodiment should not be limited in scope by any specific embodiment described herein. In fact, various modifications of the embodiments beyond those described herein will be apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims.

[0311] The above-described specification is deemed sufficient to enable those skilled in the art to carry out the embodiments. In addition to those shown and described herein, various modifications of the embodiments will be apparent to those skilled in the art from the foregoing description and are within the scope of the appended claims.

Claims

1. An FN3 domain polypeptide that binds to CD71 at a site on CD71 that does not compete with the binding of transferrin to CD71, the FN3 domain polypeptide comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:

146.

2. The FN3 domain polypeptide according to claim 1, wherein the FN3 domain polypeptide comprises two amino acid sequences selected from the group consisting of SEQ ID NOs: 146, 154, 176, 181, 271, 278, 283, and 298.

3. The FN3 domain polypeptide according to claim 1, wherein the FN3 domain polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 146, 154, 176, 181, 271, 278, 283, and 298.

4. The FN3 domain polypeptide according to claim 1, wherein the FN3 domain polypeptide is conjugated to a detectable label, an oligonucleotide, a therapeutic agent, or any combination thereof.

5. The FN3 domain polypeptide according to claim 4, wherein the detectable label is a radioisotope, magnetic beads, metal beads, colloidal particles, fluorescent dyes, high electron density reagents, enzymes, biotin, digoxigenin, or hapten.

6. The FN3 domain polypeptide according to claim 4, wherein the therapeutic agent is auristatin, monomethyl auristatin phenylalanine, dolastatin, a chemotherapeutic agent, a drug, a growth inhibitor, a toxin, or a radioisotope.

7. The aforementioned therapeutic agents include chemotherapeutic agents, drugs, antibodies, proliferation inhibitors, toxins, radioisotopes, antitubulin agents, polynucleotides, double-stranded small interfering ribonucleic acid (siRNA) molecules or their sense or antisense strands, double-stranded antisense molecules or their strands, RNA molecules, deoxyribonucleic acid (DNA) molecules, DNA minor groove binders, and DNA compound The FN3 domain polypeptide according to claim 4, which is a manufacturing inhibitor, alkylating agent, antibiotic, folic acid antimetabolite, antimetabolite, chemotherapy sensitizer, topoisomerase inhibitor, or vinca alkaloid.

8. The FN3 domain polypeptide according to claim 1, wherein the FN3 domain polypeptide is conjugated to a detectable label, an oligonucleotide, a therapeutic agent, or any combination thereof.

9. A pharmaceutical composition comprising the FN3 domain polypeptide described in claim 1 and a pharmaceutically acceptable carrier.

10. A pharmaceutical composition comprising the FN3 domain polypeptide described in claim 8 and a pharmaceutically acceptable carrier.

Images