# Patent

## Title
FN3 domain-siRNA conjugates and uses thereof

## Abstract

The present disclosure relates to compositions, such as siRNA molecules and FN3 domains conjugated to the same, as well as methods of making and using the molecules.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
The present application claims priority to U.S. Provisional Application No. 62/914,725, filed Oct. 14, 2019, U.S. Provisional Application No. 62/979,557, filed Feb. 21, 2020, and U.S. Provisional Application No. 63/054,896, filed Jul. 22, 2020, each of which is hereby incorporated by reference in its entirety.
This application is also related to U.S. Provisional Application No. 62/914,654, filed Oct. 14, 2019, U.S. Provisional Application No. 62/914,643, filed Oct. 14, 2019, U.S. Provisional Application No. 62/949,020, filed Dec. 17, 2019, U.S. application Ser. No. 17/070,020, filed Oct. 14, 2020, and PCT Application No. PCT/US20/55465, filed Oct. 14, 2020, each of which is hereby incorporated by reference in its entirety.


FIELD
The present embodiments relate to siRNA molecules that can be conjugated fibronectin type III domains (FN3) and methods of making and using the molecules.
SEQUENCE LISTING
This application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. The ASCII text file was created on Dec. 15, 2020, it is named 145965_02301_SeqList_15_Dec_2020_ST25.TXT, and it is 473 kilobytes in size.
BACKGROUND
Therapeutic nucleic acids include, e.g., small interfering RNA (siRNA), micro RNA (miRNA), antisense oligonucleotides, ribozymes, plasmids, immune stimulating nucleic acids, antisense, antagomir, antimir, microRNA mimic, supermir, U1 adaptor, and aptamer. In the case of siRNA or miRNA, these nucleic acids can down-regulate intracellular levels of specific proteins through a process termed RNA interference (RNAi). The therapeutic applications of RNAi are extremely broad, since siRNA and miRNA constructs can be synthesized with any nucleotide sequence directed against a target protein. To date, siRNA constructs have shown the ability to specifically down-regulate target proteins in both in vitro and in vivo models. In addition, siRNA constructs are currently being evaluated in clinical studies.
However, two problems currently faced by siRNA constructs are, first, their susceptibility to nuclease digestion in plasma and, second, their limited ability to gain access to the intracellular compartment where they can bind the protein RISC when administered systemically as the free siRNA or miRNA. Certain delivery systems, such as lipid nanoparticles formed from cationic lipids with other lipid components, such as cholesterol and PEG lipids, and oligonucleotides (such as siRNA) have been used to facilitate the cellular uptake of the oligonucleotides. However, these have not been shown to be successful in efficiently and effectively delivering siRNA to its intended target.
There remains a need for compositions and methods for delivering siRNA to its intended cellular target. The present embodiments fulfills these needs as well as others.
SUMMARY
In some embodiments, siRNA conjugated to FN3 domains that bind CD71 protein are provided.
In some embodiments, siRNA conjugated to FN3 domains that bind EPCAM protein are provided.
In some embodiments, siRNA conjugated to FN3 domains that bind EGFR protein are provided.
In some embodiments, FN3 domains are provided that comprise the amino acid sequence of any FN3 domain provided herein. In some embodiments, the FN3 domains bind to CD71, EPCAM, or EGFR. In some embodiments, the FN3 domains specifically bind to CD71, EPCAM, or EGFR.
In some embodiments, the composition comprises two FN3 domains connected by a linker, such as a flexible linker. In some embodiments, the two FN3 domains bind to different targets. In some embodiments, a first FN3 domain binds to one of CD71, EPCAM, or EGFR. In some embodiments, a second FN3 domain binds to one of CD71, EPCAM, or EGFR that is not the same as first FN3 domain.
In some embodiments, oligonucleotides, such as dsRNA or siRNA molecules are provided herein. In some embodiments, the oligonucleotides have the sequences as provided herein, with or without the modifications provided herein. In some embodiments, the oligonucleotides are provided in a composition, such as a pharmaceutical composition. In some embodiments, the oligonucleotides are conjugated to a polypeptide.
In some embodiments, composition comprising one or more FN3 domains conjugated to a siRNA molecule are provided.
In some embodiments, a composition having a formula of (X1)n-(X2)q-(X3)y-L-X4, wherein X1 is a first FN3 domain; X2 is second FN3 domain; X3 is a third FN3 domain or half-life extender molecule; L is a linker; and X4 is a nucleic acid molecule, wherein n, q, and y are each independently 0 or 1, are provided.
In some embodiments, a composition having a formula of C—(X1)n-(X2)q-(X3)y-L-X4, wherein C is a polymer; X1 is a first FN3 domain; X2 is second FN3 domain; X3 is a third FN3 domain or half-life extender molecule; L is a linker; and X4 is a nucleic acid molecule, wherein n, q, and y are each independently 0 or 1, are provided.
In some embodiments, a composition having a formula of (X1)n-(X2)q-(X3)y-L-X4-C, wherein X1 is a first FN3 domain; X2 is second FN3 domain; X3 is a third FN3 domain or half-life extender molecule; L is a linker; X4 is a nucleic acid molecule; and C is a polymer, wherein n, q, and y are each independently 0 or 1, are provided.
In some embodiments, a composition having a formula of X4-L-(X1)n-(X2)q-(X3)y, wherein X1 is a first FN3 domain; X2 is second FN3 domain; X3 is a third FN3 domain or half-life extender molecule; L is a linker; and X4 is a nucleic acid molecule, wherein n, q, and y are each independently 0 or 1, are provided.
In some embodiments, a composition having a formula of C—X4-L-(X1)n-(X2)q-(X3)y, wherein C is a polymer; X1 is a first FN3 domain; X2 is second FN3 domain; X3 is a third FN3 domain or half-life extender molecule; L is a linker; and X4 is a nucleic acid molecule, wherein n, q, and y are each independently 0 or 1, are provided.
In some embodiments, a composition having a formula of X4-L-(X1)n-(X2)q-(X3)y-C, wherein X1 is a first FN3 domain; X2 is second FN3 domain; X3 is a third FN3 domain or half-life extender molecule; L is a linker; X4 is a nucleic acid molecule; and C is a polymer, wherein n, q, and y are each independently 0 or 1, are provided.
In some embodiments, pharmaceutical compositions comprising one or more of the compositions provided herein are provided.
In some embodiments, methods of treating cancer in a subject in need thereof, the method comprising administering to the subject a composition provided herein are provided.
In some embodiments, a use of a composition as provided herein or of any of in the preparation of a pharmaceutical composition or medicament for treating cancer are provided.
In some embodiments, methods of reducing the expression of a target gene in a cell, the method comprising contacting the cell with a composition as provided herein are provided.
In some embodiments, isolated polynucleotides encoding the FN3 domains described herein are provided.
In some embodiments, a vector comprising the polynucleotides described herein are provided.
In some embodiments, a host cell comprising the vectors described herein are provided.
In some embodiments, methods of producing the FN3 domains are provided. In some embodiments, the method comprises culturing a host cell comprising a vector encoding or expressing the FN3 domain. In some embodiments, the method further comprises purifying the FN3 domain. In some embodiments, the FN3 domain binds CD71, EPCAM, or EGFR.
In some embodiments, pharmaceutical compositions comprising a FN3 domain that binds to CD71, EPCAM, or EGFR linked to a nucleic acid molecule and a pharmaceutically acceptable carrier are provided. In some embodiments, the composition does not comprise (e.g. is free of) a compound or protein that binds to ASGPR.
In some embodiments, kits comprising one or more of the FN3 domains with or without the nucleic acid molecules are provided.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 illustrates the knock-down of KRAS with a FN3-siRNA conjugate.
FIG. 2 illustrates the inhibition of cellular proliferation of a FN3-siRNA conjugate.
FIG. 3 illustrates various embodiments provided herein.
FIG. 4, panels A and B, illustrates various embodiments provided herein.
FIG. 5 illustrates various embodiments provided herein.
FIG. 6 illustrates various embodiments provided herein.
FIG. 7 illustrates various embodiments provided herein.
FIG. 8 illustrates various embodiments provided herein.
FIG. 9 illustrates various embodiments provided herein.
FIG. 10 illustrates various embodiments provided herein.
FIG. 11 illustrates various embodiments provided herein.



DETAILED DESCRIPTION OF THE DISCLOSURE
As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “a cell” includes a combination of two or more cells, and the like.
“Fibronectin type III (FN3) domain” (FN3 domain) refers to a domain occurring frequently in proteins including fibronectins, tenascin, intracellular cytoskeletal proteins, cytokine receptors and prokaryotic enzymes (Bork and Doolittle, Proc Nat Acad Sci USA 89:8990-8994, 1992; Meinke et al., J Bacteriol 175:1910-1918, 1993; Watanabe et al., J Biol Chem 265:15659-15665, 1990). Exemplary FN3 domains are the 15 different FN3 domains present in human tenascin C, the 15 different FN3 domains present in human fibronectin (FN), and non-natural synthetic FN3 domains as described for example in U.S. Pat. No. 8,278,419. Individual FN3 domains are referred to by domain number and protein name, e.g., the 3rd FN3 domain of tenascin (TN3), or the 10th FN3 domain of fibronectin (FN10).
The term “capture agent” refers to substances that bind to a particular type of cells and enable the isolation of that cell from other cells. Exemplary capture agents are magnetic beads, ferrofluids, encapsulating reagents, molecules that bind the particular cell type and the like.
“Sample” refers to a collection of similar fluids, cells, or tissues isolated from a subject, as well as fluids, cells, or tissues present within a subject. Exemplary samples are tissue biopsies, fine needle aspirations, surgically resected tissue, organ cultures, cell cultures and biological fluids such as blood, serum and serosal fluids, plasma, lymph, urine, saliva, cystic fluid, tear drops, feces, sputum, mucosal secretions of the secretory tissues and organs, vaginal secretions, ascites fluids, fluids of the pleural, pericardial, peritoneal, abdominal and other body cavities, fluids collected by bronchial lavage, synovial fluid, liquid solutions contacted with a subject or biological source, for example, cell and organ culture medium including cell or organ conditioned medium and lavage fluids and the like.
“Substituting” or “substituted” or “mutating” or “mutated” refers to altering, deleting of inserting one or more amino acids or nucleotides in a polypeptide or polynucleotide sequence to generate a variant of that sequence.
“Variant” refers to a polypeptide or a polynucleotide that differs from a reference polypeptide or a reference polynucleotide by one or more modifications for example, substitutions, insertions or deletions.
“Specifically binds” or “specific binding” refers to the ability of a FN3 domain to bind to its target, such as CD71, with a dissociation constant (KD) of about 1×10−6 M or less, for example about 1×10−7 M or less, about 1×10−8 M or less, about 1×10−9 M or less, about 1×10−10 M or less, about 1×10−11 M or less, about 1×10−12M or less, or about 1×10−13 M or less. Alternatively, “specific binding” refers to the ability of a FN3 domain to bind to its target (e.g. CD71) at least 5-fold above a negative control in standard ELISA assay. In some embodiments, a negative control is an FN3 domain that does not bind CD71. In some embodiment, an FN3 domain that specifically binds CD71 may have cross-reactivity to other related antigens, for example to the same predetermined antigen from other species (homologs), such as Macaca Fascicularis (cynomolgous monkey, cyno) or Pan troglodytes (chimpanzee).
“Library” refers to a collection of variants. The library may be composed of polypeptide or polynucleotide variants.
“Stability” refers to the ability of a molecule to maintain a folded state under physiological conditions such that it retains at least one of its normal functional activities, for example, binding to a predetermined antigen such as CD71.
“CD71” refers to human CD71 protein having the amino acid sequence of SEQ ID NOs: 2 or 3. In some embodiments, SEQ ID NO: 2 is full length human CD71 protein. In some embodiments, SEQ ID NO: 3 is the extracellular domain of human CD71.
“Tencon” refers to the synthetic fibronectin type III (FN3) domain having the sequence shown in SEQ ID NO:1 (SPPKDLVVTEVTEETVNLAWDNEMRVTEYLVVYTPTHEGGLEMQFRVPGDQTSTIIQE LEPGVEYFIRVFAILENKKSIPVSARVAT) and described in U.S. Pat. Publ. No. 2010/0216708.
A “cancer cell” or a “tumor cell” refers to a cancerous, pre-cancerous or transformed cell, either in vivo, ex vivo, and in tissue culture, that has spontaneous or induced phenotypic changes that do not necessarily involve the uptake of new genetic material. Although transformation can arise from infection with a transforming virus and incorporation of new genomic nucleic acid, or uptake of exogenous nucleic acid, it can also arise spontaneously or following exposure to a carcinogen, thereby mutating an endogenous gene. Transformation/cancer is exemplified by, e.g., morphological changes, immortalization of cells, aberrant growth control, foci formation, proliferation, malignancy, tumor specific markers levels, invasiveness, tumor growth or suppression in suitable animal hosts such as nude mice, and the like, in vitro, in vivo, and ex vivo (Freshney, Culture of Animal Cells: A Manual of Basic Technique (3rd ed. 1994)).
“Vector” refers to a polynucleotide capable of being duplicated within a biological system or that can be moved between such systems. Vector polynucleotides typically contain elements, such as origins of replication, polyadenylation signal or selection markers that function to facilitate the duplication or maintenance of these polynucleotides in a biological system. Examples of such biological systems may include a cell, virus, animal, plant, and reconstituted biological systems utilizing biological components capable of duplicating a vector. The polynucleotide comprising a vector may be DNA or RNA molecules or a hybrid of these.
“Expression vector” refers to a vector that can be utilized in a biological system or in a reconstituted biological system to direct the translation of a polypeptide encoded by a polynucleotide sequence present in the expression vector.
“Polynucleotide” refers to a synthetic molecule comprising a chain of nucleotides covalently linked by a sugar-phosphate backbone or other equivalent covalent chemistry. cDNA is a typical example of a polynucleotide.
“Polypeptide” or “protein” refers to a molecule that comprises at least two amino acid residues linked by a peptide bond to form a polypeptide. Small polypeptides of less than about 50 amino acids may be referred to as “peptides”.
“Valent” refers to the presence of a specified number of binding sites specific for an antigen in a molecule. As such, the terms “monovalent”, “bivalent”, “tetravalent”, and “hexavalent” refer to the presence of one, two, four and six binding sites, respectively, specific for an antigen in a molecule.
“Subject” includes any human or nonhuman animal. “Nonhuman animal” includes all vertebrates, e g, mammals and non-mammals, such as nonhuman primates, sheep, dogs, cats, horses, cows chickens, amphibians, reptiles, etc. Except when noted, the terms “patient” or “subject” are used interchangeably.
“Isolated” refers to a homogenous population of molecules (such as synthetic polynucleotides or a polypeptide such as FN3 domains) which have been substantially separated and/or purified away from other components of the system the molecules are produced in, such as a recombinant cell, as well as a protein that has been subjected to at least one purification or isolation step. “Isolated FN3 domain” refers to an FN3 domain that is substantially free of other cellular material and/or chemicals and encompasses FN3 domains that are isolated to a higher purity, such as to 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% purity.
In some embodiments, a composition comprising a polypeptide, such as a polypeptide comprising a FN3 domain, linked to a nucleic acid molecule are provided. The nucleic acid molecule can be, for example, a siRNA molecule.
Accordingly, in some embodiments, the siRNA is a double-stranded RNAi (dsRNA) agent capable of inhibiting the expression of a target gene. The dsRNA agent comprises a sense strand and an antisense strand. In some embodiments, each strand of the dsRNA agent can range from 12-40 nucleotides in length. For example, each strand can be from 14-40 nucleotides in length, 17-37 nucleotides in length, 25-37 nucleotides in length, 27-30 nucleotides in length, 17-23 nucleotides in length, 17-21 nucleotides in length, 17-19 nucleotides in length, 19-25 nucleotides in length, 19-23 nucleotides in length, 19-21 nucleotides in length, 21-25 nucleotides in length, or 21-23 nucleotides in length.
In some embodiments, the sense strand and antisense strand typically form a duplex dsRNA. The duplex region of a dsRNA agent may be from 12-40 nucleotide pairs in length. For example, the duplex region can be from 14-40 nucleotide pairs in length, 17-30 nucleotide pairs in length, 25-35 nucleotides in length, 27-35 nucleotide pairs in length, 17-23 nucleotide pairs in length, 17-21 nucleotide pairs in length, 17-19 nucleotide pairs in length, 19-25 nucleotide pairs in length, 19-23 nucleotide pairs in length, 19-21 nucleotide pairs in length, 21-25 nucleotide pairs in length, or 21-23 nucleotide pairs in length. In another example, the duplex region is selected from 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, and 27 nucleotide pairs in length.
In some embodiments, the dsRNA comprises one or more overhang regions and/or capping groups of dsRNA agent at the 3′-end, or 5′-end or both ends of a strand. The overhang can be 1-10 nucleotides in length, 1-6 nucleotides in length, for instance 2-6 nucleotides in length, 1-5 nucleotides in length, 2-5 nucleotides in length, 1-4 nucleotides in length, 2-4 nucleotides in length, 1-3 nucleotides in length, 2-3 nucleotides in length, or 1-2 nucleotides in length. The overhangs can be the result of one strand being longer than the other, or the result of two strands of the same length being staggered. The overhang can form a mismatch with the target mRNA or it can be complementary to the gene sequences being targeted or can be other sequence. The first and second strands can also be joined, e.g., by additional bases to form a hairpin, or by other non-base linkers.
In some embodiments, the nucleotides in the overhang region of the dsRNA agent can each independently be a modified or unmodified nucleotide including, but not limited to 2′-sugar modified, such as, 2-F 2′-Omethyl, thymidine (T), 2′-O-methoxyethyl-5-methyluridine (Teo), 2′-O-methoxyethyladenosine (Aeo), 2′-O-methoxyethyl-5-methylcytidine (m5Ceo), and any combinations thereof. For example, TT can be an overhang sequence for either end on either strand. The overhang can form a mismatch with the target mRNA or it can be complementary to the gene sequences being targeted or can be other sequence.
The 5′- or 3′-overhangs at the sense strand, antisense strand or both strands of the dsRNA agent may be phosphorylated. In some embodiments, the overhang region contains two nucleotides having a phosphorothioate between the two nucleotides, where the two nucleotides can be the same or different. In one embodiment, the overhang is present at the 3′-end of the sense strand, antisense strand or both strands. In one embodiment, this 3′-overhang is present in the antisense strand. In one embodiment, this 3′-overhang is present in the sense strand.
The dsRNA agent may comprise only a single overhang, which can strengthen the interference activity of the dsRNA, without affecting its overall stability. For example, the single-stranded overhang is located at the 3′-terminal end of the sense strand or, alternatively, at the 3′-terminal end of the antisense strand. The dsRNA may also have a blunt end, located at the 5′-end of the antisense strand (or the 3′-end of the sense strand) or vice versa. Generally, the antisense strand of the dsRNA has a nucleotide overhang at the 3′-end, and the 5′-end is blunt. While not bound by theory, the asymmetric blunt end at the 5′-end of the antisense strand and 3′-end overhang of the antisense strand favor the guide strand loading into RISC process. For example the single overhang comprises at least two, three, four, five, six, seven, eight, nine, or ten nucleotides in length.
In some embodiments, the dsRNA agent may also have two blunt ends, at both ends of the dsRNA duplex.
In some embodiments, every nucleotide in the sense strand and antisense strand of the dsRNA agent may be modified. Each nucleotide may be modified with the same or different modification which can include one or more alteration of one or both of the non-linking phosphate oxygens and/or of one or more of the linking phosphate oxygens; alteration of a constituent of the ribose sugar, e.g., of the 2 hydroxyl on the ribose sugar; wholesale replacement of the phosphate moiety with “dephospho” linkers; modification or replacement of a naturally occurring base; and replacement or modification of the ribose-phosphate backbone.
In some embodiments all or some of the bases in a 3′ or 5′ overhang may be modified, e.g., with a modification described herein. Modifications can include, e.g., the use of modifications at the 2′ position of the ribose sugar with modifications that are known in the art, e.g., the use of deoxyribonucleotides, 2′-deoxy-2′-fluoro (2′-F) or 2′-O-methyl modified instead of the ribosugar of the nucleobase, and modifications in the phosphate group, e.g., phosphorothioate modifications. Overhangs need not be homologous with the target sequence.
In some embodiments, each residue of the sense strand and antisense strand is independently modified with LNA, HNA, CeNA, 2′-methoxyethyl, 2′-O-methyl, 2′-O-allyl, 2′-C-allyl, 2′-deoxy, or 2′-fluoro. The strands can contain more than one modification. In one embodiment, each residue of the sense strand and antisense strand is independently modified with 2′-O-methyl or 2′-fluoro.
In some embodiments, at least two different modifications are typically present on the sense strand and antisense strand. Those two modifications may be the 2′-deoxy, 2′-O-methyl or 2′-fluoro modifications, acyclic nucleotides or others.
In one embodiment, the sense strand and antisense strand each comprises two differently modified nucleotides selected from 2′-fluoro, 2′-O-methyl or 2′-deoxy.
The dsRNA agent may further comprise at least one phosphorothioate or methylphosphonate internucleotide linkage. The phosphorothioate or methylphosphonate internucleotide linkage modification may occur on any nucleotide of the sense strand or antisense strand or both in any position of the strand. For instance, the internucleotide linkage modification may occur on every nucleotide on the sense strand and/or antisense strand; each internucleotide linkage modification may occur in an alternating pattern on the sense strand or antisense strand; or the sense strand or antisense strand comprises both internucleotide linkage modifications in an alternating pattern. The alternating pattern of the internucleotide linkage modification on the sense strand may be the same or different from the antisense strand, and the alternating pattern of the internucleotide linkage modification on the sense strand may have a shift relative to the alternating pattern of the internucleotide linkage modification on the antisense strand.
In some embodiments, the dsRNA agent comprises the phosphorothioate or methylphosphonate internucleotide linkage modification in the overhang region. For example, the overhang region comprises two nucleotides having a phosphorothioate or methylphosphonate internucleotide linkage between the two nucleotides. Internucleotide linkage modifications also may be made to link the overhang nucleotides with the terminal paired nucleotides within duplex region. For example, at least 2, 3, 4, or all the overhang nucleotides may be linked through phosphorothioate or methylphosphonate internucleotide linkage, and optionally, there may be additional phosphorothioate or methylphosphonate internucleotide linkages linking the overhang nucleotide with a paired nucleotide that is next to the overhang nucleotide. For instance, there may be at least two phosphorothioate internucleotide linkages between the terminal three nucleotides, in which two of the three nucleotides are overhang nucleotides, and the third is a paired nucleotide next to the overhang nucleotide. In some embodiments, these terminal three nucleotides may be at the 3′-end of the antisense strand.
In some embodiments, the dsRNA composition is linked by a modified base or nucleoside analogue as described in U.S. Pat. No. 7,427,672, which is incorporated herein by reference. In some embodiments, the modified base or nucleoside analogue is referred to as the linker or L in formulas described herein.
In some embodiments, the modified base or nucleoside analogue has the structure as shown in Chemical Formula I and a salt thereof.




where Base represents an aromatic heterocyclic group or aromatic hydrocarbon ring group optionally having a substituent, R1 and R2 are identical or different, and each represent a hydrogen atom, a protective group for a hydroxyl group for nucleic acid synthesis, an alkyl group, an alkenyl group, a cycloalkyl group, an aryl group, an aralkyl group, an acyl group, a sulfonyl group, a silyl group, a phosphate group, a phosphate group protected with a protective group for nucleic acid synthesis, or —P(R4)R5 where R4 and R5 are identical or different, and each represent a hydroxyl group, a hydroxyl group protected with a protective group for nucleic acid synthesis, a mercapto group, a mercapto group protected with a protective group for nucleic acid synthesis, an amino group, an alkoxy group having 1 to 5 carbon atoms, an alkylthio group having 1 to 5 carbon atoms, a cyanoalkoxy group having 1 to 6 carbon atoms, or an amino group substituted by an alky group having 1 to 5 carbon atoms, R3 represents a hydrogen atom, an alkyl group, an alkenyl group, a cycloalkyl group, an aryl group, an aralkyl group, an acyl group, a sulfonyl group, or a functional molecule unit substituent, and m denotes an integer of 0 to 2, and n denotes an integer of 1 to 3.

In some embodiments, the modified base or nucleoside analogue has the structure as shown in Chemical Formula I and salts thereof, wherein R1 is a hydrogen atom, an aliphatic acyl group, an aromatic acyl group, an aliphatic or aromatic sulfonyl group, a methyl group substituted by one to three aryl groups, a methyl group substituted by one to three aryl groups having an aryl ring substituted by a lower alkyl, lower alkoxy, halogen, or cyano group, or a silyl group.
In some embodiments, the modified base or nucleoside analogue has the structure as shown in Chemical Formula I and salts thereof, wherein R1 is a hydrogen atom, an acetyl group, a benzoyl group, a methanesulfonyl group, a p-toluenesulfonyl group, a benzyl group, a p-methoxybenzyl group, a trityl group, a dimethoxytrityl group, a monomethoxytrityl group, or a tert-butyldiphenylsilyl group.
In some embodiments, the modified base or nucleoside analogue has the structure as shown in Chemical Formula I and salts thereof, wherein R2 is a hydrogen atom, an aliphatic acyl group, an aromatic acyl group, an aliphatic or aromatic sulfonyl group, a methyl group substituted by one to three aryl groups, a methyl group substituted by one to three aryl groups having an aryl ring substituted by a lower alkyl, lower alkoxy, halogen, or cyano group, a silyl group, a phosphoroamidite group, a phosphonyl group, a phosphate group, or a phosphate group protected with a protective group for nucleic acid synthesis.
In some embodiments, the modified base or nucleoside analogue has the structure as shown in Chemical Formula I and salts thereof, wherein R2 is a hydrogen atom, an acetyl group, a benzoyl group, a methanesulfonyl group, a p-toluenesulfonyl group, a benzyl group, a p-methoxybenzyl group, a tert-butyldiphenylsilyl group, —P(OC2H4CN)(N(i-Pr)2), —P(OCH3)(N(i-Pr)2), a phosphonyl group, or a 2-chlorophenyl- or 4-chlorophenylphosphate group.
In some embodiments, the modified base or nucleoside analogue has the structure as shown in Chemical Formula I and salts thereof, wherein R3 is a hydrogen atom, a phenoxyacetyl group, an alkyl group having 1 to 5 carbon atoms, an alkenyl group having 1 to 5 carbon atoms, an aryl group having 6 to 14 carbon atoms, a methyl group substituted by one to three aryl groups, a lower aliphatic or aromatic sulfonyl group such as a methanesulfonyl group or a p-toluenesulfonyl group, an aliphatic acyl group having 1 to 5 carbon atoms such as an acetyl group, or an aromatic acyl group such as a benzoyl group.
In some embodiments, the modified base or nucleoside analogue has the structure as shown in Chemical Formula I and salts thereof, wherein the functional molecule unit substituent as R3 is a fluorescent or chemiluminescent labeling molecule, a nucleic acid incision activity functional group, or an intracellular or nuclear transfer signal peptide.
In some embodiments, the modified base or nucleoside analogue has the structure as shown in Chemical Formula I and salts thereof, wherein Base is a purin-9-yl group, a 2-oxopyrimidin-1-yl group, or a purin-9-yl group or a 2-oxopyrimidin-1-yl group having a substituent selected from the following a group: a group: A hydroxyl group, a hydroxyl group protected with a protective group for nucleic acid synthesis, an alkoxy group having 1 to 5 carbon atoms, a mercapto group, a mercapto group protected with a protective group for nucleic acid synthesis, an alkylthio group having 1 to 5 carbon atoms, an amino group, an amino group protected with a protective group for nucleic acid synthesis, an amino group substituted by an alkyl group having 1 to 5 carbon atoms, an alkyl group having 1 to 5 carbon atoms, and a halogen atom.
In some embodiments, the modified base or nucleoside analogue has the structure as shown in Chemical Formula I and salts thereof, wherein Base is 6-aminopurin-9-yl (i.e., adeninyl), 6-aminopurin-9-yl having the amino group protected with a protective group for nucleic acid synthesis, 2,6-diaminopurin-9-yl, 2-amino-6-chloropurin-9-yl, 2-amino-6-chloropurin-9-yl having the amino group protected with a protective group for nucleic acid synthesis, 2-amino-6-fluoropurin-9-yl, 2-amino-6-fluoropurin-9-yl having the amino group protected with a protective group for nucleic acid synthesis, 2-amino-6-bromopurin-9-yl, 2-amino-6-bromopurin-9-yl having the amino group protected with a protective group for nucleic acid synthesis, 2-amino-6-hydroxypurin-9-yl (i.e., guaninyl), 2-amino-6-hydroxypurin-9-yl having the amino group protected with a protective group for nucleic acid synthesis, 6-amino-2-methoxypurin-9-yl, 6-amino-2-chloropurin-9-yl, 6-amino-2-fluoropurin-9-yl, 2,6-dimethoxypurin-9-yl, 2,6-dichloropurin-9-yl, 6-mercaptopurin-9-yl, 2-oxo-4-amino-1,2-dihydropyrimidin-1-yl (i.e., cytosinyl), 2-oxo-4-amino-1,2-dihydropyrimidin-1-yl having the amino group protected with a protective group for nucleic acid synthesis, 2-oxo-4-amino-5-fluoro-1,2-dihydropyrimidin-1-yl, 2-oxo-4-amino-5-fluoro-1,2-dihydropyrimidin-1-yl having the amino group protected with a protective group for nucleic acid synthesis, 4-amino-2-oxo-5-chloro-1,2-dihydropyrimidin-1-yl, 2-oxo-4-methoxy-1,2-dihydropyrimidin-1-yl, 2-oxo-4-mercapto-1,2-dihydropyrimidin-1-yl, 2-oxo-4-hydroxy-1,2-dihydropyrimidin-1-yl (i.e., uracinyl), 2-oxo-4-hydroxy-5-methyl-1,2-dihydropyrimidin-1-yl (i.e., thyminyl), 4-amino-5-methyl-2-oxo-1,2-dihydropyrimidin-1-yl (i.e., 5-methylcytosinyl), or 4-amino-5-methyl-2-oxo-1,2-dihydropyrimidin-1-yl having the amino group protected with a protective group for nucleic acid synthesis.
In some embodiments, the modified base or nucleoside analogue has the structure as shown in Chemical Formula I and salts thereof, wherein m is 0, and n is 1.
In some embodiments, the modified base or nucleoside analogue is a DNA oligonucleotide or RNA oligonucleotide analogue, containing one or two or more of one or more types of unit structures of nucleoside analogues having the structure as shown in Chemical Formula II, or a pharmacologically acceptable salt thereof, provided that a form of linking between respective nucleosides in the oligonucleotide analogue may contain one or two or more phosphorothioate bonds [—OP(O)(S−)O—] aside from a phosphodiester bond [—OP(O2−)O—] identical with that in a natural nucleic acid, and if two or more of one or more types of these structures are contained, Base may be identical or different between these structures:




where Base represents an aromatic heterocyclic group or aromatic hydrocarbon ring group optionally having a substituent, R3 represents a hydrogen atom, an alkyl group, an alkenyl group, a cycloalkyl group, an aryl group, an aralkyl group, an acyl group, a sulfonyl group, a silyl group, or a functional molecule unit substituent, and m denotes an integer of 0 to 2, and n denotes an integer of 1 to 3.

In some embodiments, the oligonucleotide analogue or the pharmacologically acceptable salt thereof has the structure as shown in Chemical Formula II, wherein R1 is a hydrogen atom, an aliphatic acyl group, an aromatic acyl group, an aliphatic or aromatic sulfonyl group, a methyl group substituted by one to three aryl groups, a methyl group substituted by one to three aryl groups having an aryl ring substituted by a lower alkyl, lower alkoxy, halogen, or cyano group, or a silyl group.
In some embodiments, the oligonucleotide analogue or the pharmacologically acceptable salt thereof has the structure as shown in Chemical Formula II, wherein R1 is a hydrogen atom, an acetyl group, a benzoyl group, a methanesulfonyl group, a p-toluenesulfonyl group, a benzyl group, a p-methoxybenzyl group, a trityl group, a dimethoxytrityl group, a monomethoxytrityl group, or a tert-butyldiphenylsilyl group.
In some embodiments, the oligonucleotide analogue or the pharmacologically acceptable salt thereof has the structure as shown in Chemical Formula II, wherein R2 is a hydrogen atom, an aliphatic acyl group, an aromatic acyl group, an aliphatic or aromatic sulfonyl group, a methyl group substituted by one to three aryl groups, a methyl group substituted by one to three aryl groups having an aryl ring substituted by a lower alkyl, lower alkoxy, halogen, or cyano group, a silyl group, a phosphoroamidite group, a phosphonyl group, a phosphate group, or a phosphate group protected with a protective group for nucleic acid synthesis.
In some embodiments, the oligonucleotide analogue or the pharmacologically acceptable salt thereof has the structure as shown in Chemical Formula II, wherein R2 is a hydrogen atom, an acetyl group, a benzoyl group, a benzyl group, a p-methoxybenzyl group, a methanesulfonyl group, a p-toluenesulfonyl group, a tert-butyldiphenylsilyl group, —P(OC2H4CN)(N(i-Pr)2), —P(OCH3)(N(i-Pr)2), a phosphonyl group, or a 2-chlorophenyl- or 4-chlorophenylphosphate group.
In some embodiments, the oligonucleotide analogue or the pharmacologically acceptable salt thereof has the structure as shown in Chemical Formula II, wherein R3 is a hydrogen atom, a phenoxyacetyl group, an alkyl group having 1 to 5 carbon atoms, an alkenyl group having 1 to 5 carbon atoms, an aryl group having 6 to 14 carbon atoms, a methyl group substituted by one to three aryl groups, a lower aliphatic or aromatic sulfonyl group such as a methanesulfonyl group or a p-toluenesulfonyl group, an aliphatic acyl group having 1 to 5 carbon atoms such as an acetyl group, or an aromatic acyl group such as a benzoyl group.
In some embodiments, the oligonucleotide analogue or the pharmacologically acceptable salt thereof has the structure as shown in Chemical Formula II, wherein the functional molecule unit substituent as R3 is a fluorescent or chemiluminescent labeling molecule, a nucleic acid incision activity functional group, or an intracellular or nuclear transfer signal peptide.
In some embodiments, the oligonucleotide analogue or the pharmacologically acceptable salt thereof has the structure as shown in Chemical Formula II, wherein Base is a purin-9-yl group, a 2-oxopyrimidin-1-yl group, or a purin-9-yl group or a 2-oxopyrimidin-1-yl group having a substituent selected from the following a group: a group: A hydroxyl group, a hydroxyl group protected with a protective group for nucleic acid synthesis, an alkoxy group having 1 to 5 carbon atoms, a mercapto group, a mercapto group protected with a protective group for nucleic acid synthesis, an alkylthio group having 1 to 5 carbon atoms, an amino group, an amino group protected with a protective group for nucleic acid synthesis, an amino group substituted by an alkyl group having 1 to 5 carbon atoms, an alkyl group having 1 to 5 carbon atoms, and a halogen atom.
In some embodiments, the oligonucleotide analogue or the pharmacologically acceptable salt thereof has the structure as shown in Chemical Formula II, wherein Base is 6-aminopurin-9-yl (i.e. adeninyl), 6-aminopurin-9-yl having the amino group protected with a protective group for nucleic acid synthesis, 2,6-diaminopurin-9-yl, 2-amino-6-chloropurin-9-yl, 2-amino-6-chloropurin-9-yl having the amino group protected with a protective group for nucleic acid synthesis, 2-amino-6-fluoropurin-9-yl, 2-amino-6-fluoropurin-9-yl having the amino group protected with a protective group for nucleic acid synthesis, 2-amino-6-bromopurin-9-yl, 2-amino-6-bromopurin-9-yl having the amino group protected with a protective group for nucleic acid synthesis, 2-amino-6-hydroxypurin-9-yl (i.e., guaninyl), 2-amino-6-hydroxypurin-9-yl having the amino group protected with a protective group for nucleic acid synthesis, 6-amino-2-methoxypurin-9-yl, 6-amino-2-chloropurin-9-yl, 6-amino-2-fluoropurin-9-yl, 2,6-dimethoxypurin-9-yl, 2,6-dichloropurin-9-yl, 6-mercaptopurin-9-yl, 2-oxo-4-amino-1,2-dihydropyrimidin-1-yl (i.e., cytosinyl), 2-oxo-4-amino-1,2-dihydropyrimidin-1-yl having the amino group protected with a protective group for nucleic acid synthesis, 2-oxo-4-amino-5-fluoro-1,2-dihydropyrimidin-1-yl, 2-oxo-4-amino-5-fluoro-1,2-dihydropyrimidin-1-yl group having the amino group protected with a protective group for nucleic acid synthesis, 4-amino-2-oxo-5-chloro-1,2-dihydropyrimidin-1-yl, 2-oxo-4-methoxy-1,2-dihydropyrimidin-1-yl, 2-oxo-4-mercapto-1,2-dihydropyrimidin-1-yl, 2-oxo-4-hydroxy-1,2-dihydropyrimidin-1-yl (i.e., uracinyl), 2-oxo-4-hydroxy-5-methyl-1,2-dihydropyrimidin-1-yl (i.e., thyminyl), 4-amino-5-methyl-2-oxo-1,2-dihydropyrimidin-1-yl (i.e., 5-methylcytosinyl), or 4-amino-5-methyl-2-oxo-1,2-dihydropyrimidin-1-yl having the amino group protected with a protective group for nucleic acid synthesis.
In some embodiments, the oligonucleotide analogue or the pharmacologically acceptable salt thereof has the structure as shown in Chemical Formula II, wherein m is 0, and n is 1.
In some embodiments, compositions described herein further comprises a polymer (polymer moiety C). In some instances, the polymer is a natural or synthetic polymer, consisting of long chains of branched or unbranched monomers, and/or cross-linked network of monomers in two or three dimensions In some instances, the polymer includes a polysaccharide, lignin, rubber, or polyalkylen oxide (e.g., polyethylene glycol). In some instances, the at least one polymer includes, but is not limited to, alpha-, omega-dihydroxylpolyethyleneglycol, biodegradable lactone-based polymer, e.g. polyacrylic acid, polylactide acid (PLA), poly(glycolic acid) (PGA), polypropylene, polystyrene, polyolefin, polyamide, polycyanoacrylate, polyimide, polyethylenterephthalat (PET, PETG), polyethylene terephthalate (PETE), polytetramethylene glycol (PTG), or polyurethane as well as mixtures thereof. As used herein, a mixture refers to the use of different polymers within the same compound as well as in reference to block copolymers. In some cases, block copolymers are polymers wherein at least one section of a polymer is build up from monomers of another polymer. In some instances, the polymer comprises polyalkylene oxide. In some instances, the polymer comprises PEG. In some instances, the polymer comprises polyethylene imide (PEI) or hydroxy ethyl starch (HES).
In some instances, C is a PEG moiety. In some instances, the PEG moiety is conjugated at the 5′ terminus of the nucleic acid molecule while the binding moiety is conjugated at the 3′ terminus of the nucleic acid molecule. In some instances, the PEG moiety is conjugated at the 3′ terminus of the nucleic acid molecule while the binding moiety is conjugated at the 5′ terminus of the nucleic acid molecule. In some instances, the PEG moiety is conjugated to an internal site of the nucleic acid molecule. In some instances, the PEG moiety, the binding moiety, or a combination thereof, are conjugated to an internal site of the nucleic acid molecule. In some instances, the conjugation is a direct conjugation. In some instances, the conjugation is via native ligation.
In some embodiments, the polyalkylene oxide (e.g., PEG) is a polydispers or monodispers compound. In some instances, polydispers material comprises disperse distribution of different molecular weight of the material, characterized by mean weight (weight average) size and dispersity. In some instances, the monodisperse PEG comprises one size of molecules. In some embodiments, C is poly- or monodispersed polyalkylene oxide (e.g., PEG) and the indicated molecular weight represents an average of the molecular weight of the polyalkylene oxide, e.g., PEG, molecules.
In some embodiments, the molecular weight of the polyalkylene oxide (e.g., PEG) is about 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1450, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3250, 3350, 3500, 3750, 4000, 4250, 4500, 4600, 4750, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 10,000, 12,000, 20,000, 35,000, 40,000, 50,000, 60,000, or 100,000 Da.
In some embodiments, C is polyalkylene oxide (e.g., PEG) and has a molecular weight of about 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1450, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3250, 3350, 3500, 3750, 4000, 4250, 4500, 4600, 4750, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 10,000, 12,000, 20,000, 35,000, 40,000, 50,000, 60,000, or 100,000 Da. In some embodiments, C is PEG and has a molecular weight of about 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1450, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3250, 3350, 3500, 3750, 4000, 4250, 4500, 4600, 4750, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 10,000, 12,000, 20,000, 35,000, 40,000, 50,000, 60,000, or 100,000 Da. In some instances, the molecular weight of C is about 200 Da. In some instances, the molecular weight of C is about 300 Da. In some instances, the molecular weight of C is about 400 Da. In some instances, the molecular weight of C is about 500 Da. In some instances, the molecular weight of C is about 600 Da. In some instances, the molecular weight of C is about 700 Da. In some instances, the molecular weight of C is about 800 Da. In some instances, the molecular weight of C is about 900 Da. In some instances, the molecular weight of C is about 1000 Da. In some instances, the molecular weight of C is about 1100 Da. In some instances, the molecular weight of C is about 1200 Da. In some instances, the molecular weight of C is about 1300 Da. In some instances, the molecular weight of C is about 1400 Da. In some instances, the molecular weight of C is about 1450 Da. In some instances, the molecular weight of C is about 1500 Da. In some instances, the molecular weight of C is about 1600 Da. In some instances, the molecular weight of C is about 1700 Da. In some instances, the molecular weight of C is about 1800 Da. In some instances, the molecular weight of C is about 1900 Da. In some instances, the molecular weight of C is about 2000 Da. In some instances, the molecular weight of C is about 2100 Da. In some instances, the molecular weight of C is about 2200 Da. In some instances, the molecular weight of C is about 2300 Da. In some instances, the molecular weight of C is about 2400 Da. In some instances, the molecular weight of C is about 2500 Da. In some instances, the molecular weight of C is about 2600 Da. In some instances, the molecular weight of C is about 2700 Da. In some instances, the molecular weight of C is about 2800 Da. In some instances, the molecular weight of C is about 2900 Da. In some instances, the molecular weight of C is about 3000 Da. In some instances, the molecular weight of C is about 3250 Da. In some instances, the molecular weight of C is about 3350 Da. In some instances, the molecular weight of C is about 3500 Da. In some instances, the molecular weight of C is about 3750 Da. In some instances, the molecular weight of C is about 4000 Da. In some instances, the molecular weight of C is about 4250 Da. In some instances, the molecular weight of C is about 4500 Da. In some instances, the molecular weight of C is about 4600 Da. In some instances, the molecular weight of C is about 4750 Da. In some instances, the molecular weight of C is about 5000 Da. In some instances, the molecular weight of C is about 5500 Da. In some instances, the molecular weight of C is about 6000 Da. In some instances, the molecular weight of C is about 6500 Da. In some instances, the molecular weight of C is about 7000 Da. In some instances, the molecular weight of C is about 7500 Da. In some instances, the molecular weight of C is about 8000 Da. In some instances, the molecular weight of C is about 10,000 Da. In some instances, the molecular weight of C is about 12,000 Da. In some instances, the molecular weight of C is about 20,000 Da. In some instances, the molecular weight of C is about 35,000 Da. In some instances, the molecular weight of C is about 40,000 Da. In some instances, the molecular weight of C is about 50,000 Da. In some instances, the molecular weight of C is about 60,000 Da. In some instances, the molecular weight of C is about 100,000 Da.
In some embodiments, the polyalkylene oxide (e.g., PEG) is a discrete PEG, in which the discrete PEG is a polymeric PEG comprising more than one repeating ethylene oxide units. In some instances, a discrete PEG (dPEG) comprises from 2 to 60, from 2 to 50, or from 2 to 48 repeating ethylene oxide units. In some instances, a dPEG comprises about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, 26, 28, 30, 35, 40, 42, 48, 50 or more repeating ethylene oxide units. In some instances, a dPEG comprises about 2 or more repeating ethylene oxide units. In some instances, a dPEG comprises about 3 or more repeating ethylene oxide units. In some instances, a dPEG comprises about 4 or more repeating ethylene oxide units. In some instances, a dPEG comprises about 5 or more repeating ethylene oxide units. In some instances, a dPEG comprises about 6 or more repeating ethylene oxide units. In some instances, a dPEG comprises about 7 or more repeating ethylene oxide units. In some instances, a dPEG comprises about 8 or more repeating ethylene oxide units. In some instances, a dPEG comprises about 9 or more repeating ethylene oxide units. In some instances, a dPEG comprises about 10 or more repeating ethylene oxide units. In some instances, a dPEG comprises about 11 or more repeating ethylene oxide units. In some instances, a dPEG comprises about 12 or more repeating ethylene oxide units. In some instances, a dPEG comprises about 13 or more repeating ethylene oxide units. In some instances, a dPEG comprises about 14 or more repeating ethylene oxide units. In some instances, a dPEG comprises about 15 or more repeating ethylene oxide units. In some instances, a dPEG comprises about 16 or more repeating ethylene oxide units. In some instances, a dPEG comprises about 17 or more repeating ethylene oxide units. In some instances, a dPEG comprises about 18 or more repeating ethylene oxide units. In some instances, a dPEG comprises about 19 or more repeating ethylene oxide units. In some instances, a dPEG comprises about 20 or more repeating ethylene oxide units. In some instances, a dPEG comprises about 22 or more repeating ethylene oxide units. In some instances, a dPEG comprises about 24 or more repeating ethylene oxide units. In some instances, a dPEG comprises about 26 or more repeating ethylene oxide units. In some instances, a dPEG comprises about 28 or more repeating ethylene oxide units. In some instances, a dPEG comprises about 30 or more repeating ethylene oxide units. In some instances, a dPEG comprises about 35 or more repeating ethylene oxide units. In some instances, a dPEG comprises about 40 or more repeating ethylene oxide units. In some instances, a dPEG comprises about 42 or more repeating ethylene oxide units. In some instances, a dPEG comprises about 48 or more repeating ethylene oxide units. In some instances, a dPEG comprises about 50 or more repeating ethylene oxide units. In some cases, a dPEG is synthesized as a single molecular weight compound from pure (e.g., about 95%, 98%, 99%, or 99.5%) staring material in a step-wise fashion. In some cases, a dPEG has a specific molecular weight, rather than an average molecular weight. In some cases, a dPEG described herein is a dPEG from Quanta Biodesign, LMD.
In some embodiments, the dsRNA agent comprises mismatch(es) with the target, within the duplex, or combinations thereof. The mismatch can occur in the overhang region or the duplex region. The base pair can be ranked on the basis of their propensity to promote dissociation or melting (e.g., on the free energy of association or dissociation of a particular pairing, the simplest approach is to examine the pairs on an individual pair basis, though next neighbor or similar analysis can also be used). In terms of promoting dissociation: A:U is preferred over G:C; G:U is preferred over G:C; and I:C is preferred over G:C (I=inosine). Mismatches, e.g., non-canonical or other than canonical pairings (as described elsewhere herein) are preferred over canonical (A:T, A:U, G:C) pairings; and pairings which include a universal base are preferred over canonical pairings.
In some embodiments, the dsRNA agent can comprise a phosphorus-containing group at the 5′-end of the sense strand or antisense strand. The 5′-end phosphorus-containing group can be 5′-end phosphate (5′-P), 5′-end phosphorothioate (5′-PS), 5′-end phosphorodithioate (5′-PS2), 5′-end vinylphosphonate (5′-VP), 5′-end methylphosphonate (MePhos), or 5′-deoxy-5′-C-malonyl. When the 5′-end phosphorus-containing group is 5′-end vinylphosphonate (5′-VP), the 5′-VP can be either 5′-E-VP isomer, such as trans-vinylphosphate or cis-vinylphosphate, or mixtures thereof. Representative structures of these modifications can be found in, for example, U.S. Pat. No. 10,233,448, which is hereby incorporated by reference in its entirety.
In some embodiments, the dsRNA agents are 5′ phosphorylated or include a phosphoryl analog at the 5′ prime terminus. 5′-phosphate modifications include those which are compatible with RISC mediated gene silencing. Suitable modifications include: 5′-monophosphate ((HO2(O)P—O-5′); 5′-diphosphate ((HO)2(O)P—O—P(HO)(O)—O-5′); 5′-triphosphate ((HO)2(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′); 5′-guanosine cap (7-methylated or non-methylated) (7m-G-O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′); 5′-adenosine cap (Appp), and any modified or unmodified nucleotide cap structure (N—O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′); 5′-monothiophosphate (phosphorothioate; (HO)2(S)P—O-5′); 5′-monodithiophosphate (phosphorodithioate; (HO)(HS)(S)P—O-5′), 5′-phosphorothiolate ((HO)2(O)P—S-5′); any additional combination of oxygen/sulfur replaced monophosphate, diphosphate and triphosphates (e.g. 5′-alpha-thiotriphosphate, 5′-gamma-thiotriphosphate, etc.), 5′-phosphoramidates ((HO)2(O)P—NH-5′, (HO)(NH2)(O)P—O-5′), 5′-alkylphosphonates (R=alkyl=methyl, ethyl, isopropyl, propyl, etc., e.g. RP(OH)(O)—O-5′-, 5′-alkenylphosphonates (i.e. vinyl, substituted vinyl), (OH)2(O)P-5′-CH2-), 5′-alkyletherphosphonates (R=alkylether=methoxymethyl (MeOCH2-), ethoxymethyl, etc., e.g. RP(OH)(O)—O-5′-). In some embodiments, the modification can in placed in the antisense strand of a dsRNA agent.
In some embodiments, the antisense strand of the dsRNA agent is 100% complementary to a target RNA to hybridize thereto and inhibits its expression through RNA interference. The target RNA can be any RNA expressed in a cell. In another embodiment, the antisense strand of the dsRNA agent is at least 99%, at least 98%, at least 97%, at least 96%, 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, or at least 50% complementary to a target RNA. In some embodiments, the target RNA is KRAS RNA. In some embodiments, the target RNA is NRAS or HRAS. In some embodiments, the siRNA targets KRAS but does not significantly target NRAS or HRAS. In some embodiments, the siRNA molecule is a siRNA that reduces the expression of KRAS and does not significantly reduce the expression of HRAS and NRAS. In some embodiments, the siRNA molecule is a siRNA that reduces the expression of KRAS and does not reduce the expression of HRAS and NRAS by more than 50% in an assay described herein at a concentration of no more than 200 nm as described herein.
The siRNA can be targeted against any gene or RNA (e.g. mRNA) transcript of interest. In some embodiments, the KRAS transcript that is targeted can have a substitution that would encode a G12C, G12V, G12S and G12D mutation in the KRAS protein. Accordingly, in some embodiments, the siRNA targets a KRAS transcript that encodes for a KRAS mutant protein comprising a G12C, G12V, G12S and/or G12D mutation (substitution).
Other modifications and patterns of modifications can be found in, for example, U.S. Pat. No. 10,233,448, which is hereby incorporated by reference.
In some embodiments the siRNA is linked to a protein, such as a FN3 domain The siRNA can be linked to multiple FN3 domains that bind to the same target protein or different target proteins.
In some embodiments, compositions are provided herein having a formula of (X1)n-(X2)q-(X3)y-L-X4, wherein X1 is a first FN3 domain, X2 is second FN3 domain, X3 is a third FN3 domain or half-life extender molecule, L is a linker, and X4 is a nucleic acid molecule, such as, but not limited to a siRNA molecule, wherein n, q, and y are each independently 0 or 1. In some embodiments, X1, X2, and X3 bind to different target proteins. In some embodiments, y is 0. In some embodiments, n is 1, q is 0, and y is 0. In some embodiments, n is 1, q is 1, and y is 0. In some embodiments, n is 1, q is 1, and y is 1. In some embodiments, the third FN3 domain increases the half-life of the molecule as a whole as compared to a molecule without X3. In some embodiments, the half-life extending moiety is a FN3 domain that binds to albumin Examples of such FN3 domains include, but are not limited to, those described in U.S. Patent Application Publication No. 20170348397 and U.S. Pat. No. 9,156,887, which is hereby incorporated by reference in its entirety. The FN3 domains may incorporate other subunits for example via covalent interaction. In some embodiments, the FN3 domains further comprise a half-life extending moiety. Exemplary half-life extending moieties are albumin, albumin variants, albumin-binding proteins and/or domains, transferrin and fragments and analogues thereof, and Fc regions Amino acid sequences of the human Fc regions are well known, and include IgG1, IgG2, IgG3, IgG4, IgM, IgA and IgE Fc regions. In some embodiments, the FN3 domains may incorporate a second FN3 domain that binds to a molecule that extends the half-life of the entire molecule, such as, but not limited to, any of the half-life extending moieties described herein. In some embodiments, the second FN3 domain binds to albumin, albumin variants, albumin-binding proteins and/or domains, and fragments and analogues thereof.
In some embodiments, compositions are provided herein having a formula of (X1)-(X2)-L-(X4), wherein X1 is a first FN3 domain, X2 is second FN3 domain, L is a linker, and X4 is a nucleic acid molecule. In some embodiments, X4 is a siRNA molecule. In some embodiments, X1 is a FN3 domain that binds to one of CD71, EGFR, or EpCAM. In some embodiments, X2 is a FN3 domain that binds to one of CD71, EGFR, or EpCAM. In some embodiments X1 and X2 do not bind to the same target protein. In some embodiments, X1 and X2 bind to the same target protein, but at different binding sites on the protein. In some embodiments, X1 and X2 bind to the same target protein. In some embodiments, X1 and X2 are FN3 domains that bind to CD71. In some embodiments, X1 and X2 are FN3 domains that bind to EpCAM. In some embodiments, X1 is a FN3 domain that binds to CD71 and X2 is a FN3 domain that binds to EpCAM. In some embodiments, X1 is a FN3 domain that binds to EpCAM and X2 is a FN3 domain that binds to CD71. In some embodiments, any of the FN3 domains listed above or herein can be replaced or substituted with a FN3 domain that binds to EGFR. Non-limiting examples of EGFR FN3 binding domains are provided herein and can also be found in U.S. Pat. No. 9,695,228, which is hereby incorporated by reference in its entirety. In some embodiments, the composition does not comprise (e.g. is free of) a compound or protein that binds to ASGPR.
In some embodiments, compositions or complexes are provided having a formula of A1-B1, wherein A1 has a formula of C-L1-Xs and B1 has a formula of XAS-L2-F1, wherein:

    
    
        C is a polymer, such as PEG;
        L1 and L2 are each, independently, a linker;
        XS is a 5′ to 3′ oligonucleotide sense strand of a double stranded siRNA molecule;
        XAS is a 3′ to 5′ oligonucleotide antisense strand of a double stranded siRNA molecule;
        F1 is a polypeptide comprising at least one FN3 domain;
        wherein XS and XAS form a double stranded oligonucleotide molecule to form the composition/complex.
    
    


In some embodiments, the sense strand is a sense strand as provided for herein.
In some embodiments, the antisense strand is an antisense strand as provided for herein.
In some embodiments, the sense and antisense strand form a double stranded siRNA molecule that targets RAS, such as KRAS. In some embodiments, the double stranded oligonucleotide is about 21-23 nucleotides base pairs in length.
In some embodiments, C is a natural or synthetic polymer, consisting of long chains of branched or unbranched monomers, and/or cross-linked network of monomers in two or three dimensions In some instances, the polymer includes a polysaccharide, lignin, rubber, or polyalkylen oxide, which can be for example, polyethylene glycol. In some instances, the at least one polymer includes, but is not limited to, alpha-, omega-dihydroxylpolyethyleneglycol, biodegradable lactone-based polymer, e.g. polyacrylic acid, polylactide acid (PLA), poly(glycolic acid) (PGA), polypropylene, polystyrene, polyolefin, polyamide, polycyanoacrylate, polyimide, polyethylenterephthalat (PET, PETG), polyethylene terephthalate (PETE), polytetramethylene glycol (PTG), or polyurethane as well as mixtures thereof. As used herein, a mixture refers to the use of different polymers within the same compound as well as in reference to block copolymers. In some cases, block copolymers are polymers wherein at least one section of a polymer is build up from monomers of another polymer. In some instances, the polymer comprises polyalkylene oxide. In some instances, the polymer comprises PEG. In some instances, the polymer comprises polyethylene imide (PEI) or hydroxy ethyl starch (HES).
In some embodiments, the polyalkylene oxide (e.g., PEG) is a polydispers or monodispers compound. In some instances, polydispers material comprises disperse distribution of different molecular weight of the material, characterized by mean weight (weight average) size and dispersity. In some instances, the monodisperse PEG comprises one size of molecules. In some embodiments, C is poly- or monodispersed polyalkylene oxide (e.g., PEG) and the indicated molecular weight represents an average of the molecular weight of the polyalkylene oxide, e.g., PEG, molecules.
In some embodiments, the molecular weight of the polyalkylene oxide (e.g., PEG) is about 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1450, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3250, 3350, 3500, 3750, 4000, 4250, 4500, 4600, 4750, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 10,000, 12,000, 20,000, 35,000, 40,000, 50,000, 60,000, or 100,000 Da.
In some embodiments, C is polyalkylene oxide (e.g., PEG) and has a molecular weight of about 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1450, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3250, 3350, 3500, 3750, 4000, 4250, 4500, 4600, 4750, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 10,000, 12,000, 20,000, 35,000, 40,000, 50,000, 60,000, or 100,000 Da. In some embodiments, C is PEG and has a molecular weight of about 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1450, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3250, 3350, 3500, 3750, 4000, 4250, 4500, 4600, 4750, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 10,000, 12,000, 20,000, 35,000, 40,000, 50,000, 60,000, or 100,000 Da. In some instances, the molecular weight of C is about 200 Da. In some instances, the molecular weight of C is about 300 Da. In some instances, the molecular weight of C is about 400 Da. In some instances, the molecular weight of C is about 500 Da. In some instances, the molecular weight of C is about 600 Da. In some instances, the molecular weight of C is about 700 Da. In some instances, the molecular weight of C is about 800 Da. In some instances, the molecular weight of C is about 900 Da. In some instances, the molecular weight of C is about 1000 Da. In some instances, the molecular weight of C is about 1100 Da. In some instances, the molecular weight of C is about 1200 Da. In some instances, the molecular weight of C is about 1300 Da. In some instances, the molecular weight of C is about 1400 Da. In some instances, the molecular weight of C is about 1450 Da. In some instances, the molecular weight of C is about 1500 Da. In some instances, the molecular weight of C is about 1600 Da. In some instances, the molecular weight of C is about 1700 Da. In some instances, the molecular weight of C is about 1800 Da. In some instances, the molecular weight of C is about 1900 Da. In some instances, the molecular weight of C is about 2000 Da. In some instances, the molecular weight of C is about 2100 Da. In some instances, the molecular weight of C is about 2200 Da. In some instances, the molecular weight of C is about 2300 Da. In some instances, the molecular weight of C is about 2400 Da. In some instances, the molecular weight of C is about 2500 Da. In some instances, the molecular weight of C is about 2600 Da. In some instances, the molecular weight of C is about 2700 Da. In some instances, the molecular weight of C is about 2800 Da. In some instances, the molecular weight of C is about 2900 Da. In some instances, the molecular weight of C is about 3000 Da. In some instances, the molecular weight of C is about 3250 Da. In some instances, the molecular weight of C is about 3350 Da. In some instances, the molecular weight of C is about 3500 Da. In some instances, the molecular weight of C is about 3750 Da. In some instances, the molecular weight of C is about 4000 Da. In some instances, the molecular weight of C is about 4250 Da. In some instances, the molecular weight of C is about 4500 Da. In some instances, the molecular weight of C is about 4600 Da. In some instances, the molecular weight of C is about 4750 Da. In some instances, the molecular weight of C is about 5000 Da. In some instances, the molecular weight of C is about 5500 Da. In some instances, the molecular weight of C is about 6000 Da. In some instances, the molecular weight of C is about 6500 Da. In some instances, the molecular weight of C is about 7000 Da. In some instances, the molecular weight of C is about 7500 Da. In some instances, the molecular weight of C is about 8000 Da. In some instances, the molecular weight of C is about 10,000 Da. In some instances, the molecular weight of C is about 12,000 Da. In some instances, the molecular weight of C is about 20,000 Da. In some instances, the molecular weight of C is about 35,000 Da. In some instances, the molecular weight of C is about 40,000 Da. In some instances, the molecular weight of C is about 50,000 Da. In some instances, the molecular weight of C is about 60,000 Da. In some instances, the molecular weight of C is about 100,000 Da.
In some embodiments, the polyalkylene oxide (e.g., PEG) is a discrete PEG, in which the discrete PEG is a polymeric PEG comprising more than one repeating ethylene oxide units. In some instances, a discrete PEG (dPEG) comprises from 2 to 60, from 2 to 50, or from 2 to 48 repeating ethylene oxide units. In some instances, a dPEG comprises about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, 26, 28, 30, 35, 40, 42, 48, 50 or more repeating ethylene oxide units. In some instances, a dPEG comprises about 2 or more repeating ethylene oxide units. In some instances, a dPEG comprises about 3 or more repeating ethylene oxide units. In some instances, a dPEG comprises about 4 or more repeating ethylene oxide units. In some instances, a dPEG comprises about 5 or more repeating ethylene oxide units. In some instances, a dPEG comprises about 6 or more repeating ethylene oxide units. In some instances, a dPEG comprises about 7 or more repeating ethylene oxide units. In some instances, a dPEG comprises about 8 or more repeating ethylene oxide units. In some instances, a dPEG comprises about 9 or more repeating ethylene oxide units. In some instances, a dPEG comprises about 10 or more repeating ethylene oxide units. In some instances, a dPEG comprises about 11 or more repeating ethylene oxide units. In some instances, a dPEG comprises about 12 or more repeating ethylene oxide units. In some instances, a dPEG comprises about 13 or more repeating ethylene oxide units. In some instances, a dPEG comprises about 14 or more repeating ethylene oxide units. In some instances, a dPEG comprises about 15 or more repeating ethylene oxide units. In some instances, a dPEG comprises about 16 or more repeating ethylene oxide units. In some instances, a dPEG comprises about 17 or more repeating ethylene oxide units. In some instances, a dPEG comprises about 18 or more repeating ethylene oxide units. In some instances, a dPEG comprises about 19 or more repeating ethylene oxide units. In some instances, a dPEG comprises about 20 or more repeating ethylene oxide units. In some instances, a dPEG comprises about 22 or more repeating ethylene oxide units. In some instances, a dPEG comprises about 24 or more repeating ethylene oxide units. In some instances, a dPEG comprises about 26 or more repeating ethylene oxide units. In some instances, a dPEG comprises about 28 or more repeating ethylene oxide units. In some instances, a dPEG comprises about 30 or more repeating ethylene oxide units. In some instances, a dPEG comprises about 35 or more repeating ethylene oxide units. In some instances, a dPEG comprises about 40 or more repeating ethylene oxide units. In some instances, a dPEG comprises about 42 or more repeating ethylene oxide units. In some instances, a dPEG comprises about 48 or more repeating ethylene oxide units. In some instances, a dPEG comprises about 50 or more repeating ethylene oxide units. In some cases, a dPEG is synthesized as a single molecular weight compound from pure (e.g., about 95%, 98%, 99%, or 99.5%) staring material in a step-wise fashion. In some cases, a dPEG has a specific molecular weight, rather than an average molecular weight. In some cases, a dPEG described herein is a dPEG from Quanta Biodesign, LMD.
In some embodiments, L1 is any linker that can be used to link the polymer C to the sense strand XS. In some embodiments, L1 has a formula of:




In some embodiments, L2 is any linker that can be used to link the polypeptide of F1 to the antisense strand XAS. In some embodiments, L2 has a formula of in the complex of:




wherein XAS and F1 are as defined above. In some embodiments, the linker is covalently attached to F1 through a cysteine residue present on F1, which can be illustrated as follows:





In some embodiments, A1-B1 has a formula of:





wherein C1 is the polymer C, such as PEG as provided for herein, XS is a 5′ to 3′ oligonucleotide sense strand of a double stranded siRNA molecule; XAS is a 3′ to 5′ oligonucleotide antisense strand of a double stranded siRNA molecule; and F1 is a polypeptide comprising at least one FN3 domain, wherein XS and XAS form a double stranded siRNA molecule.
In some embodiments, F1 comprises polypeptide having a formula of (X1)n-(X2)q-(X3)y, wherein X1 is a first FN3 domain; X2 is second FN3 domain; X3 is a third FN3 domain or half-life extender molecule; wherein n, q, and y are each independently 0 or 1, provided that at least one of n, q, and y is 1. In some embodiments, n, q, and y are each 1. In some embodiments, n and q are 1 and y is 0. In some embodiments n and y are 1 and q is 0.
In some embodiment X1 is a CD71 FN3 binding domain, such as one provided herein. In some embodiments, X2 is a CD71 FN3 binding domain. In some embodiments, X1 and X2 are different CD71 FN3 binding domains In some embodiments, the binding domains are the same. In some embodiments, X3 is a FN3 domain that binds to human serum albumin In some embodiments, X3 is a Fc domain without effector function that extends the half-life of a protein. In some embodiments, X1 is a first CD71 binding domain, X2 is a second CD71 binding domain, and X3 is a FN3 albumin binding domain. In some embodiments, X2 is an EPCAM binding domain instead of a second CD71 binding domain. In some embodiments, X1 is an EPCAM binding FN3 domain, X2 is a CD71 FN3 binding domain, and X3 is an albumin FN3 binding domain. Examples of such polypeptides are provided herein and below. In some embodiments, compositions are provided herein having a formula of C—(X1)n-(X2)q-(X3)y-L-X4, wherein C is a polymer; X1 is a first FN3 domain; X2 is second FN3 domain; X3 is a third FN3 domain or half-life extender molecule; L is a linker; and X4 is a nucleic acid molecule, wherein n, q, and y are each independently 0 or 1.
In some embodiments, compositions are provided herein having a formula of (X1)n-(X2)q-(X3)y-L-X4-C, wherein X1 is a first FN3 domain; X2 is second FN3 domain; X3 is a third FN3 domain or half-life extender molecule; L is a linker; X4 is a nucleic acid molecule; and C is a polymer, wherein n, q, and y are each independently 0 or 1.
In some embodiments, compositions are provided herein having a formula of X4-L-(X1)n-(X2)q-(X3)y, wherein X1 is a first FN3 domain; X2 is second FN3 domain; X3 is a third FN3 domain or half-life extender molecule; L is a linker; and X4 is a nucleic acid molecule, wherein n, q, and y are each independently 0 or 1.
In some embodiments, compositions are provided herein having a formula of C—X4-L-(X1)n-(X2)q-(X3)y, wherein C is a polymer; X1 is a first FN3 domain; X2 is second FN3 domain; X3 is a third FN3 domain or half-life extender molecule; L is a linker; and X4 is a nucleic acid molecule, wherein n, q, and y are each independently 0 or 1.
In some embodiments, compositions are provided herein having a formula of X4-L-(X1)n-(X2)q-(X3)y-C, wherein X1 is a first FN3 domain; X2 is second FN3 domain; X3 is a third FN3 domain or half-life extender molecule; L is a linker; X4 is a nucleic acid molecule; and C is a polymer, wherein n, q, and y are each independently 0 or 1.
In some embodiments, the siRNA molecule comprises a sequence pair from Table 1.







Table 1







siRNA Sense and Anti-sense sequences














SEQ
Sense
SEQ
Anti-sense



siRNA
ID
Strand
ID
strand



Pair
NO
5′-3′
NO
5′-3′
















A
10
cscsUfgucUf
11
UfsGfsaauauc





CfUfugGfaua

caagaGfacagg





uUfca(invdT)

susu


 



B
12
CsasGfcuaAf
13
UfsAfsugauuc





UfUfcaGfaau

ugaauUfagcug





cAfua (invdT)

susu


 



C
14
GsasAfuuaGf
15
UfsUfsgacgau





CfufguAfucg

acagcUfaauuc





uCfaa (invdT)

susu


 



D
16
CfscsUfgUfc
17
usGfsaAfuAfU





UfCfUfuGfga

fCfcAfagaGfa





uAfuUfcAf

CfaGfgsUfsu





(invdT)




 



E
18
csAfsgCfuAf
19
usAfsuGfaUfU





aUfUfCfaGfa

fCfuGfaauUfa





auCfuAfuAf

GfcUfgsUfsu





(invdt)




 



F
20
GfsasAfuUfa
21
usUfsgAfcGf





GfCfUfgUfau

AfUfacaGfcU





cGfuCfaAf

faAfuUfcsUf





(invdt)

su


 



G
22
CfscsUfgUfc
23
usGfsaAfuAfu





UfcUfuGfgAf

CfcAfaGfaGfa





uAfuUfcAf

CfaGfgsUfsu





(invdT)




 



H
24
csAfsgCfuAf
25
UfsasUfgAfuU





aUfuCfaGfaA

fcUfgAfaUfuA





fuCfaUfa

fgCfgsUfsu





(invdt)




 



I
26
gsAfsaUfuA
27
UfsusGfaCfaU





fgCfuGfuAfu

faCfaGfeUfa





CfgUfcAfa

AfuUfcsUfsu





(invdt)




 



J
28
cscsUfgucUf
29
UfsGfsaauauc





CfUfugGfaua

caagaGfacag





uUfca(invdt)

gsusu


 



K
30
CsasGfcuaAf
31
UfsAfsugauu





UfUfcaGfaau

cugaauUfagc





cAfua(invdt)

ugsusu


 



L
32
gsasAfuuaGf
33
UfsUfsgacgau





CfUfguAfucg

acagcUfaauuc





uCfaa(invdt)

susu


 



M
34
CsasGfcuaAf
35
UfsAfsugauuc





UfUfcaGfaau

ugaauUfagcug





cAfua(invdT)

susu


 



N
36
asusAfuaaAf
37
UfsAfscuacca





CfUfugUfggu

caaguUfuauau





aGfua(invdT)

susu


 



O
38
usasAfacuUf
39
UfsCfscaacua





GfUfggUfagu

ccacaAfguuua





uGfga(invdT)

susu


 



P
40
csasAfgagUf
41
UfsAfsucguca





GfCfcuUfgac

aggcaCfucuug





gAfua(invdT)

susu


 



Q
42
gscsCfuugAf
43
UfsUfsagcugu





CfGfauAfcag

aucguCfaaggc





cUfaa(invdT)

susu


 



R
44
usgsAfcgaUf
45
UfsGfsaauuag





AfCfagCfuaa

cuguaUfcguca





uUfca(invdT)

susu


 



S
46
csgsAfuacAfG
47
UfsUfscugaauu





fCfuaAfuuc

agcuGfuaucgsu





aGfaa(invdT)

su


 



T
48
gsusGfgacGf
49
UfsUfsggauca





AfAfuaUfgau

uauucGfuccac





cCfaa(invdT)

susu


 



U
50
gsgsAfcgaAf
51
UfsGfsuuggau





UfAfugAfucc

cauauUfcgucc





aAfca(invdT)

susu


 



V
52
gsasCfgaaUf
53
UfsUfsguugga





AfUfgaUfcca

ucauaUfucguc





aCfaa(invdT)

susu


 



W
54
ascsGfaauAf
55
UfsUfsuguugg





UfGfauCfcaa

aucauAfuucgu





cAfaa(invdT)

susu


 



X
56
csgsAfauaUf
57
UfsAfsuuguug





GfAfucCfaac

gaucaUfauucg





aAfua(invdT)

susu


 



Y
58
asasUfaugAf
59
UfsCfsuauugu





UfCfcaAfcaa

uggauCfauauu





uAfga(invdT)

susu


 



Z
60
gsasUfccaAf
61
UfsAfsuccucua





CfAfauAfgag

uuguUfggaucsu





gAfua(invdT)

su


 



AA
62
cscsAfacaAf
63
UfsGfsgaauccu





UfAfgaGfgau

cuauUfguuggs





uCfca(invdT)

usu


 



BB
64
csusAfcagGf
65
UfsAfscuacuug





AfAfgcAfagu

cuucCfuguags





aGfua(invdT)

usu


 



CC
66
ascsAfggaAf
67
UfsUfsuacuacu





GfCfaaGfuag

ugcuUfccugusu





uAfaa(invdT)

su


 



DD
68
gsusAfauuGf
69
UfsGfsguuucuc





AfUfggAfgaa

caucAfauuacsu





aCfca(invdT)

su


 



EE
70
csusUfggaUf
71
UfsGfsugucgag





AfUfucUfcga

aauaUfccaagsu





cAfca(invdT)

su


 



FF
72
csasGfcagGf
73
UfsAfscuccucu





UfCfaaGfagg

ugacCfugcugsu





aGfua(invdT)

su


 



GG
74
gscsAfaugAf
75
UfsGfsuacuggu





GfGfgaCfcag

cccuCfauugcsu





uAfca(invdT)

su


 



HH
76
csasAfugaGf
77
UfsUfsguacugg





GfGfacCfagu

ucccUfcauugsu





aCfaa(invdT)

su


 



II
78
ususUfgugUf
79
UfsUfsuauggca





AfUfuuGfcca

aauaCfacaaasu





uAfaa(invdT)

su


 



JJ
80
ususGfccaUf
81
UfsUfsaguauua





AfAfauAfaua

uuuaUfggcaasu





cUfaa(invdT)

su


 



KK
82
usgsCfcauAf
83
UfsUfsuaguauu





AfAfuaAfuac

auuuAfuggcasu





uAfaa(invdT)

su


 



LL
84
cscsAfuaaAf
85
UfsAfsuuuagua





UfAfauAfcua

uuauUfuauggsu





aAfua(invdT)

su


 



MM
86
csasUfaaaUf
87
UfsGfsauuuagu





AfAfuaCfuaaa

auuaUfuuaugsu





Ufca(invdT)

su


 



NN
88
asusAfaauAf
89
UfsUfsgauuuag





AfUfacUfaaa

uauuAfuuuausu





uCfaa(invdT)

su


 



OO
90
gsasAfgauAf
91
UfsAfsuaauggu





UfUfcaCfcau

gaauAfucuucsu





uAfua(invdT)

su


 



PP
92
asgsAfuauUf
93
UfsCfsuauaaug





CfAfccAfuua

gugaAfuaucusu





uAfga(invdT)

su


 



QQ
94
asusAfuucAf
95
UfsCfsucuauaa





CfCfauUfaua

ugguGfaauausu





gAfga(invdT)

su


 



RR
96
asgsAfacaAf
97
UfsAfscucuuu





AfUfuaAfaag

uaauuUfguucu





aGfua(invdT)

susu


 



SS
98
gsasCfucuGf
99
UfsAfsgguaca





AfAfgaUfgua

ucuucAfgaguc





cCfua(invdT)

susu


 



TT
100
csusGfaagAf
101
UfsCfscauagg





UfGfuaCfcua

uacauCfuucag





uGfga(invdT)

susu


 



UU
102
asgsAfacaGf
103
UfsUfsuuugug





UfAfgaCfacaa

ucuacUfguucu





Afaa(invdT)

susu


 



VV
104
csasGfgacUf
105
UfsAfscuucuu





UfAfgcAfaga

gcuaaGfuccug





aGfua(invdT)

susu


 



WW
106
gsusUfgauGf
107
UfsAfsuagaag





AfUfgcCfuuc

gcaucAfucaac





uAfua(invdT)

susu


 



XX
108
asusGfaugCf
109
UfsAfsuguaua





CfUfucUfaua

gaaggCfaucau





cAfua(invdT)

susu


 



YY
110
usgsAfugcCf
111
UfsAfsauguau





UfUfcuAfuac

agaagGfcauca





aUfua(invdT)

susu


 



ZZ
112
gsasUfgccUf
113
UfsUfsaaugua





UfCfuaUfaca

uagaaGfgcauc





uUfaa(invdT)

susu


 



AAA
114
asusGfccuUf
115
UfsCfsuaaugu





CfUfauAfcau

auagaAfggcau





uAfga(invdT)

susu


 



BBB
116
csusUfcuaUf
117
UfsCfsgaacua





AfCfauUfagu

auguaUfagaag





uCfga(invdT)

susu


 



CCC
118
UscsUfauaCf
119
UfsCfsucgaac





AfUfuaGfuuc

uaaugUfauaga





gAfga(invdT)

susu


 



DDD
120
UsasUfacaUf
121
UfsUfsucucga





UfAfguUfcga

acuaaUfguaua





gAfaa(invdT)

susu


 



EEE
122
AsusAfcauUf
123
UfsUfsuucucg





AfGfuuCfga

aacuaAfuguau





gaAfaa(invdT)

susu


 



FFF
124
UsasCfauuAf
125
UfsAfsuuucuc





GfUfucGfaga

gaacuAfaugua





aAfua(invdT)

susu


 



GGG
126
UsusAfguuCf
127
UfsUfscgaauu





GfAfgaAfau

ucucgAfacuaa





ucGfaa(invdT)

susu


 



HHH
128
AsgsUfucgAf
129
UfsUfsuucgaa





GfAfaaUfuc

uuucuCfgaacu





gaAfaa(invdT)

susu


 



III
130
AsgsAfaauUf
131
UfsUfsuauguu





CfGfaaAfaca

uucgaAfuuucu





uAfaa(invdT)

susu


 



JJJ
132
GsasAfauuCf
133
UfsUfsuuaugu





GfAfaaAfcau

uuucgAfauuuc





aAfaa(invdT)

susu


 



KKK
134
AsasAfuucGf
135
UfsCfsuuuaug





AfAfaaCfaua

uuuucGfaauuu





aAfga(invdT)

susu


 



LLL
136
AsasUfucgAf
137
UfsUfscuuuau





AfAfacAfuaa

guuuuCfgaauu





aGfaa(invdT)

susu


 



MMM
138
AsusGfagcAf
139
UfsUfsuuacca





AfAfgaUfgg

ucuuuGfcucau





uaAfaa(invdT)

susu


 



NNN
140
AsgsCfaaaGf
141
UfsCfsuuuuua





AfUfggUfaaa

ccaucUfuugcu





aAfga(invdT)

sus







u


 



OOO
142
AsusUfucuGfU
143
UfsAfsaacccc





fCfuuGfgg

aagacAfgaaau





guUfua(invdT)

susu


 



PPP
144
GsgsGfuuuUf
145
UfsUfsgcaug





UfGfguGfca

caccaaAfaac





ugCfaa(invdT)

ccsusu


 



QQQ
146
CsgsCfacaAf
147
UfsUfsaccca





GfGfcaCfugg

gugccuUfgug





gUfaa(invdT)

cgsusu


 



RRR
148
GscsAfcaaGf
149
UfsAfsuaccc





GfCfacUfggg

agugccUfugug





uAfua(invdT)

csusu


 



SSS
150
csUfsCfUfuG
151
usGfsasAfsu





fgauAfuUfc

sAfUfCfcAfag





Af(invdT)

aGfaCfaGfgsU







fsu


 



TTT
152
AfsasUfUfCf
153
usAfsusGfsas





aGfaauCfuAf

UfUfCfuGfaau





uAf(invdt)

UfaGfcUfgsUf







su


 



UUU
154
AfsasUfUfCf
155
usAfsusGfsas





aGfaauCfuAf

UfUfCfuGfaau





uAf(invdt)

UfaGfcUfgsUf







su


 



VVV
156
csUfscUfuGf
157
usGfsaAfsusAf





gAfuAfuUfc

suCfcAfaGfaG





Af(invdT)

faCfaGfgsUfsu-


 



WWW
158
AfsasUfuCfa
159
UfsasUfsgsAfs





GfaAfuCfaUf

uUfcUfgAfaUfu





a(invdt)

AfgCfgsUfsu


 



XXX
160
AfsgsCfuGfu
161
UfsusGfsasCfs





AfuCfgUfcAf

aUfaCfaGfcUfa





a(invdt)

AfuUfcsUfsu


 



YYY
162
csUfsCfUfug
163
UfsGfsasasusa





GfauauUfca

uccaagaGfacag





(invdt)-

gsusu


 



ZZZ
164
asAfsUfUfca
165
UfsAfsusgsasu





GfaaucAfua

ucugaauUfagcu





(invdt)

gsusu


 



AAAA
166
asGfsCfUfgu
167
UfsUfsgsascsga





AfucguCfaa

uacagcUfaauucs





(invdt)

usu


 



BBBB
168
CfscsUfgUfc
169
usGfsaAfuAfUf





UfCfUfuGfga

CfcAfagaGfaCf





uAfgUfcAf

aGfgsUfsu





(invdT)-




 



CCCC
170
csAfsgCfuAf
171
usAfsuGfaUfUf





aUfUfCfaGfa

CfuGfaauUfaGf





auCfgAfuAf

cUfgsUfsu-





(invdt)-




 



DDDD
172
GfsasAfuUf
173
usUfsgAfcGfAf





aGfUfgUfau

UfacaGfcUfaAf





cGfgCfaAf

uUfcsUfsu-





(invdt)




 



EEEE
174
CfscsUfgUf
175
usGfsaAfuAfuC





cUfcUfuGfg

fcAfaGfaGfaCf





AfuAfgUfcA

aGfgsUfsu





f(invdT)-




 



FFFF
176
csAfsgCfuA
177
UfsasUfgAfuUf





faUfuCfaGf

cUfgAfaUfuAfg





aAfgCfaUfa

CfgsUfsu





(invdt)




 



GGGG
178
gsAfsaUfuA
179
UfsusGfaCfaUf





fgCfuGfuAf

aCfaGfcUfaAfu





uCfgUfcAfa

UfcsUfsu





(invdt)




 



HHHH
180
cscsUfgucU
181
UfsGfsaauaucc





fCfUfugGfa

aagaGfacaggsu





uagUfca

su





(invdt)




 



IIII
182
csasGfcuaA
183
UfsAfsugauucu





fUfUfcaGfa

gaauUfagcugsu





agcAfua

su





(invdt)




 



JJJJ
184
gsasAfuuaG
185
UfsUfsgacgaua





fCfUfguAfu

cagcUfaauucsu





cggCfaa

su





(invdt)




 



KKKK
212
CcsAcsrGrC
213
(vinyl-p)sAfs





rUrArArUrUr

uGfaUfUfCfuGf





CrArGrArArU

aauUfaGfcUfgU





rCrAsTcsAc

fsasUf


 



LLLL
214
CfsasGfcUfa
215
(vinyl-p)-





AfUfUfcAfga

sAfsuGfaUfUfC





aUfcAfua

fuGfaauUfaGfc







UfgUfsasUf


 



MMMM
216
csasrGrCrUr
217
(vinyl-p)





ArArUrUrCrA

sAfsuGfaUf





rGrArArUrCr

UfCfuGfaauU





Asusa

faGfcUfgUfs







asUf





Abbreviations Key: n = 2′-O-methyl re sidues, Nf = 2′-F residues, rN = unmodified residue, Nc = 2′,4′-BNAnc (2′-O,4′-C-aminomethylene bridged nucleic acid), s = phosphorothioate, (invdt) = inverted Dt, vinyl-p: (E)-vinylphosphonate, (n/N = anynucleotide)






As described herein, in some embodiments, the nucleic acid molecules can be modified to include a linker at the 5′ end of the of the sense strand of the dsRNA. In some embodiments, the nucleic acid molecules can be modified to include a vinyl phosphonate at the 5′ end of the of the anti-sense strand of the dsRNA. In some embodiments, the nucleic acid molecules can be modified to include a linker at the 3′ end of the of the sense strand of the dsRNA. In some embodiments, the nucleic acid molecules can be modified to include a vinyl phosphonate at the 3′ end of the of the anti-sense strand of the dsRNA. The linker can be used to link the dsRNA to the FN3 domain. The linker can covalently attach, for example, to a cysteine residue on the FN3 domain that is there naturally or that has been substituted as described herein, and for example, in U.S. Pat. No. 10,196,446, which is hereby incorporated by reference in its entirety. Non-limiting examples of such modified strands of the dsRNA are illustrated in Table 2.







TABLE 2







Pairs with Linker and/or vinyl phosphonate













SEQ

SEQ





ID

ID





NO
Sense 5′-3′
NO
Antisense 5′-3′
Linker





AB01
186
L-
187
(vinyl-p)-
mal-




cscsUfgucUfCfUfugGfaua

UfsGfsaauauccaagaGfa
NH—(CH2)6—




uUfca(invdT)

caggsusu



AB02
188
L-
189
(vinyl-p)-
mal-




csasGfcuaAfUfUfcaGfaauc

UfsAfsugauucugaauUf
NH—(CH2)6—




Afua(invdT)

agcugsusu



AB03
190
CfsasGfcUfaAfUfUfcAfga
191
(vinyl-p)-
mal- 




aUfcAfua-L

sAfsuGfaUfUfCfuGfaa
C2H4C(O)NH—(CH2)6—






uUfaGfcUfgUfsasUf



AB04
192
CfsasGfcUfaAfuUfcAfgAf
193
(vinyl-p)-
mal-




aUfcAfua-L

sAfsuGfaUfuCfuGfaAf
C2H4C(O)NH—(CH2)6—






uUfaGfcUfgUfsasUf



AB05
194
(L)cscsUfgucUfCfUfugGfa
195
(vinu)sGfsaauauccaaga
mal-




uauUfca(invdT)

Gfacaggsusu
C2H4C(O)NH—(CH2)6—


AB06
196
(L)csasGfcuaAfUfUfcaGfa
197
(vinu)sAfsugauucugaau
mal-




aucAfua

Ufagcugsusu
C2H4C(O)NH—(CH2)6—


AB07
198
(L)cscsUfgUfcUfcUfuGfg
199
(vinu)sGfsaAfuAfuCfc
mal-




AfuAfuUfcAf(invdT)

AfaGfaGfaCfaggsusu
C2H4C(O)NH—(CH2)6—


AB08
200
cscsUfgucUfCfUfugGfaua
201
(vinu)sGfsaauauccaaga
mal-




uUfca(L)

Gfacaggsusu
C2H4C(O)NH—(CH2)6—


AB09
202
(L)cscsUfgucUfCfUfugGfa
203
(vinu)sGfsaauauccaaga
(Mal-




uauUfca(invdT)

Gfacaggsusu
(PEG)12NH(CH2)6)


AB10
204
CfscsUfgUfcUfCfUfuGfga
205
(vinu)sGfsaAfuAfUfCf
mal-




uAfuUfcAf(L)-

cAfagaGfaCfaGfgsUfs
C2H4C(O)NH—(CH2)6—






u



AB11
206
CfsasGfcUfaAfUfUfcAfga
207
vinu)sAfsuGfaUfUfCfu
mal-




aUfcAfuAf(L)-

GfaaufaGfcfgsUfsu-
C2H4C(O)NH—(CH2)6—


AB12
208
usUfsgAfcGfaUfaCfAfGfc
209
vinu)sGfsaAfuUfAfGfc
mal-




UfaauUfcAfuAf(L)

fguaUfcGfuCfaAfsgsG
C2H4C(O)NH—(CH2)6—






f



AB13
210
(vinu)CfsasGfcUfaAfUfUf
211
AfsuGfaUfUfCfuGfaau
mal-




cAfgaaUfcAfua

UfaGfcUfgUfsasUf-L
C2H4C(O)NH—(CH2)6—


AB14
218
CCsACsrGrCrUrArArUrUr
219
(vinyl-





CrArGrArArU

p)sAfsuGfaUfUfCfuGf





rCrAsTCsAC

aauUfaGfcUfgUfsasUf



AB15
220
X-
221
(vinyl-p)-
mal-




CfsasGfcUfaAfUfUfcAfga

sAfsuGfaUfUfCfuGfaa
C2H4C(O)(NH)—(CH2)6




aUfcAfua-L

uUfaGfcUfgUfsasUf



AB16
222
csasrGrCrUrArArUrUrCrA
223
(vinyl-
mal-




rGrArArUrCrAsusa-(L)

p)sAfsuGfaUfUfCfuGf
C2H4C(O)(NH)—(CH2)6






aauUfaGfcUfgUfsasUf





Abbreviations Key: n = 2′-O-methyl residues, Nf = 2′-F residues, rN = unmodified residue,


NC = 2′,4′-BNANC (2′-O,4′-C-aminomethylene bridged nucleic acid), s = phosphorothioate,


(invdt) = inverted Dt, Vinu = vinylphosphonate, vinyl-p = (E)-vinylphosphonate, (L) is a linker, and












Structure of the linkers (L) are as follows:
mal-C2H4C(O)(NH)-(CH2)6— is




(Mal-(PEG)12)(NH)CH2)6) is





and

Mal-NH—(CH2)6—, which can also be referred to as aminohexyl linker-(CH2)6—, is





When connected to the siRNA, the structures, L-(X4) can be represented by the following formulas:




Although certain siRNA sequences are illustrated herein with certain modified nucleobases, the sequences without such modifications are also provided herein. That is, the sequence can comprise the sequences illustrated in the tables provided herein without any modifications. The unmodified siRNA sequences can still comprise, in some embodiments, a linker at the 5′ end of the of the sense strand of the dsRNA. In some embodiments, the nucleic acid molecules can be modified to include a vinyl phosphonate at the 5′ end of the of the anti-sense strand of the dsRNA. In some embodiments, the nucleic acid molecules can be modified to include a linker at the 3′ end of the of the sense strand of the dsRNA. In some embodiments, the nucleic acid molecules can be modified to include a vinyl phosphonate at the 3′ end of the of the anti-sense strand of the dsRNA. The linker can be as provided herein.
In some embodiments, the FN3 proteins comprise a polypeptide comprising a polypeptide that binds CD71 are provided. In some embodiments, the polypeptide comprises a FN3 domain that binds to CD71. In some embodiments, the polypeptide comprises a sequence of SEQ ID NOs: 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, or 328 are provided. In some embodiments, the polypeptide that binds CD71 comprises a sequence of SEQ ID NOs: 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, or 328. The sequence of CD71 protein that the polypeptides can bind to can be, for example, SEQ ID Nos: 2 or 3. In some embodiments, the FN3 domain that binds to CD71 specifically binds to CD71.
In some embodiments, the FN3 domain that binds CD71 is based on Tencon sequence of SEQ ID NO:1 or Tencon 27 sequence of SEQ ID NO:4 (LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFLIQYQESEKVGEAIVLTVPGSERSYDLT GLKPGTEYTVSIYGVKGGHRSNPLSAIFTT), optionally having substitutions at residues positions 11, 14, 17, 37, 46, 73, or 86 (residue numbering corresponding to SEQ ID NO:4).
In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NOs: 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, or 317.
In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NOs: 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, or 328.
In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NOs: 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, or 623.
In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:300.
In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:301.
In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:302.
In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:303.
In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:304.
In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:305.
In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:306.
In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:307.
In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:308.
In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:309.
In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:310.
In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:311.
In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:312.
In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:313.
In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:314.
In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:315.
In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:316.
In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:317.
In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:318.
In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:319.
In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:320.
In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:321.
In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:322.
In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:323.
In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:324.
In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:325.
In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:326.
In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:327.
In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO:328.
In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 395. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 396. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 397. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 398. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 399. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 400. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 401. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 402. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 403. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 404. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 405. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 406. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 407. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 408. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 409. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 410. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 411. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 412. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 413. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 414. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 415. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 416. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 417. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 418. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 419. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 420. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 421. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 422. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 423. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 424. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 425. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 426. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 427. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 428. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 429. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 430. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 431. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 432. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 433. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 434. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 435. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 436. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 437. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 438. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 439. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 440. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 441. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 442. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 443. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 444. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 445. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 446. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 447. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 448. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 449. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 450. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 451. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 452. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 453. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 454. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 455. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 456. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 457. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 458. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 459. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 460. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 461. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 462. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 463. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 464. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 465. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 466. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 467. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 468. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 469. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 470. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 471. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 472. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 473. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 474. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 475. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 476. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 477. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 478. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 479. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 480. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 481. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 482. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 483. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 484. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 485. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 486. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 487. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 488. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 489. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 490. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 491. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 492. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 493. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 494. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 495. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 496. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 497. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 498. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 499. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 500. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 501. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 502. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 503. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 504. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 505. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 506. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 507. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 508. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 509. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 510. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 511. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 512. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 513. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 514. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 515. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 516. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 517. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 518. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 519. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 520. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 521. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 522. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 523. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 524. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 525. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 526. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 527. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 528. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 529. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 530. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 531. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 532. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 533. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 534. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 535. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 536. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 537. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 538. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 539. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 540. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 541. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 542. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 543. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 544. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 545. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 546. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 547. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 548. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 549. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 550. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 551. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 552. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 553. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 554. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 555. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 556. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 557. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 558. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 559. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 560. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 561. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 562. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 563. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 564. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 565. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 566. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 567. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 568. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 569. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 570. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 571. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 572. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 573. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 574. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 575. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 576. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 577. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 578. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 579. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 580. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 581. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 582. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 583. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 584. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 585. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 586. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 587. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 588. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 589. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 590. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 591. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 592. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 593. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 594. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 595. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 596. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 597. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 598. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 599. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 600. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 601. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 602. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 603. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 604. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 605. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 606. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 607. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 608. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 609. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 610. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 611. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 612. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 613. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 614. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 615. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 616. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 617. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 618. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 619. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 620. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 621. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 622. In some embodiments, an isolated FN3 domain that binds CD71 comprises the amino acid sequence of SEQ ID NO: 623.
In some embodiments, the isolated FN3 domain that binds CD71 comprises an initiator methionine (Met) linked to the N-terminus of the molecule.
In some embodiments, the isolated FN3 domain that binds CD71 comprises an amino acid sequence that is 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to one of the amino acid sequences of SEQ ID NOs: 300-317. In some embodiments, the isolated FN3 domain that binds CD71 comprises an amino acid sequence that is 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to one of the amino acid sequences of SEQ ID NOs: 318-328. In some embodiments, the isolated FN3 domain that binds CD71 comprises an amino acid sequence that is 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to one of the amino acid sequences of SEQ ID NOs: 395-623. Percent identity can be determined using the default parameters to align two sequences using BlastP available through the NCBI website. The sequences of the FN3 domains that bind to CD71 can be found, for example, in Table 3.







TABLE 3







CD71-binding FN3 domain sequences








SEQ ID
Amino Acid sequence of FN3 domains that bind to CD71











300
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFTIQYEELTTVGEAIYLR



VPGSERSYDLTGLKPGTEYVVWIEGVKGGLRSNPLGAAFTT


 


301
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFAITYIEWWDVGEAIGL



KVPGSERSYDLTGLKPGTEYRVHIQGVKGGNNSYPLDALFTT


 


302
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFEIAYFEAIWNGEAIYLT



VPGSERSYDLTGLKPGTEYQVEIRGVKGGPTSRPLFAWFTT


 


303
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFTITYIEWWENGEAIALS



VPGSERSYDLTGLKPGTEYQVGIAGVKGGYKSYPLWALFTT


 


304
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFHIIYTEEEKEGEAIYLRV



PGSERSYDLTGLKPGTEYLVEIEGVKGGKRSVPLNASFTT


 


305
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFHIAYEESHTTGEAIFLR



VPGSERSYDLTGLKPGTEYSVSIEGVKGGHYSPPLTAKFTT


 


306
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFDIDYREWWTLGEAIVL



TVPGSERSYDLTGLKPGTEYYVNIQGVKGGLRSYPLSAIFTT


 


307
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFVIEYWEYVGHGEAIVL



TVPGSERSYDLTGLKPGTEYSVGIYGVKGGSLSRPLSAIFTT


 


308
MLPAPKNLVISRVTEDSARLSWTAPDAAFDSFFIYYIESYPAGEAIVLTV



PGSERSYDLTGLKPGTEYWVGIDGVKGGRWSTPLSAIFTT


 


309
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFTIEYYESFYGGEAIVLT



VPGSERSYDLTGLKPGTEYYVSIYGVKGGWLSRPLSAIFTT


 


310
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFYIEYYESYPGGEAIVLT



VPGSERSYDLTGLKPGTEYDVYIYGVKGGYWSRPLSAIFTT


 


311
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFYIEYYESLPDGEAIVLT



VPGSERSYDLTGLKPGTEYAVYIYGVKGGYYSRPLSAIFTT


 


312
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFAIYYLESYPEGEAIVLT



VPGSERSYDLTGLKPGTEYWVGIDGVKGGTWSSPLSAIFTT


 


313
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFTIEYFEFTGTGEAIVLTV



PGSERSYDLTGLKPGTEYYVSIYGVKGGLLSAPLSAIFTT


 


314
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFTIWYAEALGDGEAIVL



TVPGSERSYDLTGLKPGTEYFVDIYGVKGGFWSLPLSAIFTT


 


315
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFVIEYFEQFNLGEAIVLT



VPGSERSYDLTGLKPGTEYWVGIYGVKGGWLSHPLSAIFTT


 


316
MLPAPKNLVVSRVTEDSARLSWTAPDAAFSFGISYLEWWEDGEAIVL



TVPGSERSYDLTGLKPGTEYWVSIAGVKGGKRSYPLSAIFTT


 


317
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFVIEYREGAWYGEAIVL



TVPGSERSYDLTGLKPGTEYFVDITGVKGGWWSDPLSAIFTT


 


318
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFFIKYIEWWADGEAIVLT



VPGSERSYDLTGLKPGTEYLVEIYGVKGGKWSWPLSAIFTT


 


319
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFKISYQEWWEDGEAIVL



TVPGSERSYDLTGLKPGTEYWVNISGVKGGVQSYPLSAIFTT


 


320
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFFISYIEWWDLGEAIVLT



VPGSERSYDLTGLKPGTEYHVEIFGVKGGTQSYPLSAIFTT


 


321
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFQILYQENAFEGEAIVLT



VPGSERSYDLTGLKPGTEYWVYIYGVKGGYPSVPLSAIFTT


 


322
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFIIEYWEFVGYGEAIVLT



VPGSERSYDLTGLKPGTEYWVAIYGVKGGDLSKPLSAIFTT


 


323
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFVIEYFEALEGGEAIVLT



VPGSERSYDLTGLKPGTEYFVGIYGVKGGPLSKPLSAIFTT


 


324
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFSIKYLEWWQDGEAIVL



TVPGSERSYDLTGLKPGTEYYVHIAGVKGGYRSYPLSAIFTT


 


325
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFTIWYAEADGWGEAIVL



TVPGSERSYDLTGLKPGTEYFVDIYGVKGGYLSVPLSAIFTT


 


326
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFTIWYAEWEDEGEAIVL



TVPGSERSYDLTGLKPGTEYRVEIYGVKGGYPSKPLSAIFTT


 


327
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFTIWYAEAIGHGEAIVLT



VPGSERSYDLTGLKPGTEYWVDIWGVKGGQQSKPLSAIFTT


 


328
MLPAPKNLVVSRVTEDSARLSWRVESRTFDSFLIQYQESEKVGEAIVLT



VPGSERSYDLTGLKPGTEYTVSIYGVVWDTRDNPISNPLSAIFTT


 


3
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFPILYLELNHHGEEIVLT



VPGSERSYDLTGLKPGTEYWVYIFGVKGGMYSAPLSAIFTTGG


 


395
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFVIEYREGAWYGEAIVL



TVPGSERSYDLTGLKPGTEYAVYIPGVKGGPRSFPLSAIFTT


 


396
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFAIAYVEWWKLGEAIVL



TVPGSERSYDLTGLKPGTEYVVPIPGVKGGGHSSPLSAIFTT


 


397
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFYIYYYESSGTGEAIVLT



VPGSERSYDLTGLKPGTEYFVDIGGVKGGSYSLPLSAIFTT


 


398
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFPIYYWEVFPAGEAIELD



VPGSERSYDLTGLKPGTEYFVRIEGVKGGASSYPLRAEFTT


 


399
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFFIWYWEKSVDGEAIVL



TVPGSERSYDLTGLKPGTEYNVGIQGVKGGTPSDPLSAIFTT


 


400
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFIIWYAEWVNDGEAIVL



TVPGSERSYDLTGLKPGTEYRVEITGVKGGTWSRPLSAIFTT


 


401
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFAIEYYEPVPAGEAIYLD



VPGSERSYDLTGLKPGTEYDVTIYGVKGGYYSHPLFASFTT


 


402
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIEYFEWTVGGEAIVL



TVPGSERSYDLTGLKPGTEYYVSIYGVKGGWLSPPLSAIFTT


 


403
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFHISYEETPVVGEAIYLR



VPGSERSYDLTGLKPGTEYTVAIHGVKGGRESTPLIAPFTT


 


404
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFTIHYWEFDPPGEAIVLT



VPGSERSYDLTGLKPGTEYTVYIEGVKGGWWSKPLSAIFTT


 


405
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFVIEYWERTQPGEAIVLT



VPGSERSYDLTGLKPGTEYDVWISGVKGGKWSEPLSAIFTT


 


406
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFLIRYWEWYVLGEAIVL



TVPGSERSYDLTGLKPGTEYYVEISGVKGGWQSWPLSAIFTT


 


407
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFYIGYLEPGDNGEAIVLT



VPGSERSYDLTGLKPGTEYNVSIGGVKGGLGSYPLSAIFTT


 


408
MLPAPKNLVVSRITEDSARLSWTAPDAAFDSFGIYYYEWWSTGEAIVLT



VPGSERSYDLTGPKPGTEYYVKISGVKGGYRSYPLSAIFTT


 


409
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFRISYYEWYDLGEAIVLT



VPGSERSYDLTGLKPGTEYWVDIAGVKGGYYSYPLSAIFTT


 


410
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFFIGYLEPQPPGEAIVLTV



PGSERSYDLTGLKPGTEYNVTIQGVKGGFPSMPLSAIFTT


 


411
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFFISYFEGWASGEAIHLY



VPGSERSYDLTGLKPGTEYSVHIQGVKGGQPSTPLSAIFTT


 


412
MLPAPKNLVVSRITEDSARLSWTAPDAAFDSFDIPYGEFDTIGEAIVLTV



PGSERSYDLTGLKPGTEYDVYIEGVKGGHLSWPLSAIFTT


 


413
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFGIQYNEFVFRGEAIVLT



VPGSERSYDLTGLKPGTEYFVPISGVKGGDDSRPLSAIFTT


 


414
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIEYWEVVGFGEAIVL



TVPGSERSYDLTGLKPGTEYWVGIYGVKGGNPSVPLSAIFTT


 


415
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFAIDYDEPINSGEAIVLT



VPGSERSYDLTGPKPGTEYEVEIYGVKGGYLSRPLSAIFTT


 


416
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFSIDYDEPQPVGEAIVLT



VPGSERSYDLTGLKPGTEYRVDIWGVKGGPTSGPLRATFTT


 


417
MLLAPKNLVVSRVTEDSARLSWTAPDAAFDSFAIEYFEYTGEGEAIVLT



VPGSERSYDLTGLKPGTEYYVGIYGVKGGYLSRPLSAIFTT


 


418
MLPAPKNLVVSHVTEDSARLSWTAPDAAFDSFDIEYYELVGSGEAIVLT



VPGSERSYDLTGLKPGTEYYVAIYGVKGGYLSRPLSAIFTT


 


419
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFGIAYYERSGAGEAIVLT



VPGSERSYDLTGLKPGTEYMVYINGVKGGFVSSPLSAIFTT


 


420
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFTIAYEEHGLVGEAIYLR



VPGSERSYDLTGLKPGTEYHVGIMGVKGGVFSSPLSAIFTT


 


421
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFFIQYTESHWVGEAIVLT



VPGSERSYDLTGLKPGTEYAVPIEGVKGGDSSTPLSAIFTT


 


422
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFTIIYGEVNPYGEAIVLT



VPGSERSYDLTGLKPGTEYDVFIEGVKGGHLSWPLSAIFTT


 


423
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFHIAYEELVTEGEAIYLR



VPGSERSYDLTGLKPGTEYLVDIEGVKGGHLSSPLSAIFTT


 


424
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFFIHYHEWWEAGEAIVL



TVPGSERSYDLTGLKPGTEYLVDIPGVKGGDLSVPLSAIFTT


 


425
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIYYYESVGTGEAIVLT



VPGSERSYDLTGLKPGTEYFVDISGVKVGTYSLPLSAIFTT


 


426
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFFIAYFEFANPGEAIVLT



VPGSERSYDLTGLKPGTEYKVVIQGVKGGTPSEPLSAISTT


 


427
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFDIHYKEHSWWGEAIVL



TVPGSERSYDLTGLKPGTEYIVPIPGVKGGGISRPLSAIFTT


 


428
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFAIEYWEAVGSGEAIVLT



VPGSERSYDLTGLKPGTEYHVYIYGVKGGYLSLPLSAIFTT


 


429
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFFIGYLEPQPPGEAIVLTV



PGSERSYDLTGLKPGTEYNVTIQGVKGGFPSMPLSAIFTTT


 


430
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFSIAYSEVRYDGEAIVLT



VPGSERSYDLTGLKPGTEYVVPIGGVKGGGSSSPLSAIFTT


 


431
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFTIPYGEAFNPGEAIVLT



VPGSERSYDLTGLKPGTEYDVFIEGVKGGTLSWPLSAIFTT


 


432
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFRILYGEVDPWGEAIVLT



VPGSERSYDLTGLKPGTEYDVWIEGVKGGKLSWPLSAIFTT


 


433
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFHIEYEETPQKGEAIFLR



VPGSERSYDLTGLKPGTEYVVNIRGVKGGDLSSPLGALFTT


 


434
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFRIEYIEWWVGGEAIVLT



VPGSERSYDLTGLKPGTEYWVDIKGVKGGKRSYPLSAIFTT


 


435
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFSIDYPEFPVRGEAIVLT



VPGSERSYDLTGPKPGTEYNVTIQGVKGGFPSMPLSAIFTT


 


436
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFQIPYWEQSLGGEAIVLT



VPGSERSYDLTGLKPGTEYEVWIEGVKGGDLSFPLSAISTT


 


437
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFVIPYEEYLYTGEAIVLT



VPGSERSYDLTGLKPGTEYDVWIEGVKGGLTSWPLSAIFTT


 


438
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFSIDYPEFPVRGEAIVLT



VPGSERSYDLTGLKPGTEYAVTIWGVKGGFTSQPLSAIFTT


 


439
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFVIEYFEFVGEGEAIVLT



VPGSERSYDLTGLKPGTEYDVGIYGVKGGSLSSPLSAIFTT


 


440
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFSIDYLELGESGEAIVLT



VPGSERSYDLTGLKPGTEYWVYIFGVKGGYPSAPLSAIFTT


 


441
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFPIPYGESPPSGEAIVLTV



PGSERSYDLTGLKPGTEYVVIIRGVKGGGRSGPLSAISTT


 


442
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFIINYIEIVQYGEAIVLTV



PGSERSYDLTGLKPGTEYPESIWGVKGGGASSPLSAIFTT


 


443
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFDIEYYEAVGAGEAIVLT



VPGSERSYDLTGLKPGTEYTVGIYGVKGGWLSKPLSVIFTT


 


444
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFHIPYVEAEVPGEAIQLH



VPGSERSYDLTGLKPGTEYYVEIWGVKGGFYSPPLIAEFTT


 


445
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFSIDYYEGKGYGEAIVLT



VPGSERSYDLTGLKPGTEYQVLISGVKGGKYSLPLSAIFTT


 


446
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFTIVYAEVTYDGEAIVLT



VPGSERSYDLTGLKPGTEYDVFIEGVKGGELSWPLSAIFTT


 


447
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFPIVYGEAWVTGEAIVLT



VPGSERSYDLTGLKPGTEYDVWIEGVKGGELSWPLSAIFTT


 


448
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIDYYERKYVGEAIVL



TVPGSERSYDLTGLKPGTEYEVTIYGVKGGWYSDPLSAIFTT


 


449
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFPISYYEMSGLGEAIVLT



VPGSERSYDLTGLKPGTEYMVYIFGVKGGLNSLPLSAIFTT


 


450
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFFIYYIESYPAGEAIVLTV



PGSERSYDLTGLKPGTEYWMGIDGVKGGRWSTPLSAIFTT


 


451
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFEIEYDEPSVAGEAIVLT



VPGSERSYDLTGLKPGTEYRVFIWGVKGGNQSWPLSAIFTT


 


452
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFFIKYIEWWADGEAIVLT



VPGSERSYDLTGLKPGTEYLVEIYGVKGGRQSYPLSAIFTT


 


453
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFDISYWESGKYGEAIVLT



VPGSESSYDLTGLKPGTEYLVDIFGVKGGYPSEPLSAIFTT


 


454
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWISYEESDTEGEAIYLR



VPGSERSYDLTGLKPGTEYNVTIQGVKGGFPSMPLSAIFTT


 


455
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFVIEYFEQFNLGEAIVLT



VPGSERSYDLTGLKPGTEYLVGIYGVKGGWLSHPLSAIFTT


 


456
MLPAPKNLVVSRVTKDSARLSWTAPDAAFDSFHIAYEEATTYGEAIFLR



VPGSERSYDLTGLKPGTEYEVKIHGVKGGADSKPLVAPFTT


 


457
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFHIAYEEADSEGEAIYLR



VPGSERSYDLTGLKPGTEYSVNIQGVKGGIVSFPLHAEFTT


 


458
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFIIPYAEVRPDGEAIVLTV



PGSERSYDLTGLKPGTEYSVLIHGVKGGKLSLPLSAIFTT


 


459
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFFIPYAEPSPTGEAIVLTV



PGSERSYDLTGLKPGTEYDVWIEGVKGGTLSWPLSAIFTT


 


460
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFYIAYAEPRPDGEAIVLT



VPGSERSYDLTGLKPGTEYSVLIHGVKGGLLSSPLSAIFTT


 


461
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFFIGYLEPQPPGEAIVLTV



PGSERSYDLTGLKPGTEYSVLIHGVKGGRNSDPLSAISTT


 


462
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFHIEYEEQYSTGEAIYLR



VPGSERSYDLTGLKPGTEYHVDIEGVKGGRRSFPLNAFFTT


 


463
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFIIPYAEVRPDGEAIVLTV



PGSERSYDLTGLKPGTEYSVLIHGVKGGKLSEPLSAIFTT


 


464
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFPIAYAEPSPTGEAIVLTV



PGSERSYDLTGLKPGTEYSVLIHGVKGGHLSDPLSAIFTT


 


465
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFFIPYAEPSPTGEAIVLTV



PGSERSYDLTGLKPGTEYGVVILGVKGGYGSDPLSAIFTT


 


466
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFFIGYLEPQPPGEAIVLTV



PGSERSYDLTGLKPGTEYNVTIQGVKGGFPSSPLSAIFTT


 


467
MLPAPKNLVVSRVTEDSARLSWTAPDAALDSFRIAYTEYFVGGEAIVLT



VPGSERSYDLTGLKPGTEYGVGIYGVKGGAGSSPLSAIFTT


 


468
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFFIPYAEPRPDGEAIVLT



VPGSERSYDLTGLKPGTEYSVLIHGVKGGLLSSPLSAIFTT


 


469
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFPITYRERSQYGEAIVLT



VPGSERSYDLTGLKPGTEYVVPIEGVKGGRGSKPLSAIFTT


 


470
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFAIEYFENLGIGEAIVLTV



PGSERSYDLTGLKPGTEYVVNIYGVKGGWLSSPLSAIFTT


 


471
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFAIEYYEYVGNGEAIVLT



VPGSERSYDLTGLKPGTEYQVGIYGVKGGYYSRPLSAIFTT


 


472
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFIIDYLELDDYGEAIVLT



VPGSERSYDLTGLKPGTEYPVYIYGVKGGLPSTPLSAIFTT


 


473
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFLIPYAETSPSGEAIVLTV



PGSERSYDLTGLKPGTEYSVLIHGVKGGRNSDPLSAIFTT


 


474
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFNIAYGEWRQHGEAIVL



TVPGSERSYDLTGLKPGTEYDVFIDGVKGGNLSWPLSAIFTT


 


475
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFTIRYWEELPTGEAIVLT



VPGSERSYDLTGLKPGTEYTVEIFGVKGGYLSRPLSAISTT


 


476
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFHIAYEEATTYGEAIFLR



VPGSERSYDLTGLKPGTEYDVWIEGVKGGTISGPLSAIFTT


 


477
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFPIAYAEPRPDGEAIVLT



VPGSERSYDLTGLKPGTEYFVDIFGVKGGTLSRPLSAIFTT


 


478
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFLIPYAETSPSGEAIVLTV



PGSERSYDLTGLKPGTEYSVLIHGVKGGHLSDPLSAIFTT


 


479
MLPARKNLVVSRVTEDSARLSWTAPDAAFDSFFIPYAEPSPTGEAIVLTV



PGSERSYDLTGLKPGTEYSVLIHGVKGGHLSDPLSAISTT


 


480
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFTILYNEIQNVGEAIVLT



VPGSERSYDLTGLKPGTEYDVWIEGVKGGELSWPLSAIFTT


 


481
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFFIGYLEPQPPGEAIVLTV



PGSERSYDLTGLKPGTEYNVTIQGVKGGTPSEPLSAIFTT


 


482
MLPAPKNLVVSRVTEDSARLSWTTPDAAFDSFFIGYLEPYPPGEAIVLTV



PGSERSYDLTGLKPGTEYVVSIQGVKGGKPSDPLSAIFTT


 


483
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFFIGYLEPQPPGEAIVLTV



PGSERSYDLTGLKPGTEYNVTIQGVKGGFPSVPLSAIFTT


 


484
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFFIGYPEYPATGEAIVLT



VPGSERSYDLTGLKPGTEYFVDINGVKGGSLSYPLSAIFTT


 


485
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFAIRYLEWWDVGEAIVL



TVPGSERSYDLTGLKPGTEYLVEIKGVKGGKFSYPLSAIFTT


 


486
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFPIEYDEWWALGEAITLI



VPASERSYDLTGLKPGTEYVVKIHGVKGGQRSYPLIAFFTT


 


487
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFTIHYRELYVQAIVLTVP



GSERSYDLTGLKPGTEYLVMIPGVKGGPTSVPLSAIFTT


 


488
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFFIGYLEPQPPGEAIVLTV



PGSERSYDLTGLKPGTEYKVVIQGVKGGTPSEPLSAIFTT


 


489
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFFIGYLEPQPPGEAIVLTV



PGSERSYDLTGLKPGTEYSVVIQGVKGGFPSDPLSAIFTT


 


490
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFPIAYAEPRPDGEAIVLT



VPGSERSYDLTGLKPGTEYSVGIHGVKGGHDSSPLSAIFTT


 


491
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFFIGYLEPQPPGEAIVLTV



PGSERSYDLTGLKPGTEYNVTIQGVKGGRASGPLSAIFTT


 


492
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFDIAYAEPIPRGEAIVLTV



PGSERSYDLTGLKPGTEYSVLIHGVKGGRRSVPLSAIFTT


 


493
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFYIAYAEPRPDGEAIVLT



VPGSERSYDLTGLKPGTEYPVPIPGVKGGPGSSPLSAIFTT


 


494
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFEISYYEMRGYGEAIVLT



VPGSERSYDLTGLKPGTEYSVLIHGVEGGDYSSPLSAISTT


 


495
MLPAPKNLVVSHVTEDSARLSWTAPDAAFDSFPIAYAEPRPDGEAIVLT



VPGSERSYDLTGLKPGTEYSVLIHGVKGGLLSSPLSAIFTT


 


496
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFFIGYLEPYPPGEAIVLTV



PGSERSYDLTGLKPGTEYVVSIQGVKGGTPSQPLSAIFTT


 


497
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFLIPYAETSPSGEAIVLTV



PGSERSYDLTGLKPGTEYSVLIHGVKGGRPSNPLVAAFTT


 


498
MLPAPKNLVVSRITEDSARLSWTAPDAAFDSFGIGYYEHKRFGEAIQLS



VPGSERSYDLTGLKPGTEYEVDIEGVKGGVLSWPLFAEFTT


 


499
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFEIDYDELAIYGEAIVLT



VPGSERSYDLTGLKPGTEYGVMIIGVKGGLPSDPLSAIFTT


 


500
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFFIGYLESAEAIVLTVPGS



ERSYDLTGLKPGTEYLVTIQGVKGGIASDPLSAIFTT


 


501
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDFVIEYFEFVGYGEAIVLT



VPGSERSYDLTGLKPGTEYSVGIYGVKGGKLSPPLSAIFTT


 


502
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFLIPYAETSPSGEAIVLTV



PGSERSYDLTGLKPGTEYSVLIHGVKGGKLSLPLSAIFTT


 


503
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFHEWVYFGEAIVLTVPG



SERSYDLTGLKPGTEYFVDIWGVKGGTVSKPLSAIFTT


 


504
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFFIGYPEYPATGEAITLFV



PGSERSYDLTGLKPGTEYNVVIQGVKGGRPSNPLVVAFTT


 


505
MLPAPENLVVSRVTEDSARLSWTAPDAAFDSFEITYEENWRRGEAIVLT



VPGSERSYDLTGPKPGTEYIVIIQGVKGGAESWPLSAIFTT


 


506
MLPAPKNLVVSRVTEDSARLSWTALDAAFDSFFIGYLEPQPPGEAIVLT



VPGSERSYDLTGLKPGTEYNVTIQGVKGGFPSMPLSAIFTT


 


507
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFTIWYAEAVGNGEAIVL



TVPGSERSYDLTGLKPGTEYWVDIWGVKGGEFSSPLSAIFTT


 


508
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFEIDYDELAIYGEAIVLT



VPGSERSYDLTGLKPGTEYRVFIYGVKGGWTSWPLSTIFTT


 


509
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFPIEYDEIPFWGEAIVLTV



PGSERSYDLTGLKPGTEYRVWIHGVKGGNSSWPLSAIFTT


 


510
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFNIHYVEWWVLGEAIVL



TVPGSERSYDLTGLKPGTEYPVYIYGVKGGPKSIPLSAIFTT


 


511
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFKIDYLEINDNGEAIVLT



VPGSERSYDLTGLKPGTEYPVYIWGVKGGYPSSPLSAIFTT


 


512
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFLIAYNEDRKFGEAIVLT



VPGSERSYDLTGLKPGTEYDVWIEGVKGGSLSFPLSAIFTT


 


513
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFGIRYFEWWDLGEAIVL



TVPGSERSYDPTGLKPGTEYNVTIQGVKGGFPSMPLSAIFTT


 


514
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFYIEYYEWMHTGEAIVL



TVPGSERSYDLTGLKPGTEYSVYIYGVKGGYPSSPLSAIFTT


 


515
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFNIDYWETWVIGEAIVLT



VPGSERSYDLTGLKPGTEYEVIIPGVKGGTISPPLSAIFTT


 


516
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFYIDYLELTYSGEAIVLT



VPGSERSYDLTGLKPGTEYYVYIYGVKGGYPSSPLSAIFTT


 


517
MLPAPKNLVVSRVTEDSARLSWTAPDAALDSFRIEYYESYGHGEAIVLT



VPGSERSYDLTGLKPGTEYDVGIYGVKGGYYSRPLSAIFTT


 


518
MLPAPKNLVVSRVTEDSARLPWTAPDAAFDSFWISYYESVGYGEAIVLT



VPGSERSYDLTGLKPGTEYYVDISGVKGGVYSLPLSAIFTT


 


519
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFDIDYDEPAWNGEAIVL



TVPGSERSYDLTGLKPGTEYRVFIYGVKGGNTSWPLSAIFTT


 


520
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFDIEYDELWKNGEAIVL



TVPGSERSYDLTGLKPGTEYRVFIYGVKGGYGSFPLSAIFTT


 


521
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFFIGYLEPQPPGEAIVLTV



PGSERSYDLTGLKPGTEYNVTIQGVKGGTPSEPLSAISTT


 


522
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFGIVYREPYVGGEAIVLT



VPGSERSYDLTGLKPGTEYGVPIPGVKGGYDSGPLSAIFTT


 


523
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFTIPYIEYVWWGEAIVLT



VQGSERSYDLTGLKPGTEYPVTIGGVKGGSRSHPLHAHFTT


 


524
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFSIVYGERFVNGEAIVLT



VPGSERSYDLTGLKPGTEYHVYIDGVKGGDLSWPLSAIFTT


 


525
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWINYYEAQPDGEAIVL



TVPGSERSYDLTGLKPGTEYDVEIAGVKGGTASLPLSAIFTT


 


526
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFIIEYWEQIGVGEAIVLT



VPGSERSYDLTGLKPGTEYWVGIYGVKGGLLSSPLSAIFTT


 


527
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIYYWEIERAGEAIRLD



VPGSERSYDLTGLKPGTEYRVDIWGVKGGPTSGPLRATFTT


 


528
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFAIPYGERQELGEAIVLT



VPGSERSYDLTGLKPGTEYFVVIQGVKGGQPSYPLSAIFTT


 


529
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFLIPYAETSPTGEAIVLTV



PGSERSYDLTGLKPGTEYSVLIHGVKGGYPSSPLSAIFTT


 


530
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFPIAYAEPTPSGEAIVLTV



PGSERSYDLTGLKPGTEYSVLIHGVKGGGLSLPLSAIFTT


 


531
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFIIEYWEWYFAGEAIVLT



VPGSERSYDLTGLKPGTEYTVWITGVKGGTWSEPLSAIFTT


 


532
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFTILYYEMVGEGEAIVLT



VPGSERSYDLTGPKPGTEYWVDIYGVKGGGWSRPLSAIFTT


 


533
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFHIDYLELTYAGEAIVLT



VPGSERSYDLTGLKPGTEYYVTIYGVKGGYPSSPLSAIFTT


 


534
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFHIIYEEDGTEGEAIYLR



VPGSERSYDLTGLKPGTEYEVDIEGVKGGVLSWPLFAEFTT


 


535
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFHISYQEVVAEGEAIYLR



VPGSERSYDLTGLKPGTEYYVLIHGVKGGYESKPLDASFTT


 


536
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFYIEYFEWTGSGEAIVLT



VPGSERSYDLTGLKPGTEYNVAIYGVKGGAVSYPLSAIFTT


 


537
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFTIWYAEALGDGEAIVL



TVPGSERSYDLTGLKPGTEYFVDIPGVKGGTRSSPLSAISTT


 


538
MLLAPKNLVVSRVTEDSARLSWTAPDAAFDSFRYLEQGLYGEAIVLTV



PGSERSYDLTGLKPGTEYWVEIIGVKGGEYSTPLSAIFTT


 


539
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFKIEYFEYVGYGEAIVLT



VPGSERSYDLTGLKPGTEYYVAIYGVKGGWYSRPLSAIFTT


 


540
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFHIIYEEVLTEGEAIYLRV



PGSERSYDLTGLKPGTEYGVTIKGVKGGAYSIPLIATFTT


 


541
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFRIRYLEWWNIGEAIVLT



VPGSERSYDLTGLKPGTEYHVDIWGVKGGYSSYPLSAIFTT


 


542
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFEIYYVEWSEAGEAIVLT



VPGSERSYDLTGLKPGTEYRVEIRGVKGGSWSSPLSAIFTT


 


543
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFSIHYDEDWRRGEAIVLT



VPGSERSYDLTGLKPGTEYLVEIPGVKGGKASYPLSAIFTT


 


544
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFQIRYPKRWISGEAIVLT



VPGSERSYDLTGLKPGTEYEVVIRGVKGGEYSWPLSAIFTT


 


545
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFEIPYIETVALGEAIVLTV



PGSERSYDLTGLKPGTEYYVEIYGVKGGSYSYPLSAISTT


 


546
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFTIAYDETLNLGEAIVLT



VPGSERSYDLTGLKPGTEYIVGIFGVKGGTHSWPLSAIFTT


 


547
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFYIVYAEPIPNGEAIVLT



VPGSERSYDLTGLKPGTEYSVLIHGVKGGRNSDPLSAIFTT


 


548
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFYITYWETWDYGEAIVL



TVPGSERSYDLTGLKPGTEYKVPITGVKGGGPSVPLSAIFTT


 


549
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFSINYREWWSDGEAIYL



PVPGSERSYDLTGLKPGTEYAVYIQGVKGGSRSFPLHAWFTT


 


550
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIEYYEELGSGEAIVLT



VPGSERSYDLTGLKPGTEYRVYIYGVKGGYPSSPLSAIFTT


 


551
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFTILYGEMGTTGEAIVLT



VPGSERSYDLTGLKPGTEYDVFIEGVKGGELSWPLSAIFTT


 


552
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFKIFYQEFGGEAIVLTVP



GSERSYDLTGLKPGTEYWVDIYGVKGGYTSSPLSAIFTT


 


553
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFAITYYEGRWRGEAIVL



TVPGSERSYDLTGLKPGTEYGVPIRGVKGGTGSLPLSAIFTT


 


554
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFRIKYLEWWLGGEAIVL



TVPGSERSYDLTGLKPGTEYWVDIQGVKGGVLSWPLSAIFTT


 


555
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFNIYYYEWFVSGEAIVLT



VPGSERSYDLTGLKPGTEYFVDIDGVKGGYRSRPLSAIFTT


 


556
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFPIKYLEWWSWGEAIVL



TVPGSERSYDLTGLKPGTEYRVPISGVKGGGMSGPLSAIFTT


 


557
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFAIPYYEWVNHGEAIVL



TVPGSERSYDLTGLKPGTEYPVGIDGVKGGGPSWPLSAIFTT


 


558
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFHIDYSEFHLRGEAIVLT



VPGSERSYDLTGLKPGTEYLGIFGVKGGEQSGPLSAIFTT


 


559
MLPAPKNLVVSRITEDSARLSWTAPDAAFDSFGIAYNEGDHYGEAIVLT



VPGSERSYDLTGLKPGTEYSVWIEGVKGGNLSYPLSAIFTT


 


560
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFTIAYNEQNHYGEAIVLT



VPGSERSYDLTGLKPGTEYGVWIEGVKGGTLSWPLSAIFTT


 


561
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFYIEWTYKGEAIVLTVPG



SERSYDLTGLKPGTEYFVGIPGVKGGKSSYPLSAIFTTNPKGDTP


 


562
MGSLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFAIPYAEPSPTGEAIVL



TVPGSERSYDLTGLKPGTEYPVWIQGVKGGSPSAPLSAEFTT


 


563
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFSIDYFESVGFGEAIVLT



VPGSERSYDLTGLKPGTEYDVQITGVKGGPHSLPLSAIFTT


 


564
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFFIGYLEPYPPGEAIVLTV



PGSERSYDLTGLKPGTEYAVEIAGVKGGLLSSPLSAISTT


 


565
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFFIGYLEPQPPGEAIVLTV



PGSERSYDLTGLKPGTEYNVTIQGVKGGFPSMPLSAIVTT


 


566
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFDIGYTEYGGYGEAIVLT



VPGSERSYDLTGLKPGTEYWVLIQGVKGGGSSVPLSAIFTT


 


567
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFTIEYWETIGGGEAIVLT



VPGSERSYDLTGLKPGTEYYVGIYGVKGGWWSRPLSAIFTT


 


568
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFFIPYAEPSPTGEAIVLTV



PGSERSYDLTGLKPGTEYSVLIHGVKGGHLSDPLSAISTT


 


569
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFDIEYYELIGRGEAIVLT



VPGSERSYDLTGLKPGTEYWVGIYGVKGGWLSRPLSAIFTT


 


570
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFHIVYHEPRPSGEAIVLT



VPGSERSYDLTGLKPGTEYEVGIVGVKGGDLSVPLSAIFTT


 


571
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFHIVYHEPRPSGEAIVLT



VPGSERSYDLTGLKPGTEYEVGIVSVKGGDLSVPLSAIFTT


 


572
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFFIPYAEPSPTGEAIVLTV



PGSERSYDLTGLKPGTEYDVWIEGVKGGVLSWPLSAIFTT


 


573
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFTIEYFEFVDAGEAIVLT



VPGSERSYDLTGLKPGTEYWVEIWGVKGGSWSKPLSAIFTT


 


574
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFNISYYEYFVHGEAIVLT



VPGSERSYDLTGLKPGTEYYVIDGVKGGDPSEPLSAIFTT


 


575
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFTIVYGEWGVPGEAIVLT



VPGSERSYDLTGLKPGTEYDVWIEGVKGGDLSWPLSAIVTT


 


576
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFAIEYFEYTGEGEAIVLT



VPGSERSYDLTGLKPGTEYYVGIYGVKGGYLSRPLSAIFTT


 


577
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFFIGYLEPQPPGEAIVLTV



PGSERSYDLTGLKPGTEYNVTIQGVKGGFPSMPLSAISTT


 


578
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFRIKYQEWWVEGEAIVL



TVPGSERSYDLTGLKPGTEYVVQIAGVKGGLSSYPLSAIFTT


 


579
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWIYYIETSHQGEAIVLT



VPGSERSYDLTGLKPGTEYFVLIKGVKGGYDSVPLSAIFTT


 


580
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFMIRYQEGTRWGEAIVL



TVPGSERSYDLTGLKPGTEYIVMIAGVKGGQISLPLSAIFTT


 


581
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFPIVYSEIHVIGEAIVLTV



PGSERSYDLTGLKPGTEYDVWIEGVKGGHLSEPLSAIFTT


 


582
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFTIVYGEAGAFGEAIVLT



VPGSERSYDLTGLKPGTEYDVLIEGVKGGNLSWPLSAIFTT


 


583
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFHINYAEVYTKGEAILLT



VPGSERSYDLTGLKPGTEYEVYIPGVKGGPFSRPLNAQFTT


 


584
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFLIRYQEWQRWGEAIVL



TVPGSERSYDLTGLKPGTEYTVHIAGVKGGMLSLPLSAIFTT


 


585
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFYIPYAETRDDGEAIVLT



VPGSERSYDLTGLKPGTEYSVLIHGVKGGDLSSPLSAIFTT


 


586
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFGIPYAESTPTGEAIVLT



VPGSERSYDLTGLKPGTEYSVLIHGVKGGHLSDPLSAIFTT


 


587
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFTIFKDGEAIVLTVPGSE



RSYDLTGLKPGTEYYVYIYGVKGGYPSKPLSAIFTT


 


588
MLPAPKNLVVSRVTEDSVRLSWTAPDAAFDSFAISYEEWWVHGEAIVL



TVPGSERSYDLTGLKPGTEYSVVIPGVKGGLYSWTLSAISTT


 


589
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFLIAYAEVTLHGEAIVLT



VPGSERSYDLTGLKPGTEYSVLIHGVKGGRNSDPLSAIFTT


 


590
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFRIDYLELTSLGEAIVLT



VPGSERSYDLTGLKPGTEYPVPILGVKGGLSSWPLSAIFTT


 


591
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFWINYYEGIGEGEAIVLT



VPGSERSYDLTGLKPGTEYYVDISGVKGGSYSLPLSAIFTT


 


592
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFPIAYAEPRPDGEAIVLT



VPGSERSYDLTGLKPGTEYSVLIHGVKGGHLSDPLSAIFTT


 


593
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFSIEYYESVGLGEAIVLT



VPGSERSYDLTGLKPGTEYDVSIYGVKGGYLSRPLSAIFTT


 


594
MLPAPKNLVVRXVTEDSARLSWTAPDAAFDSFEIEYDEPYRGGEAIVLT



VPGSERSYDLTSLKPGTEYPVSIGGVKGGITSDPLSAIFTT


 


595
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFYIDYDEIHDWGEAIVLT



VPGSERSYDLTGLKPGTEYAVQIGGVKGGSFSWTLSAIFTT


 


596
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFAIVYHEPRPDGEAIVLT



VPGSERSYDLTGLKPGTEYEVVILGVKGGVHSYPLSAIFTT


 


597
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFPIAYAEPRPDGEAIVLT



VPGSERSYDLTGLKPGTEYSVLIHGVKGGLLSSPLSAIFTT


 


598
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFFIGYLEPQPPGEAIVLTV



PGSERSYDLTGLKPGTEYNVTIQGVKGGFPSMPLSAIFTT


 


599
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFLIPYAETSPSGEAIVLTV



PGSERSYDLTGLKPGTEYSVLIHGVKGGDYSSPLSAIFTT


 


600
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFNIYYPEFPVRGEAIVLT



VPGSERSYDLTGLKPGTEYVVSIWGVKGGTQSWPLSAIFTT


 


601
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFTIEYHESGPVGEAIVLT



VPGSERSYDLTGLKPGTEYMVWIFGVKGGFVSRPLSAIFTT


 


602
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFLIPYAETSPSGEAIVLTV



PGSERSYDLTGLKPGTEYSVLIHGVKGGDYSSPLSAISTT


 


603
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFAIPYYEDTNDGEAIVLT



VPGSERSYDLTGLKPGTEYWVSIQGVKGGTVSGPLSAIFTT


 


604
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFFYLEQAWGGEAIVLTV



PGSERSYDLTGLKPGTEYWVEITGVKGGYASSPLSAIFTT


 


605
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFHIEYEEPETEGEAIYLH



VPGSERSYDLTGLKPGTEYKVLIRGVKGGSYSIPLQAPFTT


 


606
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFDIAYWELTPSGEAIELL



VPGSERSYDLTGLKPGTEYRVDIIGVKGGFISEPLGATFTT


 


607
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFTIEYWEFTGSGEAIVLT



VPGSERSYDLTGLKPGTEYDVSIYGVKGGWLSYPLSAIFTT


 


608
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFSIIYSEWNVTGEAIVLT



VPGSERSYDLTGLKPGTEYDVWIEGVKGGGMSKPLSAISTT


 


609
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFFIGYLEPIPSGEAIVLTV



PGSERSYDLTGLKPGTEYPVVIQGVKGGHPSQPLSAIFTT


 


610
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFFIGYLEPQPPGEAIILTV



PGSERSYDLTGLKPGTEYNVTIQGVKGGFPSMPLSAIFTT


 


611
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFLIPYAETSPSGEAITLFV



PGSERSYDLTGLKPGTEYNVVIQGVKGGRPSNPLVAASTT


 


612
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFPIAYAEPRPDGEAIVLT



VPGSERSYDLTGLKPGTEYSVLIHGVKGGLLSSPLSAISTT


 


613
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFLIEYWESVGYGEAIVLT



VPGSERSYDLTGLKPGTEYWVGIYGVKGGYYSRPLSAIFTT


 


614
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFFIGYLEPQPPGEAIVLTV



PGSERSYDLTGLKPGTEYNVTIHGVKGGTPSMPLSAIFTT


 


615
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFEIEYDEPYRGGEAIVLT



VPGSERSYDLTSLKPGTEYPVSIGGVKGGITSDPLSAIFTT


 


616
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFDIYYPEYYDRGEAIVLT



VPGSERSYDLTGLKPGTEYTVYIDGVKGGGGSGPLSAIFTT


 


617
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFFIAYFEFANPGEAIVLT



VPGSERSYDLTGLKPGTEYKVVIQGVKGGTPSEPLSAIFTT


 


618
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFIITYWEHVGDGEAIVLT



VPGSERSYDLTGLKPGTEYFVEIYGVKGGYLSKPLSAIFTT


 


619
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFEIDYDEPFVYGEAIVLT



VPGSERSYDLTGLKPGTEYRVFIFGVKGGNGSWPLSAIFTT


 


620
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFYIEYFETQGYGEAIVLT



VPGSERSYDLTGLKPGTEYYVAIYGVKGGYLSRPLSAIFTT


 


621
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFPITYSEPAHYGEAIVLT



VPGSERSYDLTGLKPGTEYHVGIMGVKGGVFSSPLSAIFTT


 


622
MLPAPKNLVVSEVTEDSARLSWQGVARAFDSFLITYREQIFAGEVIVLT



VPGSERSYDLTGLKPGTEYPVWIQGVKGGSPSAPLSAISTT


 


623
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFIIDYLELDQEGEAIVLT



VPGSERSYDLTGLKPGTEYAVYIFGVKGGYPSTPLSAIFTT









As provided herein, in some embodiments, the FN3 domain that binds to CD71 binds to SEQ ID NO: 2 (human mature CD71) or SEQ ID NO: 5 (human mature CD71 extracellular domain), sequence of each provided below:













2
MTKEYQDLQHLDNEESDHHQLRKGPPPPQPLLQRLCSGPRLLLLSLGL



SLLLLVVVCVIGSQNSQLQEELRGLRETFSNFTASTEAQVKGLSTQGG



NVGRKMKSLESQLEKQQKDLSEDHSSLLLHVKQFVSDLRSLSCQMAAL



QGNGSERTCCPVNWVEHERSCYWFSRSGKAWADADNYCRLEDAHLVVV



TSWEEQKFVQHHIGPVNTWMGLHDQNGPWKWVDGTDYETGFKNWRPEQ



PDDWYGHGLGGGEDCAHFTDDGRWNDDVCQRPYRWVCETELDKASQEP



PLL


 


5
QNSQLQEELRGLRETFSNFTASTEAQVKGLSTQGGNVGRKMKSLESQL



EKQQKDLSEDHSSLLLHVKQFVSDLRSLSCQMAALQGNGSERTCCPVN



WVEHERSCYWFSRSGKAWADADNYCRLEDAHLVVVTSWEEQKFVQHHI



GPVNTWMGLHDQNGPWKWVDGTDYETGFKNWRPEQPDDWYGHGLGGGE



DCAHFTDDGRWNDDVCQRPYRWVCETELDKASQEPPLL









In some embodiments, the FN3 domain that binds to EpCAM comprises a polypeptide comprising an amino acid sequence of SEQ ID NOs: 329, 330, 331, 332, 333, 334, or 335 are provided.
In some embodiments, fibronectin type III (FN3) domains that bind or specifically bind human EpCAM protein (SEQ ID NO: 336) are provided. As provided herein, these FN3 domains can bind to the EpCAM protein. Also provided, even if not explicitly stated is that the domains can also specifically bind to the EpCAM protein. Thus, for example, a FN3 domain that binds to EpCAM would also encompass a FN3 domain protein that specifically binds to EpCAM. In some embodiments, an isolated FN3 domain that binds or specifically binds EpCAM is provided.
In some embodiments, the FN3 domain may bind EpCAM at least 5-fold above the signal obtained for a negative control in a standard ELISA assay.
In some embodiments, the FN3 domain that binds or specifically binds EpCAM comprises an initiator methionine (Met) linked to the N-terminus of the molecule. In some embodiments, the FN3 domain that binds or specifically binds EpCAM comprises a cysteine (Cys) linked to a C-terminus of the FN3 domain. The addition of the N-terminal Met and/or the C-terminal Cys may facilitate expression and/or conjugation of half-life extending molecules.
In some embodiments, the FN3 domain that binds EpCAM is based on Tencon sequence of SEQ ID NO:1 or Tencon 27 sequence of SEQ ID NO:4, optionally having substitutions at residues positions 11, 14, 17, 37, 46, 73, or 86 (residue numbering corresponding to SEQ ID NO:4).
In some embodiments, an isolated FN3 domain that binds EpCAM comprises the amino acid sequence of SEQ ID NOs:329, 330, 331, 332, 333, 334, or 335.
In some embodiments, an isolated FN3 domain that binds EpCAM comprises the amino acid sequence of SEQ ID NO:329.
In some embodiments, an isolated FN3 domain that binds EpCAM comprises the amino acid sequence of SEQ ID NO:330.
In some embodiments, an isolated FN3 domain that binds EpCAM comprises the amino acid sequence of SEQ ID NO:331.
In some embodiments, an isolated FN3 domain that binds EpCAM comprises the amino acid sequence of SEQ ID NO:332.
In some embodiments, an isolated FN3 domain that binds EpCAM comprises the amino acid sequence of SEQ ID NO:333.
In some embodiments, an isolated FN3 domain that binds EpCAM comprises the amino acid sequence of SEQ ID NO:334.
In some embodiments, an isolated FN3 domain that binds EpCAM comprises the amino acid sequence of SEQ ID NO:335.
In some embodiments, the isolated FN3 domain that binds EpCAM comprises an initiator methionine (Met) linked to the N-terminus of the molecule.
In some embodiments, the isolated FN3 domain that binds EpCAM comprises an amino acid sequence that is 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to one of the amino acid sequences of SEQ ID NOs: 329-335. Percent identity can be determined using the default parameters to align two sequences using BlastP available through the NCBI website.
The sequences of FN3 domains that can bind to EpCAM are provided in Table 4.







TABLE 4







EpCAM-binding FN3 domain sequences








SEQ
Amino Acid sequences of FN3 domains


ID NO:
that bind to EpCAM





329
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFSISYRERS



AWGEAIALVVPGSERSYDLTGLKPGIEYIVGIIGVKGGLRS



NPLRADFTT


 


330
MLPAPKNLVVSRVTEDSARLSWTAPYAAFDSFAISYRERS



REGEVIALTVPGSERSYDLTGLKPGTEYIVGILGVKGGRRS



KPLRAQFTT


 


331
MLPAPKNLVVSRVTEDSARLSWEGYRNNAHFDSFLIQYQ



ESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVVAA



VPRNYYSNPLSAIFTT


 


332
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFYIRYYEGS



GYGEAIVLTVPGSERSYDLTGLKPGTEYYVYIGGVKGGSP



SSPLSAIFTTG


 


333
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFKIGYWEW



RKYGEAIELNVPGSERSYDLTGLKPGTEYRVLIYGVKGGA



GSHPLRALFTT


 


334
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFSISYRERS



AWGEAIALVVPGSERSYDLTGLKPGIEYIVGIIGVKGGLRS



NPLRADFTTGGGGSGGGGSGGGGSGGGGSLPAPKNLVVS



RVTEDSARLSWTAPDAAFDSFHIEYWEQSIVGEAIVLTVPG



SERSYDLTGLKPGTEYRVWIYGVKGGNDSWPLSAIFTT


 


335
MLPAPKNLVVSRVTEDSARLSWTAPYAAFDSFAISYRERS



REGEVIALTVPGSERSYDLTGLKPGTEYIVGILGVKGGRRS



KPLRAQFTTGGGGSGGGGSGGGGSGGGGSLPAPKNLVVS



RVTEDSARLSWTAPDAAFDSFHIEYWEQSIVGEAIVLTVPG



SERSYDLTGLKPGTEYRVWIYGVKGGNDSWPLSAIFTT









In some embodiments, the sequences provided herein, including those that bind to EpCAM or CD71, does not comprise the initial methionine. The methionine, for example, can be removed when the FN3 domain is linked to another domain, such as a linker or other FN3 domain.
The sequence of EpCAM is as follows:













SEQ ID
QEECVCENYKLAVNCFVNNNRQCQCTSVGAQNTVICSKL


NO:
AAKCLVMKAEMNGSKLGRRAKPEGALQNNDGLYDPDCD


336
ESGLFKAKQCNGTSMCWCVNTAGVRRTDKDTEITCSERV



RTYWIIIELKHKAREKPYDSKSLRTALQKEITTRYQLDPKFI



TSILYENNVITIDLVQNSSQKTQNDVDIADVAYYFEKDVK



GESLFHSKKMDLTVNGEQLDLDPGQTLIYYVDEKAPEFSM



QGLK









In some embodiments, the FN3 domain that binds to EGFR comprises a polypeptide comprising an amino acid sequence of SEQ ID NOs: 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, or 368 are provided.
As provided herein, these FN3 domains can bind to the EGFR protein. Also provided, even if not explicitly stated is that the domains can also specifically bind to the EGFR protein. Thus, for example, a FN3 domain that binds to EGFR would also encompass a FN3 domain protein that specifically binds to EGFR. In some embodiments, an FN3 domain that binds or specifically binds EGFR is provided.
In some embodiments, the FN3 domain may bind EGFR at least 5-fold above the signal obtained for a negative control in a standard ELISA assay.
In some embodiments, the FN3 domain that binds or specifically binds EGFR comprises an initiator methionine (Met) linked to the N-terminus of the molecule. In some embodiments, the FN3 domain that binds or specifically binds EGFR comprises a cysteine (Cys) linked to a C-terminus of the FN3 domain The addition of the N-terminal Met and/or the C-terminal Cys may facilitate expression and/or conjugation of half-life extending molecules.
In some embodiments, the FN3 domain that binds EGFR comprises the amino acid sequence of SEQ ID NOs: 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, or 368.
In some embodiments, the isolated FN3 domain that binds EGFR comprises an initiator methionine (Met) linked to the N-terminus of the molecule.
In some embodiments, the isolated FN3 domain that binds EGFR comprises an amino acid sequence that is 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to one of the amino acid sequences of SEQ ID NOs: 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, or 368. Percent identity can be determined using the default parameters to align two sequences using BlastP available through the NCBI website. The sequences of the FN3 peptides that bind to EGFR can be, for example, found in Table 5.







TABLE 5







EGFR-binding FN3 domain sequences








SEQ



ID



NO:
EGFR Binding FN3 Domains (Sequences)





337
LPAPKNLVVSEVTEDSLRLSWADPHGFYDSFLIQYQESEKVGEAI



NLTVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNMRGLPLS



AEFTT


 


338
LPAPKNLVVSEVTEDSLRLSWTYDRDGYDSFLIQYQESEKVGEAI



NLTVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNMRGLPLS



AEFTT


 


339
LPAPKNLAASEVTEDSLRLSWGYNGDHPDSFLIQYQESEKVGEAI



NLTVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNMRGLPLS



AEFTT


 


340
LPAPKNLVVSEVTEDSLRLSWDDPRGFYESFLIQYQESEKVGEAI



NLTVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNMRGLPLS



AEFTT


 


341
LPAPKNLVVSEVTEDSLRLSWTWPYADLDSFLIQYQESEKVGEAI



NLTVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNMRGLPLS



AEFTT


 


342
LPAPKNLVVSEVTEDSLRLSWGYNGDHPDSFLIQYQESEKVGEAI



NLTVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNMRGLPLS



AEFTT


 


343
LPAPKNLVVSEVTEDSLRLSWDYDLGVYFDSFLIQYQESEKVGE



AINLTVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNMRGLP



LSAEFTT


 


344
LPAPKNLVVSEVTEDSLRLSWDDPWAFYESFLIQYQESEKVGEAI



NLTVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNMRGLPLS



AEFTT


 


345
LPAPKNLVVSEVTEDSLRLSWTAPDAAFDSFLIQYQESEKVGEAI



NLTVPGSERSYDLTGLKPGTEYTVSIYGVLGSYVFEHDVMLPLSA



EFTT


 


346
LPAPKNLVVSEVTEDSARLSWDDPWAFYESFLIQYQESEKVGEAI



VLTVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNMRGLPLS



AIFTT


 


347
LPAPKNLVVSEVTEDSARLSWTAPDAAFDSFLIQYQESEKVGEAI



VLTVPGSERSYDLTGLKPGTEYTVSIYGVLGSYVFEHDVMLPLSA



IFTT


 


348
LPAPKNLVVSEVTEDSLRLSWTWPYADLDSFLIQYQESEKVGEAI



NLTVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNMRGLPLS



AEFTT


 


349
LPAPKNLVVSEVTEDSARLSWADPHGFYDSFLIQYQESEKVGE



AIVLTVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNMRGL



PLSAIFTT


 


350
LPAPKNLVVSEVTEDSARLSWDDPWAFYESFLIQYQESEKVGE



AIVLTVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNIRGLP



LSAIFTT


 


351
LPAPKNLVVSEVTEDSARLSWDDPHAFYESFLIQYQESEKVGEA



IVLTVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNIRGLPLS



AIFTT


 


352
LPAPKNLVVSEVTEDSARLSWADPHGFYDSFLIQYQESEKVGE



AIVLTVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNIRGLP



LSAIFTT


 


353
MLPAPKNLVVSEVTEDSLRLSWADPHGFYDSFLIQYQESEKVGE



AINLTVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNMRGLP



LSAEFTT


 


354
MLPAPKNLVVSEVTEDSLRLSWTYDRDGYDSFLIQYQESEKVGE



AINLTVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNMRGLP



LSAEFTT


 


355
MLPAPKNLVVSEVTEDSLRLSWGYNGDHFDSFLIQYQESEKVGE



AINLTVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNMRGLP



LSAEFTT


 


356
MLPAPKNLVVSEVTEDSLRLSWDDPRGFYESFLIQYQESEKVGE



AINLTVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNMRGLP



LSAEFTT


 


357
MLPAPKNLVVSEVTEDSLRLSWTWPYADLDSFLIQYQESEKVGE



AINLTVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNMRGLP



LSAEFTT


 


358
MLPAPKNLVVSEVTEDSLRLSWGYNGDHFDSFLIQYQESEKVGE



AINLTVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNMRGLP



LSAEFTT


 


359
MLPAPKNLVVSEVTEDSLRLSWDYDLGVYFDSFLIQYQESEKVG



EAINLTVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNMRGL



PLSAEFTT


 


360
MLPAPKNLVVSEVTEDSLRLSWDDPWAFYESFLIQYQESEKVGE



AINLTVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNMRGLP



LSAEFTT


 


361
MLPAPKNLVVSEVTEDSLRLSWTAPDAAFDSFLIQYQESEKVGE



AINLTVPGSERSYDLTGLKPGTEYTVSIYGVLGSYVFEHDVMLPL



SAEFTT


 


362
MLPAPKNLVVSEVTEDSARLSWDDPWAFYESFLIQYQESEKVGE



AIVLTVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNMRGLP



LSAIFTT


 


363
MLPAPKNLVVSEVTEDSARLSWTAPDAAFDSFLIQYQESEKVGE



AIVLTVPGSERSYDLTGLKPGTEYTVSIYGVLGSYVFEHDVMLPL



SAIFTT


 


364
MLPAPKNLVVSEVTEDSLRLSWTWPYADLDSFLIQYQESEKVGE



AINLTVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNMRGLP



LS AEFTT


 


365
MLPAPKNLVVSEVTEDSARLSWADPHGFYDSFLIQYQESEKVG



EAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNMRG



LPLSAIFTT


 


366
MLPAPKNLVVSEVTEDSARLSWDDPWAFYESFLIQYQESEKVG



EAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNIRGL



PLSAIFTT


 


367
MLPAPKNLVVSEVTEDSARLSWDDPHAFYESFLIQYQESEKVG



EAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNIRGL



PLSAIFTT


 


368
MLPAPKNLVVSEVTEDSARLSWADPHGFYDSFLIQYQESEKVG



EAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNIRGL



PLSAIFTT









In some embodiments, the FN3 domain comprises two FN3 domains connected by a linker. The linker can be a flexible linker. The linker can be a short peptide sequence, such as those described herein. For example, the linker can be a G/S or G/A linker and the like. As provided herein, the linker can be, for example, (GS)2, (SEQ ID NO:369), (GGGS)2 (SEQ ID NO:370), (GGGGS)5 (SEQ ID NO:371), (AP)2 (SEQ ID NO:372), (AP)5 (SEQ ID NO:373), (SEQ ID NO:374), (AP)20 (SEQ ID NO:375) and A(EAAAK)5AAA (SEQ ID NO:376). These are non-limiting examples and other linkers can also be used. The number of GGGGS or GGGGA repeats can also be 1, 2, 3, 4, or 5. In some embodiments, the linker comprises one or more GGGGS repeats and one or more GGGGA repeats.
In some embodiments, the FN3 domains may bind CD71, EpCAM, or EGFR, as applicable, with a dissociation constant (KD) of less than about 1×10−7 M, for example less than about 1×10−8 M, less than about 1×10−9 M, less than about 1×10−10 M, less than about 1×10−11 M, less than about 1×10−12M, or less than about 1×10−13 M as determined by surface plasmon resonance or the Kinexa method, as practiced by those of skill in the art. The measured affinity of a particular FN3 domain-antigen interaction can vary if measured under different conditions (e.g., osmolarity, pH). Thus, measurements of affinity and other antigen-binding parameters (e.g., KD, Kon, Koff) are made with standardized solutions of protein scaffold and antigen, and a standardized buffer, such as the buffers described herein.
In some embodiments, the FN3 domain may bind to its target protein at least 5-fold above the signal obtained for a negative control in a standard ELISA assay.
In some embodiments, the FN3 domain that binds or specifically binds its target protein comprises an initiator methionine (Met) linked to the N-terminus of the molecule. In some embodiments, the FN3 domain that binds or specifically binds to its target protein comprises a cysteine (Cys) linked to a C-terminus of the FN3 domain The addition of the N-terminal Met and/or the C-terminal Cys may facilitate expression and/or conjugation of half-life extending molecules.
The FN3 domain can also contain cysteine substitutions, such as those that are described in U.S. Pat. No. 10,196,446, which is hereby incorporated by reference in its entirety. Briefly, in some embodiments, the polypeptide comprising an FN3 domain can have an FN3 domain that has a residue substituted with a cysteine, which can be referred to as a cysteine engineered fibronectin type III (FN3) domain In some embodiments, the FN3 domain comprises at least one cysteine substitution at a position selected from the group consisting of residues 6, 8, 10, 11, 14, 15, 16, 20, 30, 34, 38, 40, 41, 45, 47, 48, 53, 54, 59, 60, 62, 64, 70, 88, 89, 90, 91, and 93 of the FN3 domain based on SEQ ID NO: 1 (LPAPKNLVVSEVTEDSLRLSWTAPDAAFDSFLIQYQESEKVGEAINLTVPGSERSYDLTG LKPGTEYTVSIYGVKGGHRSNPLSAEFTT, SEQ ID NO: 624) of U.S. Pat. No. 10,196,446, which is hereby incorporated by reference in its entirety, and the equivalent positions in related FN3 domains. A cysteine substitution at a position in the domain or protein comprises a replacement of the existing amino acid residue with a cysteine residue. Other examples of cysteine modifications can be found in, for example, U.S. Patent Application Publication No. 20170362301, which is hereby incorporated by reference in its entirety. The alignment of the sequences can be performed using BlastP using the default parameters at, for example, the NCBI website.
In some embodiments, the FN3 domain that binds to the target protein is internalized into a cell. In some embodiments, internalization of the FN3 domain may facilitate delivery of a detectable label or therapeutic into a cell. In some embodiments, internalization of the FN3 domain may facilitate delivery of a cytotoxic agent into a cell. The cytotoxic agent can act as a therapeutic agent. In some embodiments, internalization of the FN3 domain may facilitate the delivery of any detectable label, therapeutic, and/or cytotoxic agent disclosed herein into a cell. In some embodiments, the cell is a tumor cell. In some embodiments, the cell is a liver cell or a lung cell. In some embodiments, the therapeutic is a siRNA molecule as provided for herein.
As provided herein, the different FN3 domains that are linked to the siRNA molecule can also be conjugated or linked to another FN3 domain that binds to a different target. This would enable the molecule to be multi-specific (e.g. bi-specific, tri-specific, etc.), such that it binds to a first target and another, for example, target. In some embodiments, the first FN3 binding domain is linked to another FN3 domain that binds to an antigen expressed by a tumor cell (tumor antigen).
In some embodiments, FN3 domains can be linked together by a linker to form a bivalent FN3 domain The linker can be a flexible linker. In some embodiments, the linker is a G/S linker. In some embodiments the linker has 1, 2, 3, or 4 G/S repeats. A G/S repeat unit is four glycines followed by a serine, e.g. GGGGS. Other examples of linkers are provided herein and can also be used.
Without being bound to any particular theory, in some embodiments, the FN3 domains that are linked to the nucleic acid molecule may be used in the targeted delivery of the therapeutic agent to cells that express the binding partner of the one or more FN3 domains (e.g. tumor cells), and lead intracellular accumulation of the nucleic acid molecule therein. This can allow the siRNA molecule to properly interact with the cell machinery to inhibit the expression of the target gene and also avoid, in some embodiments, toxicity that may arise with untargeted administration of the same siRNA molecule.
The FN3 domain described herein that bind to their specific target protein may be generated as monomers, dimers, or multimers, for example, as a means to increase the valency and thus the avidity of target molecule binding, or to generate bi- or multispecific scaffolds simultaneously binding two or more different target molecules. The dimers and multimers may be generated by linking monospecific, bi- or multispecific protein scaffolds, for example, by the inclusion of an amino acid linker, for example a linker containing poly-glycine, glycine and serine, or alanine and proline. Exemplary linker include (GS)2, (SEQ ID NO:369), (GGGS)2 (SEQ ID NO:370), (GGGGS)5 (SEQ ID NO:371), (AP)2 (SEQ ID NO:372), (AP)5 (SEQ ID NO:373), (AP)10 (SEQ ID NO:374), (AP)20 (SEQ ID NO:375) and A(EAAAK)5AAA (SEQ ID NO:376). The dimers and multimers may be linked to each other in a N- to C-direction. The use of naturally occurring as well as artificial peptide linkers to connect polypeptides into novel linked fusion polypeptides is well known in the literature (Hallewell et al., J Biol Chem 264, 5260-5268, 1989; Alfthan et al., Protein Eng. 8, 725-731, 1995; Robinson & Sauer, Biochemistry 35, 109-116, 1996; U.S. Pat. No. 5,856,456). The linkers described in this paragraph may be also be used to link the domains provided in the formula provided herein and above.
Half-Life Extending Moieties
The FN3 domains may also, in some embodiments, incorporate other subunits for example via covalent interaction. In some embodiments, the FN3 domains that further comprise a half-life extending moiety. Exemplary half-life extending moieties are albumin, albumin variants, albumin-binding proteins and/or domains, transferrin and fragments and analogues thereof, and Fc regions Amino acid sequences of the human Fc regions are well known, and include IgG1, IgG2, IgG3, IgG4, IgM, IgA and IgE Fc regions. In some embodiments, the FN3 domains that specifically bind CD22 may incorporate a second FN3 domain that binds to a molecule that extends the half-life of the entire molecule, such as, but not limited to, any of the half-life extending moieties described herein. In some embodiments, the second FN3 domain binds to albumin, albumin variants, albumin-binding proteins and/or domains, and fragments and analogues thereof.
All or a portion of an antibody constant region may be attached to the FN3 domain to impart antibody-like properties, especially those properties associated with the Fc region, such as Fc effector functions such as C1q binding, complement dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, down regulation of cell surface receptors (e.g., B cell receptor; BCR), and may be further modified by modifying residues in the Fc responsible for these activities (for review; see Strohl, Curr Opin Biotechnol. 20, 685-691, 2009).
Additional moieties may be incorporated into the FN3 domains such as polyethylene glycol (PEG) molecules, such as PEG5000 or PEG20,000, fatty acids and fatty acid esters of different chain lengths, for example laurate, myristate, stearate, arachidate, behenate, oleate, arachidonate, octanedioic acid, tetradecanedioic acid, octadecanedioic acid, docosanedioic acid, and the like, polylysine, octane, carbohydrates (dextran, cellulose, oligo- or polysaccharides) for desired properties. These moieties may be direct fusions with the protein scaffold coding sequences and may be generated by standard cloning and expression techniques. Alternatively, well known chemical coupling methods may be used to attach the moieties to recombinantly produced molecules disclosed herein.
A pegyl moiety may for example be added to the FN3 domain t by incorporating a cysteine residue to the C-terminus of the molecule, or engineering cysteines into residue positions that face away from the binding face of the molecule, and attaching a pegyl group to the cysteine using well known methods.
FN3 domains incorporating additional moieties may be compared for functionality by several well-known assays. For example, altered properties due to incorporation of Fc domains and/or Fc domain variants may be assayed in Fc receptor binding assays using soluble forms of the receptors, such as the FcγRI, FcγRII, FcγRIII or FcRn receptors, or using well known cell-based assays measuring for example ADCC or CDC, or evaluating pharmacokinetic properties of the molecules disclosed herein in in vivo models.
The compositions provided herein can be prepared by preparing the FN3 proteins and the nucleic acid molecules and linking them together. The techniques for linking the proteins to a nucleic acid molecule are known and any method can be used. For example, in some embodiments, the nucleic acid molecule is modified with a linker, such as the linker provided herein, and then the protein is mixed with the nucleic acid molecule comprising the linker to form the composition. For example, in some embodiments, a FN3 domains is conjugated to a siRNA a cysteine using thiol-maleimide chemistry. In some embodiments, a cysteine-containing FN3 domain can be reduced in, for example, phosphate buffered saline (or any other appropriate buffer) with a reducing agent (e.g. tris(2-carboxyethyl) phosphine (TCEP)) to yield a free thiol. Then, in some embodiments, the free thiol containing FN3 domain was mixed with a maleimide linked-modified siRNA duplex and incubated under conditions to form the linked complex. In some embodiments, the mixture is incubated for 0-5 hr, or about 1, 2, 3, 4 or 5 hr at RT. The reaction can be, for example, quenched with N-ethyl maleimide. In some embodiments, the conjugates can be purified using affinity chromatography and ion exchange. Other methods can also be used and this is simply one non-limiting embodiment.
Methods of making FN3 proteins are known and any method can be used to produce the protein. Examples are provided in the references incorporated by reference herein.
Kits
In some embodiments, a kit comprising the compositions described herein are provided.
The kit may be used for therapeutic uses and as a diagnostic kit.
In some embodiments, the kit comprises the FN3 domain conjugated to the nucleic acid molecule.
Uses of the Conjugates FN3 Domains
The compositions provided for herein may be used to diagnose, monitor, modulate, treat, alleviate, help prevent the incidence of, or reduce the symptoms of human disease or specific pathologies in cells, tissues, organs, fluid, or, generally, a host.
In some embodiments, a method of treating a subject having cancer is provided, the method comprising administering to the subject a composition provided for herein.
In some embodiments, the subject has a solid tumor.
In some embodiments, the solid tumor is a melanoma.
In some embodiments, the solid tumor is a lung cancer. In some embodiments, the solid tumor is a non-small cell lung cancer (NSCLC). In some embodiments, the solid tumor is a squamous non-small cell lung cancer (NSCLC). In some embodiments, the solid tumor is a non-squamous NSCLC. In some embodiments, the solid tumor is a lung adenocarcinoma.
In some embodiments, the solid tumor is a renal cell carcinoma (RCC).
In some embodiments, the solid tumor is a mesothelioma.
In some embodiments, the solid tumor is a nasopharyngeal carcinoma (NPC).
In some embodiments, the solid tumor is a colorectal cancer.
In some embodiments, the solid tumor is a prostate cancer. In some embodiments, the solid tumor is castration-resistant prostate cancer.
In some embodiments, the solid tumor is a stomach cancer.
In some embodiments, the solid tumor is an ovarian cancer.
In some embodiments, the solid tumor is a gastric cancer.
In some embodiments, the solid tumor is a liver cancer.
In some embodiments, the solid tumor is pancreatic cancer.
In some embodiments, the solid tumor is a thyroid cancer.
In some embodiments, the solid tumor is a squamous cell carcinoma of the head and neck.
In some embodiments, the solid tumor is a carcinomas of the esophagus or gastrointestinal tract.
In some embodiments, the solid tumor is a breast cancer.
In some embodiments, the solid tumor is a fallopian tube cancer.
In some embodiments, the solid tumor is a brain cancer.
In some embodiments, the solid tumor is an urethral cancer.
In some embodiments, the solid tumor is a genitourinary cancer.
In some embodiments, the solid tumor is an endometriosis.
In some embodiments, the solid tumor is a cervical cancer.
In some embodiments, the solid tumor is a metastatic lesion of the cancer.
In some embodiments, the subject has a hematological malignancy.
In some embodiments, the hematological malignancy is a lymphoma, a myeloma or a leukemia. In some embodiments, the hematological malignancy is a B cell lymphoma. In some embodiments, the hematological malignancy is Burkitt's lymphoma. In some embodiments, the hematological malignancy is Hodgkin's lymphoma. In some embodiments, the hematological malignancy is a non-Hodgkin's lymphoma.
In some embodiments, the hematological malignancy is a myelodysplastic syndrome.
In some embodiments, the hematological malignancy is an acute myeloid leukemia (AML). In some embodiments, the hematological malignancy is a chronic myeloid leukemia (CML). In some embodiments, the hematological malignancy is a chronic myelomoncytic leukemia (CMML).
In some embodiments, the hematological malignancy is a multiple myeloma (MM).
In some embodiments, the hematological malignancy is a plasmacytoma.
In some embodiments, methods of treating cancer in a subject in need thereof are provided. In some embodiments, the methods comprise administering to the subject any composition provided herein. In some embodiments, a use of a composition as provided herein are provided in the preparation of a pharmaceutical composition or medicament for treating cancer. In some embodiments, the composition can be used for treating cancer.
In some embodiments, methods of reducing the expression of a target gene in a cell are provided. In some embodiments, the methods comprise contacting the cell with a composition a composition as provided herein. In some embodiments, the cell is contacted ex-vivo. In some embodiments, the cell is contacted in-vivo. In some embodiments, the target gene is KRAS. The target gene, however, can be any target gene as the evidence provided herein demonstrates that siRNA molecules can be delivered efficiently when conjugated to a FN3 domain.
In some embodiments, methods of delivering a siRNA molecule to a cell in a subject are provided. In some embodiments, the methods comprise administering to the subject a pharmaceutical composition comprising a composition as provided for herein. In some embodiments, the cell is a CD71 positive cell. In some embodiments, the cell is an EpCAM positive cell. In some embodiments, the cell is an EGFR positive cell. In some embodiments, the cell is a CD71 positive cell and an EpCAM positive cell. In some embodiments, the cell is also positive for EGFR. The term “positive cell” in reference to a protein refers to a cell that expresses the protein. In some embodiments, the protein is expressed on the cell surface. In some embodiments, the cell is a muscle cell, a brain cell, or a cell inside the blood brain barrier. In some embodiments, the siRNA downregulates the expression of a target gene in the cell. In some embodiments, the target gene is KRAS. In some embodiments, the KRAS has a mutation. In some embodiments, the mutation in KRAS is a G12C, G12V, G12S or G12D mutation.
In some embodiments, the compositions provided herein may be used to diagnose, monitor, modulate, treat, alleviate, help prevent the incidence of, or reduce the symptoms of human disease or specific pathologies in cells, tissues, organs, fluid, or, generally, a host, also exhibit the property of being able to cross the blood brain barrier. The blood-brain barrier (BBB) prevents most macromolecules (e.g., DNA, RNA, and polypeptides) and many small molecules from entering the brain. The BBB is principally composed of specialized endothelial cells with highly restrictive tight junctions, consequently, passage of substances, small and large, from the blood into the central nervous system is controlled by the BBB. This structure makes treatment and management of patients with neurological diseases and disorders (e.g., brain cancer) difficult as many therapeutic agents cannot be delivered across the BBB with desirable efficiency. Additional conditions that involve disruptions of the BBB include: stroke, diabetes, seizures, hypertensive encephalopathy, acquired immunodeficiency syndrome, traumatic brain injuries, multiple sclerosis, Parkinson's disease (PD) and Alzheimer disease. This ability is especially useful for treating brain cancers including for example: astrocytoma, medulloblastoma, glioma, ependymoma, germinoma (pinealoma), glioblastoma multiform, oligodendroglioma, schwannoma, retinoblastoma, and congenital tumors; or a cancer of the spinal cord, e.g., neurofibroma, meningioma, glioma, and sarcoma. In certain embodiments, the compositions provided for herein can be used to deliver a therapeutic or cytotoxic agent, for example, across the blood brain barrier. In certain embodiments, the compositions provided for herein can be used to deliver a therapeutic or cytotoxic agent, for example, across the blood brain barrier.
In some embodiments, the compositions or pharmaceutical compositions provided herein may be administered alone or in combination with other therapeutics, that is, simultaneously or sequentially. In some embodiments, the other or additional therapeutics are other anti-tumor agent or therapeutics. Different tumor types and stages of tumors can require the use of various auxiliary compounds useful for treatment of cancer. For example, the compositions provided herein can be used in combination with various chemotherapeutics such as taxol, tyrosine kinase inhibitors, leucovorin, fluorouracil, irinotecan, phosphatase inhibitors, MEK inhibitors, among others. The composition may also be used in combination with drugs which modulate the immune response to the tumor such as anti-PD-1 or anti-CTLA-4, among others. Additional treatments can be agents that modulate the immune system, such antibodies that target PD-1 or PD-L1.
“Treat” or “treatment” refers to the therapeutic treatment and prophylactic measures, wherein the object is to prevent or slow down (lessen) an undesired physiological change or disorder, such as the development or spread of cancer. In some embodiments, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the condition or disorder as well as those prone to have the condition or disorder or those in which the condition or disorder is to be prevented.
A “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result. A therapeutically effective amount of the compositions provided herein may vary according to factors such as the disease state, age, sex, and weight of the individual. Exemplary indicators of an effective amount is improved well-being of the patient, decrease or shrinkage of the size of a tumor, arrested or slowed growth of a tumor, and/or absence of metastasis of cancer cells to other locations in the body.
Administration/Pharmaceutical Compositions
In some embodiments, pharmaceutical compositions of the compositions provided herein and a pharmaceutically acceptable carrier, are provided. For therapeutic use, the compositions may be prepared as pharmaceutical compositions containing an effective amount of the domain or molecule as an active ingredient in a pharmaceutically acceptable carrier. “Carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the active compound is administered. Such vehicles can be liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. For example, 0.4% saline and 0.3% glycine can be used. These solutions are sterile and generally free of particulate matter. They may be sterilized by conventional, well-known sterilization techniques (e.g., filtration). The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, stabilizing, thickening, lubricating and coloring agents, etc. The concentration of the molecules disclosed herein in such pharmaceutical formulation can vary widely, i.e., from less than about 0.5%, usually at least about 1% to as much as 15 or 20% by weight and will be selected primarily based on required dose, fluid volumes, viscosities, etc., according to the particular mode of administration selected. Suitable vehicles and formulations, inclusive of other human proteins, e.g., human serum albumin, are described, for example, in e.g. Remington: The Science and Practice of Pharmacy, 21st Edition, Troy, D. B. ed., Lipincott Williams and Wilkins, Philadelphia, Pa. 2006, Part 5, Pharmaceutical Manufacturing pp 691-1092, See especially pp. 958-989.
The mode of administration for therapeutic use of the compositions disclosed herein may be any suitable route that delivers the agent to the host, such as parenteral administration, e.g., intradermal, intramuscular, intraperitoneal, intravenous or subcutaneous, pulmonary; transmucosal (oral, intranasal, intravaginal, rectal), using a formulation in a tablet, capsule, solution, powder, gel, particle; and contained in a syringe, an implanted device, osmotic pump, cartridge, micropump; or other means appreciated by the skilled artisan, as well known in the art. Site specific administration may be achieved by for example intrarticular, intrabronchial, intraabdominal, intracapsular, intracartilaginous, intracavitary, intracelial, intracelebellar, intracerebroventricular, intracolic, intracervical, intragastric, intrahepatic, intracardial, intraosteal, intrapelvic, intrapericardiac, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravascular, intravesical, intralesional, vaginal, rectal, buccal, sublingual, intranasal, or transdermal delivery.
Pharmaceutical compositions can be supplied as a kit comprising a container that comprises the pharmaceutical composition as described herein. A pharmaceutical composition can be provided, for example, in the form of an injectable solution for single or multiple doses, or as a sterile powder that will be reconstituted before injection. Alternatively, such a kit can include a dry-powder disperser, liquid aerosol generator, or nebulizer for administration of a pharmaceutical composition. Such a kit can further comprise written information on indications and usage of the pharmaceutical composition.
EXAMPLES
The following examples are illustrative of the embodiments disclosed herein. These examples are provided for the purpose of illustration only and the embodiments should in no way be construed as being limited to these examples, but rather should be construed to encompass any and all variations which become evidence as a result of the teaching provided herein. Those of skill in the art will readily recognize a variety of non-critical parameters that could be changed or modified to yield essentially similar results.
Example 1: The siRNA sequence pairs, provided in Table 6, were made according to routine synthetic methods. The oligonucleotides were ordered from Axolabs GmbH, and Bio-Synthesis Inc. The oligonucleotides were confirmed by routine techniques.







TABLE 6







siRNA Sense and Anti-sense Sequence Pairs











siRNA
SEQ

SEQ



Pair
ID NO
Sense Strand 5′-3 
ID NO
Anti-sense strand 5′-3′














A
10
cscsUfgucUfCfUfugGfauauUf
11
UfsGfsaauauccaagaGfaca




ca(invdT)

ggsusu


 


B
12
CsasGfcuaAfUfUfcaGfaaucAf
13
UfsAfsugauucugaauUfagc




ua(invdT)

ugsusu


 


C
14
GsasAfuuaGfCfufguAfucguCf
15
UfsUfsgacgauacagcUfaau




aa(invdT)

ucsusu


 


D
16
CfscsUfgUfcUfCfUfuGfgauAf
17
usGfsaAfuAfUfCfcAfaga




uUfcAf(invdT)

GfaCfaGfgsUfsu


 


E
18
csAfsgCfuAfaUfUfCfaGfaauC
19
usAfsuGfaUfUfCfuGfaau




fuAfuAf(invdt)

UfaGfcUfgsUfsu


 


F
20
GfsasAfuUfaGfCfUfgUfaucGf
21
usUfsgAfcGfAfUfacaGfc




uCfaAf(invdt)

UfaAfuUfcsUfsu


 


G
22
CfscsUfgUfcUfcUfuGfgAfuAf
23
usGfsaAfuAfuCfcAfaGfa




uUfcAf(invdT)

GfaCfaGfgsUfsu


 


H
24
csAfsgCfuAfaUfuCfaGfaAfuC
25
UfsasUfgAfuUfcUfgAfaU




faUfa(invdt)

fuAfgCfgsUfsu


 


I
26
gsAfsaUfuAfgCfuGfuAfuCfg
27
UfsusGfaCfaUfaCfaGfcU




UfcAfa(invdt)

faAfuUfcsUfsu


 


J
28
cscsUfgucUfCfUfugGfauauUf
29
UfsGfsaauauccaagaGfaca




ca(invdt)

ggsusu


 


K
30
csasGfcuaAfUfUfcaGfaaucAf
31
UfsAfsugauucugaauUfagc




ua(invdt)

ugsusu


 


L
32
gsasAfuuaGfCfUfguAfucguCf
33
UfsUfsgacgauacagcUfaau




aa(invdt)

ucsusu


 


M
34
csasGfcuaAfUfUfcaGfaaucAf
35
UfsAfsugauucugaauUfagc




ua(invdT)

ugsusu


 


N
36
asusAfuaaAfCfUfugUfgguaGf
37
UfsAfscuaccacaaguUfuau




ua(invdT)

aususu


 


O
38
usasAfacuUfGfUfggUfaguuGf
39
UfsCfscaacuaccacaAfguu




ga(invdT)

uasusu


 


P
40
csasAfgagUfGfCfcuUfgacgAf
41
UfsAfsucgucaaggcaCfucu




ua(invdT)

ugsusu


 


Q
42
gscsCfuugAfCfGfauAfcagcUf
43
UfsUfsagcuguaucguCfaag




aa(invdT)

gcsusu


 


R
44
usgsAfcgaUfAfCfagCfuaauUf
45
UfsGfsaauuagcuguaUfcgu




ca(invdT)

casusu


 


S
46
csgsAfuacAfGfCfuaAfuucaGf
47
UfsUfscugaauu agcuGfu au




aa(invdT)

cgsusu


 


T
48
gsusGfgacGfAfAfuaUfgaucCf
49
UfsUfsggaucauauucGfucc




aa(invdT)

acsusu


 


U
50
gsgsAfcgaAfUfAfugAfuccaAf
51
UfsGfsuuggaucauauUfcgu




ca(invdT)

ccsusu


 


V
52
gsasCfgaaUfAfUfgaUfccaaCfa
53
UfsUfsguuggaucauaUfucg




a(invdT)

ucsusu


 


W
54
ascsGfaauAfUfGfauCfcaacAfa
55
UfsUfsuguuggaucauAfuu




a(invdT)

cgususu


 


X
56
csgsAfauaUfGfAfucCfaacaAf
57
UfsAfsuuguuggaucaUfau




ua(invdT)

ucgsusu


 


Y
58
asasUfaugAfUfCfcaAfcaauAf
59
UfsCfsuauuguuggauCfaua




ga(invdT)

uususu


 


Z
60
gsasUfccaAfCfAfauAfgaggAf
61
UfsAfsuccucuauuguUfgga




ua(invdT)

ucsusu


 


AA
62
cscsAfacaAfUfAfgaGfgauuCf
63
UfsGfsgaauccucuauUfguu




ca(invdT)

ggsusu


 


BB
64
csusAfcagGfAfAfgcAfaguaGf
65
UfsAfscuacuugcuucCfugu




ua(invdT)

agsusu


 


CC
66
ascsAfggaAfGfCfaaGfuaguAf
67
UfsUfsuacuacuugcuUfccu




aa(invdT)

gususu


 


DD
68
gsusAfauuGfAfUfggAfgaaaCf
69
UfsGfsguuucuccaucAfauu




ca(invdT)

acsusu


 


EE
70
csusUfggaUfAfUfucUfcgacAf
71
UfsGfsugucgagaauaUfcca




ca(invdT)

agsusu


 


FF
72
csasGfcagGfUfCfaaGfaggaGf
73
UfsAfscuccucuugacCfugc




ua(invdT)

ugsusu


 


GG
74
gscsAfaugAfGfGfgaCfcaguAf
75
UfsGfsuacuggucccuCfauu




ca(invdT)

gcsusu


 


HH
76
csasAfugaGfGfGfacCfaguaCf
77
UfsUfsguacuggucccUfcau




aa(invdT)

ugsusu


 


II
78
ususUfgugUfAfUfuuGfccauAf
79
UfsUfsuauggcaaauaCfaca




aa(invdT)

aasusu


 


JJ
80
ususGfccaUfAfAfauAfauacUf
81
UfsUfsaguauuauuuaUfggc




aa(invdT)

aasusu


 


KK
82
usgsCfcauAfAfAfuaAfuacuAf
83
UfsUfsuaguauuauuuAfug




aa(invdT)

gcasusu


 


LL
84
cscsAfuaaAfUfAfauAfcuaaAf
85
UfsAfsuuuaguauuauUfua




ua(invdT)

uggsusu


 


MM
86
csasUfaaaUfAfAfuaCfuaaaUfc
87
UfsGfsauuuaguauuaUfuua




a(invdT)

ugsusu


 


NN
88
asusAfaauAfAfUfacUfaaauCf
89
UfsUfsgauuuaguauuAfuu




aa(invdT)

uaususu


 


OO
90
gsasAfgauAfUfUfcaCfcauuAf
91
UfsAfsuaauggugaauAfucu




ua(invdT)

ucsusu


 


PP
92
asgsAfuauUfCfAfccAfuuauAf
93
UfsCfsuauaauggugaAfuau




ga(invdT)

cususu


 


QQ
94
asusAfuucAfCfCfauUfauagAf
95
UfsCfsucuauaaugguGfaau




ga(invdT)

aususu


 


RR
96
asgsAfacaAfAfUfuaAfaagaGf
97
UfsAfscucuuuuaauuUfgu




ua(invdT)

ucususu


 


SS
98
gsasCfucuGfAfAfgaUfguacCf
99
UfsAfsgguacaucuucAfgag




ua(invdT)

ucsusu


 


TT
100
csusGfaagAfUfGfuaCfcuauGf
101
UfsCfscauagguacauCfuuc




ga(invdT)

agsusu


 


UU
102
asgsAfacaGfUfAfgaCfacaaAfa
103
UfsUfsuuugugucuacUfgu




a(invdT)

ucususu


 


VV
104
csasGfgacUfUfAfgcAfagaaGf
105
UfsAfscuucuugcuaaGfucc




ua(invdT)

ugsusu


 


WW
106
gsusUfgauGfAfUfgcCfuucuAf
107
UfsAfsuagaaggcaucAfuca




ua(invdT)

acsusu


 


XX
108
asusGfaugCfCfUfucUfauacAf
109
UfsAfsuguauagaaggCfauc




ua(invdT)

aususu


 


YY
110
usgsAfugcCfUfUfcuAfuacaUf
111
UfsAfsauguauagaagGfcau




ua(invdT)

casusu


 


ZZ
112
gsasUfgccUfUfCfuaUfacauUf
113
UfsUfsaauguauagaaGfgca




aa(invdT)

ucsusu


 


AAA
114
asusGfccuUfCfUfauAfcauuAf
115
UfsCfsuaauguauagaAfggc




ga(invdT)

aususu


 


BBB
116
csusUfcuaUfAfCfauUfaguuCf
117
UfsCfsgaacuaauguaUfaga




ga(invdT)

agsusu


 


CCC
118
UscsUfauaCfAfUfuaGfuucgAf
119
UfsCfsucgaacuaaugUfaua




ga (invdT)

gasusu


 


DDD
120
UsasUfacaUfUfAfguUfcgagAf
121
UfsUfsucucgaacuaaUfgua




aa(invdT)

uasusu


 


EEE
122
AsusAfcauUfAfGfuuCfgagaA
123
UfsUfsuucucgaacuaAfugu




faa(invdT)

aususu


 


FFF
124
UsasCfauuAfGfUfucGfagaaAf
125
UfsAfsuuucucgaacuAfaug




ua(invdT)

uasusu


 


GGG
126
UsusAfguuCfGfAfgaAfauucG
127
UfsUfscgaauuucucgAfacu




faa(invdT)

aasusu


 


HHH
128
AsgsUfucgAfGfAfaaUfucgaA
129
UfsUfsuucgaauuucuCfgaa




faa(invdT)

cususu


 


III
130
AsgsAfaauUfCfGfaaAfacauAf
131
UfsUfsuauguuuucgaAfuu




aa(invdT)

ucususu


 


JJJ
132
GsasAfauuCfGfAfaaAfcauaAf
133
UfsUfsuuauguuuucgAfau




aa(invdT)

uucsusu


 


KKK
134
AsasAfuucGfAfAfaaCfauaaAf
135
UfsCfsuuuauguuuucGfaau




ga(invdT)

uususu


 


LLL
136
AsasUfucgAfAfAfacAfuaaaGf
137
UfsUfscuuuauguuuuCfgaa




aa(invdT)

uususu


 


MMM
138
AsusGfagcAfAfAfgaUfgguaA
139
UfsUfsuuaccaucuuuGfcuc




faa(invdT)

aususu


 


NNN
140
AsgsCfaaaGfAfUfggUfaaaaAf
141
UfsCfsuuuuuaccaucUfuug




ga(invdT)

cususu


 


OOO
142
AsusUfucuGfUfCfuuGfggguU
143
UfsAfsaaccccaagacAfgaa




fua(invdT)

aususu


 


PPP
144
GsgsGfuuuUfUfGfguGfcaugC
145
UfsUfsgcaugcaccaaAfaac




faa(invdT)

ccsusu


 


QQQ
146
CsgsCfacaAfGfGfcaCfugggUf
147
UfsUfsacccagugccuUfgug




aa(invdT)

cgsusu


 


RRR
148
GscsAfcaaGfGfCfacUfggguAf
149
UfsAfsuacccagugccUfugu




ua(invdT)

gcsusu


 


SSS
150
csUfsCfUfuGfgauAfuUfcAf
151
usGfsasAfsusAfUfCfcAfa




(invdT)

gaGfaCfaGfgsUfsu


 


TTT
152
AfsasUfUfCfaGfaauCfuAfuAf
153
usAfsusGfsasUfUfCfuGfa




(invdt)

auUfaGfcUfgsUfsu


 


UUU
154
AfsasUfUfCfaGfaauCfuAfuAf
155
usAfsusGfsasUfUfCfuGfa




(invdt)

auUfaGfcUfgsUfsu


 


VVV
156
csUfscUfuGfgAfuAfuUfcAf
157
usGfsaAfsusAfsuCfcAfa




(invdT)

GfaGfaCfaGfgsUfsu-


 


WWW
158
AfsasUfuCfaGfaAfuCfaUfa
159
UfsasUfsgsAfsuUfcUfgAf




(invdt)

aUfuAfgCfgsUfsu


 


XXX
160
AfsgsCfuGfuAfuCfgUfcAfa
161
UfsusGfsasCfsaUfaCfaGf




(invdt)

cUfaAfuUfcsUfsu


 


YYY
162
csUfsCfUfugGfauauUfca
163
UfsGfsasasusauccaagaGfa




(invdt)-

caggsusu


 


ZZZ
164
asAfsUfUfcaGfaaucAfua(invdt)
165
UfsAfsusgs asuucugaauUf






agcugsusu


 


AAAA
166
asGfsCfUfguAfucguCfaa(invdt)
167
UfsUfsgsascsgauacagcUfa






auucsusu


 


BBBB
168
CfscsUfgUfcUfCfUfuGfgauAf
169
usGfsaAfuAfUfCfcAfaga




gUfcAf(invdT)-

GfaCfaGfgsUfsu


 


CCCC
170
csAfsgCfuAfaUfUfCfaGfaauC
171
usAfsuGfaUfUfCfuGfaau




fgAfuAf(invdt)-

UfaGfcUfgsUfsu-


 


DDDD
172
GfsasAfuUfaGfUfgUfaucGfg
173
usUfsgAfcGfAfUfacaGfc




CfaAf(invdt)

UfaAfuUfcsUfsu-


 


EEEE
174
CfscsUfgUfcUfcUfuGfgAfuAf
175
usGfsaAfuAfuCfcAfaGfa




gUfcAf(invdT)-

GfaCfaGfgsUfsu


 


FFFF
176
csAfsgCfuAfaUfuCfaGfaAfgC
177
UfsasUfgAfuUfcUfgAfaU




faUfa(invdt)

fuAfgCfgsUfsu


 


GGGG
178
gsAfsaUfuAfgCfuGfuAfuCfg
179
UfsusGfaCfaUfaCfaGfcU




UfcAfa(invdt)

faAfuUfcsUfsu


 


HHHH
180
cscsUfgucUfCfUfugGfauagUf
181
UfsGfsaauauccaagaGfaca




ca(invdt)

ggsusu


 


IIII
182
csasGfcuaAfUfUfcaGfaagcAf
183
UfsAfsugauucugaauUfagc




ua(invdt)

ugsusu


 


JJJJ
184
gsasAfuuaGfCfUfguAfucggCf
185
UfsUfsgacgauacagcUfaau




aa(invdt)

ucsusu


 


KKKK
212
CcsAcsrGrCrUrArArUrUrCrA
213
(vinyl-




rGrArArU rCrAsTCsAC

p)sAfsuGfaUfUfCfuGfaa






uUfaGfcUf gUfsasUf


 


LLLL
214
CfsasGfcUfaAfUfUfcAfgaaUf
215
(vinyl-p)-




cAfua

sAfsuGfaUfUfCfuGfaauU






faGfcUfgUfsasUf


 


MMMM
216
csasrGrCrUrArArUrUrCrArGr
217
(vinyl-




ArArUrCrAsusa

p)sAfsuGfaUfUfCfuGfaa






uUfaGfcUfgUfsasUf





Abbreviations Key:


n = 2′-O-methyl residues,


Nf = 2′-F residues,


rN = unmodified residue,


NC = 2′,4′-BNANC (2′-O,4′-C-aminomethylene bridged nucleic acid),


s = phosphorothioate,


(invdt) = inverted Dt,


vinyl-p: (E)-vinylphosphonate,


(n/N = any nucleotide)






Certain siRNAs were evaluated in a HEK-293 rLUC-KRAS reporter assay at 24 hours. siRNAs were delivered by lipofection. EnduRen luciferase substrate was used to generate the luminescent signal. Briefly, a synthetic lentiviral expression vector was constructed so that the DNA sequence encoding the human KRAS open reading frame was fused to the DNA sequence encoding Renilla luciferase. This results in a fusion mRNA from which the luciferase protein can be translated. Candidate siRNA sequences targeting the KRAS open reading frame are evaluated for their ability to induce RNAi in the luciferase-KRAS fusion mRNA. Luciferase signal is quantified using EnduRen live cell luciferase substrate (Promega). Stable cell lines expressing the luciferase reporter in HEK-293 and H358 cells were used to assess candidate siRNAs (Table 7), siRNAs attached to linker sequences (Table 8) and FN3-siRNA conjugates (Table 10).







TABLE 7







Results of siRNA Knockdown of Luciferase













SEQ 

Percent 




ID 
SEQ ID 
knockdown 




NO
NO
of luciferase 



siRNA 
Sense 
Antisense
signal 



Pair
Strand
strand
at 10 pM
















N
36
37
>30



O
38
39
<30



P
40
41
>30



Q
42
43
<30



R
44
45
<30



S
46
47
>30



M
34
35
>30



T
48
49
>30



U
50
51
>30



V
52
53
>30



W
54
55
<30



X
56
57
>30



Y
58
59
<30



Z
60
61
<30



AA
62
63
>30



BB
64
65
>30



CC
66
67
>30



DD
68
69
<30



EE
70
71
<30



FF
72
73
<30



GG
74
75
<30



HH
76
77
<30



II
78
79
<30



JJ
80
81
>30



KK
82
83
>30



LL
84
85
>30



MM
86
87
>30



NN
88
89
<30



OO
90
91
>30



PP
92
93
>30



QQ
94
95
<30



RR
96
97
<30



SS
98
99
<30



TT
100
101
<30



UU
102
103
>30



VV
104
105
<30



WW
106
107
<30



XX
108
109
<30



YY
110
111
<30



ZZ
112
113
<30



AAA
114
115
<30



BBB
116
117
<30



CCC
118
119
<30



DDD
120
121
<30



EEE
122
123
<30



FFF
124
125
<30



GGG
126
127
<30



HHH
128
129
<30



III
130
131
<30



JJJ
132
133
<30



KKK
134
135
<30



LLL
136
137
<30



MMM
138
139
<30



NNN
140
141
<30



SSS
150
151
<30



TTT
152
153
>30



UUU
154
155
<30



VVV
156
157
<30



WWW
158
159
>30



XXX
160
161
<30



YYY
162
163
>30



ZZZ
164
165
<30



AAAA
166
167
>30



BBBB
168
169
<30



CCCC
170
171
>30



DDDD
172
173
>30



EEEE
174
175
<30



FFFF
176
177
>30



GGGG
178
179
>30



HHHH
180
181
>30



IIII
182
183
>30



JJJJ
184
185
>30










siRNA linker and vinyl phosphonates were generated according to known methods. The siRNA linker and modified strands made are provided in Table 8.







TABLE 8







Pairs with Linker and/or Vinyl Phophosphonate













SEQ







ID

SEQ





NO
Sense 5-3
ID NO
Antisense 5-3
Linker





AB01
186
L-
187
(vinyl-p)-
mal-




cscsUfgucUfCfUfugGfa

UfsGfsaauauccaaga
NH—(CH2)6—




uauUfca(invdT)

Gfacaggsusu



AB02
188
L-
189
(vinyl-p)-
mal-




csasGfcuaAfUfUfcaGfa

UfsAfsugauucugaa
NH—(CH2)6—




aucAfua(invdT)

uUfagcugsusu



AB03
190
CfsasGfcUfaAfUfUfcAf
191
(vinyl-p)-
mal-




gaaUfcAfua-L

sAfsuGfaUfUfCfu
C2H4CONH—(CH2)6—






GfaauUfaGfcUfgUf







sasUf



AB04
192
CfsasGfcUfaAfuUfcAfg
193
(vinyl-p)-
mal-




AfaUfcAfua-L

sAfsuGfaUfuCfuGf
C2H4CONH—(CH2)6—






aAfuUfaGfcUfgUfs







asUf



AB05
194
(L)cscsUfgucUfCfUfug
195
(vinu)sGfsaauaucca
mal-




GfauauUfca(invdT)

agaGfacaggsusu
C2H4CONH—(CH2)6—


AB06
196
(L)csasGfcuaAfUfUfca
197
(vinu)sAfsugauucu
mal-




GfaaucAfua

gaauUfagcugsusu
C2H4CONH—(CH2)6—


AB07
198
(L)cscsUfgUfcUfcUfuG
199
(vinu)sGfsaAfuAfu
mal-




fgAfuAfuUfcAf(invdT)

CfcAfaGfaGfaCfag
C2H4CONH—(CH2)6—






gsusu



AB08
200
cscsUfgucUfCfUfugGfa
201
(vinu)sGfsaauaucca
mal-




uauUfca(L)

agaGfacaggsusu
C2H4CONH—(CH2)6—


AB09
202
(L)cscsUfgucUfCfUfug
203
(vinu)sGfsaauaucca
(Mal-




GfauauUfca(invdT)

agaGfacaggsusu
PEG12)(NHC6)


AB10
204
CfscsUfgUfcUfCfUfuGf
205
(vinu)sGfsaAfuAfU
Propyl_linker




gauAfuUfcAf(L)-

fCfcAfagaGfaCfaG







fgsUfsu



AB11
206
CfsasGfcUfaAfUfUfcAf
207
vinu)sAfsuGfaUfUf
Propyl_linker




gaaUfcAfuAf(L)-

CfuGfaaufaGfcfgs







Ufsu-



AB12
208
usUfsgAfcGfaUfaCfAf
209
vinu)sGfsaAfuUfAf
Propyl_linker




GfcUfaauUfcAfuAf(L)

GfcfguaUfcGfuCfa







AfsgsGf



AB13
210
(vinu)CfsasGfcUfaAfUf
211
AfsuGfaUfUfCfuG
(Amc6-




UfcAfgaaUfcAfua

faauUfaGfcUfgUfs
Glen)[BMPS-






asUf-L
Mal]


AB14
218
CCsACsrGrCrUrArArUr
219
(vinyl-





UrCrArGrArArU

p)sAfsuGfaUfUfCf





rCrAsTCsAC

uGfaauUfaGfcUf







gUfsasUf



AB15
220
X-
221
(vinyl-p)-
mal-




CfsasGfcUfaAfUfUfcAf

sAfsuGfaUfUfCfu
C2H4C(O)(NH)—(CH2)6




gaaUfcAfua-L

GfaauUfaGfcUfgUf







sasUf



AB16
222
csasrGrCrUrArArUrUr
223
(vinyl-
mal-




CrArGrArArUrCrAsusa-

p)sAfsuGfaUfUfCf
C2H4C(O)(NH)—(CH2)6




(L)

uGfaauUfaGfcUfg







UfsasUf





Abbreviations Key: n = 2′-O-methyl residues, Nf = 2′-F residues, rN = unmodified residue,


NC = 2′,4′-BNANC (2′-O,4′-C-aminomethylene bridged nucleic acid), s = phosphorothioate,


(invdt) = inverted Dt, Vinu = vinylphosphonate, vinyl-p = (E)-vinylphosphonate, (L) is a linker,












The sequences with the linkers and/or vinyl phosphonate modified sequences were then evaluated in HEK-293 rLUC-KRAS reporter assay at 24 hours as described above. NAC-quenched linkers were delivered to cells by lipofection. EnduRen luciferase substrate was used to generate the luminescent signal. EC50s and Emax were calculated using Graphpad Prism software. Results provided in Table 9.







TABLE 9







siRNA linker and vinyl phosphonates Results









siRNA Pair
EC50



Identifier
(pM)
Emax (%)












AB03
66.98
72.37


AB05
94.6
80.92


AB06
217.2
69.69


AB07
377.9
79.96


AB08
157.2
79.56


AB09
137.5
77.42


AB10
263.7
73.61


AB11
125
73.75


AB12
167
62.97









FN3-siRNA Conjugates are active. FN3-siRNA conjugates are prepared in H358 KRAS-luciferase reporter line. H358 cells expressing the Renilla luciferase-KRAS reporter were treated with FN3-siRNA conjugates for 72 hours. The luciferase assay is described above. EnduRen luciferase substrate was used to generate the luminescent signal. EC50s and Emax were calculated using Graphpad Prism software. FN3 domains were conjugated to siRNA via unique cysteines using thiol-maleimide chemistry. Cysteine-containing FN3 domains in PBS were reduced with tris(2-carboxyethyl) phosphine (TCEP) to yield free thiol. Free thiol containing the FN3 domain was mixed with maleimide linked-modified siRNA duplex, incubated for 2 hr incubation at RT and quenched with N-ethyl maleimide. Conjugates were purified using affinity chromatography and ion exchange. FN3-siRNA conjugate homogeneity was confirmed by SDS-PAGE, analytical SEC and liquid chromatography/mass spectrometry (LC/MS). Results provided in Table 10.







TABLE 10







FN3-siRNA Conjugate Results










FN3 

H358-rLuc-
H358-rLuc-


Domain

KRAS EC50
KRAS Emax


SEQ ID
FN3-siRNA Conjugate
(nM)
(%)













377
EGFR-KRAS siRNA (AB03)
0.13
77.3


378
CD71-KRAS (AB03)
3.51
88.4


379
EPCAM12/H9-KRAS siRNA
0.12
88.0



(AB03)




380
TENCON (Control)-KRAS
9.20
90.5



siRNA (AB03)




381
CD71/CD71 (CD71_32)-
0.059
91.9



KRAS siRNA (AB03)




382
CD71/CD71/ABD-KRAS
0.41
92.6



siRNA (AB03)




383
EPCAM/EPCAM/ABD-KRAS
0.32
77.0



siRNA (AB03)




384
EPCAM/CD71/ABD-KRAS
0.20
88.8



siRNA (AB03)v1




385
EPCAM/CD71/ABD-KRAS
0.12
89.7



siRNA (AB03)v2




386
EPCAM/EPCAM_ABDcon
N.D.
N.D.



KRAS siRNA (AB03)




387
EPCAM/EPCAM/ABD-KRAS
N.D.
N.D.



siRNA (AB03)




388
EpCAM/CD71/ABD_V2_
N.D.
N.D.



KRAS siRNA (AB03)




389
CD71/EpCAM/ABD_KRAS
N.D.
N.D.



siRNA (AB03)




390
EpCAM_CD71_ABD
N.D.
N.D.


391
EpCAM/EpCAM/CD71/ABD_
N.D.
N.D.



KRAS siRNA (AB03)




392
EPCAM/CD71/EPCAM-
N.D.
N.D.



KRAS siRNA (AB03)











Sequences of FN3 Domains referenced in the table above that were conjugated to the siRNA or as shown as controls are provided in Table 11.







TABLE 11







Sequences of FN3 domains








SEQ ID
SEQUENCE





377
MLPAPKNLVVSEVTEDSARLSWDDPWAFYESFLIQYQESEKVGEAIVLTV



PGSERSYDLTGLKPGTEYTVSIYGVHNVYKDTNIRGLPLSAIFTT


 


378
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFTIWYAEAIGHGEAIVLTV



PGSERSYDLTGLKPGTEYWVDIWGVKGGQQSKPLSAIFTT


 


379
MLPAPKNLVVSRVTEDSARLSWTAPYAAFDSFAISYRERSREGEVIALTVP



GSERSYDLTGLKPGTEYIVGILGVKGGRRSKPLRAQFTTGGGGSGGGGSG



GGGSGGGGSLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFHIEYWEQSIV



GEAIVLTVPGSERSYDLTGLKPGTEYRVWIYGVKGGNDSWPLSAIFTT


 


380
MLPAPKNLVVSEVTEDSARLSWTAPDAAFDSFLIQYQESEKVGEAIVLTVP



GSERSYDLTGLKPGTEYTVSIYGVKGGHRSNPLSAIFTT


 


381
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFTIWYAEAIGHGEAIVLTV



PGSERSYDLTGLKPGTEYWVDIWGVKGGQQSKPLSAIFTTGGGGSGGGGS



GGGGSGGGGSLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFTIWYAEAIG



HGEAIVLTVPGSERSYDLTGLKPGTEYWVDIWGVKGGQQSKPLSAIFTT


 


382
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFTIWYAEAIGHGEAIVLTV



PGSERSYDLTGLKPGTEYWVDIWGVKGGQQSKPLSAIFTTGGGGSGGGGS



GGGGSGGGGSLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFTIWYAEAIG



HGEAIVLTVPGSERSYDLTGLKPGTEYWVDIWGVKGGQQSKPLSAIFTTA



PAPAPAPAPTIDEWLLKEAKEKAIEELKKAGITSDYYFDLINKAKTVEGVN



ALKDEILKA


 


383
MLPAPKNLVVSRVTEDSARLSWTAPYAAFDSFAISYRERSREGEVIALTVP



GSERSYDLTGLKPGTEYIVGILGVKGGRRSKPLRAQFTTGGGGSGGGGSG



GGGSGGGGSLPAPKNLVVSRVTEDSARLSWTAPYAAFDSFAISYRERSRE



GEVIALTVPGSERSYDLTGLKPGTEYIVGILGVKGGRRSKPLRAQFTTAPA



PAPAPAPTIDEWLLKEAKEKAIEELKKAGITSDYYFDLINKAKTVEGVNAL



KDEILKA


 


384
MLPAPKNLVVSRVTEDSARLSWTAPYAAFDSFAISYRERSREGEVIALTVP



GSERSYDLTGLKPGTEYIVGILGVKGGRRSKPLRAQFTTGGGGSGGGGSG



GGGSGGGGSLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFTIWYAEAIGH



GEAIVLTVPGSERSYDLTGLKPGTEYWVDIWGVKGGQQSKPLSAIFTTAP



APAPAPAPTIDEWLLKEAKEKAIEELKKAGITSDYYFDLINKAKTVEGVNA



LKDEILKA


 


385
MLPAPKNLVVSRVTEDSARLSWTAPYAAFDSFAISYRERSREGEVIALTVP



GSERSYDLTGLKPGTEYIVGILGVKGGRRSKPLRAQFTTAPAPAPAPAPLP



APKNLVVSRVTEDSARLSWTAPDAAFDSFTIWYAEAIGHGEAIVLTVPGSE



RSYDLTGLKPGTEYWVDIWGVKGGQQSKPLSAIFTTAPAPAPAPAPTIDE



WLLKEAKEKAIEELKKAGITSDYYFDLINKAKTVEGVNALKDEILKA


 


386
MLPAPKNLVVSRVTEDSARLSWTAPYAAFDSFAISYRERSREGEVIALTVP



GSERSYDLTGLKPGTEYIVGILGVKGGRRSKPLRAQFTTGGGGSGGGGSG



GGGSGGGGSLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFSISYRERSAW



GEAIALVVPGSERSYDLTGLKPGIEYIVGIIGVKGGLRSNPLRADFTTAPAP



APAPAPTIDEWLLKEAKEKAIEELKKAGITSDYYFDLINKAKTVEGVNALK



DEILKA


 


387
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFSISYRERSAWGEAIALVV



PGSERSYDLTGLKPGIEYIVGIIGVKGGLRSNPLRADFTTGGGGSGGGGSG



GGGSGGGGSLPAPKNLVVSRVTEDSARLSWTAPYAAFDSFAISYRERSRE



GEVIALTVPGSERSYDLTGLKPGTEYIVGILGVKGGRRSKPLRAQFTTAPA



PAPAPAPTIDEWLLKEAKEKAIEELKKAGITSDYYFDLINKAKTVEGVNAL



KDEILKA


 


388
MLPAPKNLVVSRVTEDSARLSWTAPYAAFDSFAISYRERSREGEVIALTVP



GSERSYDLTGLKPGTEYIVGILGVKGGRRSKPLRAQFTTGGGGSGGGGSG



GGGSGGGGSLPAPKNLVISRVTEDSARLSWTAPDAAFDSFFIYYIESYPAG



EAIVLTVPGSERSYDLTGLKPGTEYWVGIDGVKGGRWSTPLSAIFTTAPAP



APAPAPTIDEWLLKEAKEKAIEELKKAGITSDYYFDLINKAKTVEGVNALK



DEILKA


 


389
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFAIYYLESYPEGEAIVLTVP



GSERSYDLTGLKPGTEYWVGIDGVKGGTWSSPLSAIFTTGGGGSGGGGSG



GGGSGGGGSLPAPKNLVVSRVTEDSARLSWTAPYAAFDSFAISYRERSRE



GEVIALTVPGSERSYDLTGLKPGTEYIVGILGVKGGRRSKPLRAQFTTAPA



PAPAPAPTIDEWLLKEAKEKAIEELKKAGITSDYYFDLINKAKTVEGVNAL



KDEILKA


 


390
MLPAPKNLVVSRVTEDSARLSWTAPYAAFDSFAISYRERSREGEVIALTVP



GSERSYDLTGLKPGTEYIVGILGVKGGRRSKPLRAQFTTGGGGSGGGGSG



GGGSGGGGSLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFAIYYLESYPE



GEAIVLTVPGSERSYDLTGLKPGTEYWVGIDGVKGGTWSSPLSAIFTTAPA



PAPAPAPTIDEWLLKEAKEKAIEELKKAGITSDYYFDLINKAKTVEGVNAL



KDEILKA


 


391
MLPAPKNLVVSRVTEDSARLSWTAPYAAFDSFAISYRERSREGEVIALTVP



GSERSYDLTGLKPGTEYIVGILGVKGGRRSKPLRAQFTTGGGGSGGGGSG



GGGSGGGGSLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFSISYRERSAW



GEAIALVVPGSERSYDLTGLKPGIEYIVGIIGVKGGLRSNPLRADFTTGGGG



SGGGGSGGGGSGGGGSLPAPKNLVISRVTEDSARLSWTAPDAAFDSFFIYY



IESYPAGEAIVLTVPGSERSYDLTGLKPGTEYWVGIDGVKGGRWSTPLSAI



FTTAPAPAPAPAPTIDEWLLKEAKEKAIEELKKAGITSDYYFDLINKAKTV



EGVNALKDEILKA


 


392
MLPAPKNLVVSRVTEDSARLSWTAPYAAFDSFAISYRERSREGEVIALTVP



GSERSYDLTGLKPGTEYIVGILGVKGGRRSKPLRAQFTTAPAPAPAPAPLP



APKNLVVSRVTEDSARLSWTAPDAAFDSFTIWYAEAIGHGEAIVLTVPGSE



RSYDLTGLKPGTEYWVDIWGVKGGQQSKPLSAIFTTAPAPAPAPAPLPAP



KNLVVSRVTEDSARLSWTAPYAAFDSFAISYRERSREGEVIALTVPGSERS



YDLTGLKPGTEYIVGILGVKGGRRSKPLRAQFTT


 


393
MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFAIYYLESYPEGEAIVLTVP



GSERSYDLTGLKPGTEYWVGIDGVKGGTWSSPLSAIFTTGGGGSGGGGSG



GGGSGGGGSLPAPKNLVVSRVTEDSARLSWTAPYAAFDSFAISYRERSRE



GEVIALTVPGSERSYDLTGLKPGTEYIVGILGVKGGRRSKPLRAQFTTGGG



GSGGGGSGGGGSGGGGSLPAPKNLVASRVTEDSARLSWTAPDAAFDSFNI



AYWEPGIGGEAIWLRVPGSERSYDLTGLKPGTEYKVWIHGVKGGASSPPL



IARFTTGGHHHHHHC









SEQ ID NO: 388 was also made N-ethyl maleimide reacted with the C-terminus, which is done to keep the FN3 domain in a monomeric form. The structure can be represented by the following formula:




Example 2. FN3-siRNA conjugate (SEQ ID NO:385) specifically lowers endogenous KRAS mRNA. A431 cells (wild-type KRAS, non-KRAS dependent) were treated with the FN3-siRNA (SEQ ID NO: 385 linked to AB03) conjugates for 96 hours. cDNA from the cells was generated and quantitative RT-PCR was performed using Taqman primer/probe assays specific for KRAS, HRAS, and NRAS. Ubiquitin C (UBC) was the endogenous control. The delta-delta Ct method was used to quantify expression of each gene in cells that were treated with FN3-siRNA conjugates. SEQ 41 showed dose-dependent, specific knockdown of KRAS. The corresponding FN3 construct alone (No siRNA Ctrl) did not produce knockdown of KRAS. The non-targeting FN3 control (Tencon) conjugated to the KRAS siRNA did not produce significant knockdown of KRAS. This is illustrated in FIG. 1. The data is also illustrated in tabular form in Table 12.







TABLE 12







Results of FN3-siRNA conjugates












nM
SEQ ID
No siRNA




Conjugate
NO: 385
Ctrl
(AB03)











Relative KRAS mRNA levels












0.132
1.502
1.976
1.894



1.6
1.399
1.595
2.068



8
1.231
1.906
2.008



40
0.554
2.289
1.504



200
0.105
1.544
1.13







Relative HRAS mRNA levels












0.132
1.335883
1.107742
1.167772



1.6
1.246747
1.126604
1.181024



8
1.119824
1.147848
1.0595



40
1.131173
1.14322
1.182572



200
1.048989
1.056286
1.099616







Relative NRAS mRNA levels












0.132
1.3048
1.089474
1.164604



1.6
1.068661
1.052238
1.258119



8
1.000139
0.974874
1.051284



40
1.043022
1.164602
1.239046



200
1.121631
1.062723
1.17195










Quantitative polymerase chain reaction (qPCR) for quantification of gene knockdown. Quantitative reverse transcription polymerase chain reactions (RT-PCR) were performed on cellular samples in order to directly measure knockdown of endogenous KRAS mRNA. After treatment with FN3-siRNA conjugates, A431 cells are lysed and cDNA is generated using Cells-to-Ct kits (ThermoFisher). cDNAs were quantitated using TaqMan gene expression assays specific for KRAS, HRAS, NRAS, or an endogenous control (ubiquitin C).
Example 3. FN3-siRNA conjugates reduce proliferation of KRAS dependent cells. SEQ ID 385 and SEQ ID 381 conjugated to (AB03) reduce proliferation in KRAS-dependent H358 cells in vitro. H358 grown in 3D spheroid conditions were treated with FN3 constructs with or without a conjugated KRAS siRNA. Both the EPCAM/CD71 and CD71/CD71 FN3 domains without the siRNA showed  ̃25-40% inhibition of proliferation after 7 days of treatment while FN3-KRAS conjugates inhibited proliferation up to 100%. The data is illustrated in FIG. 2.
3-dimensional proliferation assay. Cells are grown as 3-dimensional spheroids using 3D Spheroid Microplates (Corning). These plates favor the formation of 3-D spheroids of tumor cells, a format that is known to support KRAS-driven cell growth. Cell proliferation is measured using CellTiterGlo-3D assays (Promega), which use cellular ATP levels as an indicator of cell number. Following treatment with FN3-siRNA conjugates, H358 spheroids are lysed with CellTiterGlo-3D reagent and quantified using a plate reader to measure luminescence. Percent inhibition of proliferation is calculated by comparing the ATP signal present at the end of the conjugate treatment to the ATP signal present in the starting number of cells immediately prior to treatment (FIG. 2).
These examples demonstrate the surprising and unexpected ability of FN3-siRNA conjugates to reduce a target gene and also inhibit cellular proliferation. The results also demonstrate that it can be done with a composition comprising more than one FN3 domain and still effectively deliver a siRNA molecule, which has not previously been demonstrated. Furthermore, the examples and embodiments provided herein demonstrate FN3 Domain-siRNA conjugates enable receptor specific delivery of siRNA to extra-hepatic cell types; intracellular trafficking and an endosomal depot for FN3 contributes to an extended duration of activity of FN3-siRNA conjugates; FN3-siRNA conjugates have demonstrated potent reduction of mRNA and protein and inhibition of proliferation in epithelial tumor cell lines; and bispecific binding of FN3 domains to tumor cells expressing high levels of targeted receptors improves avidity and activity and can improve selectivity
Example 4. siRNA sequences directed against KRAS conjugated with malemide were found to inhibit KRAS expression. Various linker site and linkage chemistry of KRAS siRNAs were evaluated by transfection using a HEK293 luciferase cell line. Each of the molecules were found to inhibit KRAS expression by this assay, which are illustrated in FIG. 3.
Example 5. KRAS-FN3 domain conjugates inhibit cancer cell growth. H358, NSCLC, cell line in 3D spheroid culture was treated with KRAS FN3 conjugates for 15 days (FIG. 4, Panel A). The siRNA-FN3 domain conjugate was conjugated to either a CD71 or EPCAM FN3 binding domain. Cells were subsequently treated with CellTiter-Glo to assess proliferation MIA-PaCa, pancreatic cancer, cell line in 3D spheroid culture was treated with KRAS FN3 conjugates for 7 days CellTiter-Glo to assess proliferation (FIG. 4, Panel B). The conjugates were found to be effective in inhibit cell growth. The results are illustrated in FIG. 4.
Example 6. A H358 luciferase line that can be used measure KRAS expression was treated with a monomeric CD71 FN3 binding, EpCam FN3 binding KRAS siRNA conjugates. The plates were read at 24 h, 48 h, and 72 h. A time dependent effect was observed consistent with receptor mediated uptake and accumulation of the conjugate in the cell. These results are illustrated in FIG. 5. These results demonstrate that the FN3-siRNA conjugates can be internalized into the cell.
Example 7. A H358 3D spheroids were treated with FN3 domain KRAS siRNA conjugates for 72 h. The cells were washed after 6 h and 24 h and then measured for fluorescent signal. The 6 h and 24 h washout experiment demonstrates a lasting effects for the FN3 accumulation in the early endosome and siRNA silencing on KRAS mRNA. These results are illustrated in FIG. 6. These results demonstrate the persistence of the effect.
Example 8. FN3 Binding Domains-siRNA conjugates can inhibit the expression of more than one KRAS mutant. H358-NSCLC (G12C), MIA PaCa-2-pancreatic (G12C), HPAF II-pancreatic (G12D), A549-NSCLC (G12S), H460-NSCLC (Q61H) and A431-skin (KRAS WT) cancer lines were treated with KRAS2 EPCAM/CD71 FN3 conjugates for 72 h. The cells from each experiment were measured for residual KRAS mRNA using qPCR. The conjugates were found to decrease the expression of each variant. These results are illustrated in FIG. 7.
Example 9. EPCAM/CD71-FN3 Bispecific Binding Domain siRNA conjugates decreases KRAS protein levels. A431 and H358 cells were treated with bispecific FN3 domains conjugated to KRAS siRNA at 2, 20 and 200 nM concentrations. After 72 h the cells were compared by Western blot and for the presence of KRAS protein. A good correlation between mRNA silencing and protein was observed. These results are illustrated in FIG. 8.
Example 10. FN3-siRNA conjugates reduce proliferation of KRAS dependent cells. SEQ ID 393 conjugated to (AB03) reduce proliferation in KRAS-G12D dependent cells in vitro. The cells grown in 3D spheroid conditions were treated with the constructs with or without a conjugated KRAS siRNA as described in Example 3. A control, AMG-510, that targets G12C was used as a negative control. The FN3-KRAS conjugate inhibited proliferation up to 100%, which was significantly more than the control without the siRNA or AMG-510, which is G12C RAS inhibitor. The data is illustrated in FIG. 9. SEQ ID NO: 393 is illustrated as a polypeptide that comprises 3 FN3 domains that bind to CD71 (SEQ ID NO: 312), EpCAM (SEQ ID NO: 330-without the initial methionine) and an albumin binding domain comprising the sequence of: (LPAPKNLVASRVTEDSARLSWTAPDAAFDSFNIAYWEPGIGGEAIWLRVPGSERSYDLT GLKPGTEYKVWIHGVKGGASSPPLIARFTTGG (SEQ ID NO: 394). Each of these domains are exemplary only and linked by various peptide linkers. The domains can be swapped with other CD71, EpCAM and albumin binding domains, such as those provided herein or referenced herein.
Example 11. FN3 Domain Conjugation, PEG Modifier and siRNA
FIG. 10 illustrates a non-limiting example of how a FN3 domain was linked to a siRNA and PEG molecule. Briefly, a polypeptide as provided herein was conjugated to a siRNA linker with the distal 5′ disulfide 4 through cysteine maleimide chemistry. The reaction was passed through a desalting column (7 kD molecular weight cutoff-MCWO) to afford product 5. The conjugate was purified in two steps. Step I affinity chromatography; to remove un-reacted siRNA linker using a Ni-NTA column. Step II-Ion exchange chromatography (CaptoQ or DEAE); to remove un-reacted Centyrin. Fractions containing pure conjugate (determined by SDS gel) were pooled, exchanged into PBS by desalting using Zeba desalting columns (Thermo), and concentrated if necessary.
The cysteine group was removed using 10 mM TCEP. The reaction was monitored by LC-MS. After completion of the reduction TCEP was removed by desalting (7 kD MWCO) to yield 6. Intermediate 6 with was then stirred with the maleimide-PEG moiety (10 equivalents with respect to 6) in PBS. The reaction was incubated at room temperature ( ̃20-25 C) for 6-12 hrs. The reaction was monitored by LC/MS. After completion of reaction the product 7 was purified by passing the reaction mixture through desalting column (7 kD MCWO) to remove excess maleimide-PEG.
Analytical Characterization of CENTYRIN Domain-siRNA Conjugates
FN3-siRNA conjugates were characterized by a combination of analytical techniques. SDS-PAGE was used to compare amounts of conjugate to free protein. For SDS-PAGE, 4-20% Mini-PROTEAN® TGX Stain-FreeTM Protein Gels (BioRad) were run in SDS buffer for one hour at 100 V. Gels were visualized under UV light. Analytical SEC (Superdex-75 5/150 GL column-GE) was used to analyze purity and aggregation state of Centyrin-siRNA conjugates. Liquid chromatography/mass spectrometry (LC/MS) was used to confirm identity and purity of the conjugates. Samples were analyzed using a Waters Acuity UPLC/Xevo G2-XS TOF mass spectrometer system. The instrument was operated in negative electro-spray ionization mode and scanned from m/z 200 to 3000. Conjugate was seen as two fragments; Antisense and Sense-FN3 polypeptide.
Example 12. Mice were dosed via intravenous administration of a FN3 domain conjugated according to Example 11 at 5 mg/kg. Serum was collected and analyzed via stem-loop PCR to quantify antisense RNA strand of the siRNA molecule. LLOQ for assay determined to be 1 nM. Two FN3-siRNA conjugates were tested in pK studies and in vitro luciferase assays. The pK of the antisense RNA of the siRNA molecules were found to have adequate stability in the blood of the mice. This is illustrated in FIG. 11. The open triangles were SEQ ID NO: 393 linked to siRNA AB03 and found to have an AUC (1 nm baseline) of 1251. The closed circles were SEQ ID NO: 393 linked to siRNA AB15 and found to have an AUC (1 nm baseline) of 4470. This data demonstrates that the siRNA molecule was stable and detectable.
Luciferase assays of the same molecules were performed as described herein and were read using EnduRen substrate 72 hours following administration of the FN3-siRNA conjugates. Proliferation assays were read using CellTiterGlo 14 days following administration of FN3-siRNA conjugates. The activities in various assays is shown in the table below.















H358-
SW620-
A549-




LUC
LUC
LUC
H358



EC50
EC50
EC50
Proliferation


FN3-siRNA Conjugate#
(nM)
(nM)
(nM)
(nM)



















SEQ ID NO: 393 linked 
0.55
1.28
11.35
6.44


to siRNA AB03






SEQ ID NO: 393 linked 
0.95
2.07
17.27
37.35


to siRNA AB15













These data demonstrate that the FN3-siRNA conjugates were active.

These examples demonstrate the surprising and unexpected ability of the FN3-siRNA conjugates to reduce different mutant forms of a target gene and also inhibit cellular proliferation. The results also demonstrate that it can be done with a composition comprising more than one FN3 domain and still effectively deliver a siRNA molecule, which has not previously been demonstrated. Furthermore, the examples and embodiments provided herein demonstrate FN3 Domain-siRNA conjugates enable receptor specific delivery of siRNA to extra-hepatic cell types; intracellular trafficking and an endosomal depot for FN3 contributes to an extended duration of activity of FN3-siRNA conjugates; FN3-siRNA conjugates have demonstrated potent reduction of mRNA and protein and inhibition of proliferation in epithelial tumor cell lines; and bispecific binding of FN3 domains to tumor cells expressing high levels of targeted receptors improves avidity and activity and can improve selectivity
EXAMPLE 12. Knockdown of mRNA in muscle cells using CD71 FN3 domain-oligonucleotide conjugates. muCD71 binding FN3 domains are conjugated to siRNA oligonucleotides or antisense oligonucleotides (ASOs) using maleimide chemistry via a cysteine that is uniquely engineered into the FN3 domain. The cysteine substitutions can be one such as those provided for herein and also as provided for in U.S. Patent Application Publication No. 20150104808, which is hereby incorporated by reference in its entirety. siRNAs or ASOs are modified with standard chemical modifications and confirmed to enable knockdown of the targeted mRNA in vitro. FN3 domain-oligonucleotide conjugates are dosed intravenously in mice at doses up to 10 mg/kg oligonucleotide payload. At various time points following dosing, mice are sacrificed; skeletal muscle, heart muscle and various other tissues will be recovered and stored in RNAlaterTM (Sigma Aldrich) until needed. Target gene knockdown is assessed using standard qPCR ΔΔCT methods and primers specific for the target gene and a control gene. The target gene is found to be knock downed in the muscles and such knockdown is enhanced by conjugating the siRNA or ASO to the CD71 FN3 binding domain.
The results and embodiments provided herein demonstrate that the FN3-siRNA conjugates can provide receptor specific delivery of KRAS siRNA. They provide high potency against tumor cell lines and provide FN3 domain conjugates demonstrate differentiated trafficking vs. antibodies facilitating siRNA delivery. These results also demonstrate that the FN3 domains can be used for delivery of any siRNA payloads or other payloads into tumor cells or other cells that have internalizing receptor positive cells.
General Methods
Standard methods in molecular biology are described Sambrook, Fritsch and Maniatis (1982 & 1989 2nd Edition, 2001 3rd Edition) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Sambrook and Russell (2001) Molecular Cloning, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Wu (1993) Recombinant DNA, Vol. 217, Academic Press, San Diego, Calif.). Standard methods also appear in Ausbel, et al. (2001) Current Protocols in Molecular Biology, Vols. 1-4, John Wiley and Sons, Inc. New York, N.Y., which describes cloning in bacterial cells and DNA mutagenesis (Vol. 1), cloning in mammalian cells and yeast (Vol. 2), glycoconjugates and protein expression (Vol. 3), and bioinformatics (Vol. 4).
Methods for protein purification including immunoprecipitation, chromatography, electrophoresis, centrifugation, and crystallization are described (Coligan, et al. (2000) Current Protocols in Protein Science, Vol. 1, John Wiley and Sons, Inc., New York). Chemical analysis, chemical modification, post-translational modification, production of fusion proteins, glycosylation of proteins are described (see, e.g., Coligan, et al. (2000) Current Protocols in Protein Science, Vol. 2, John Wiley and Sons, Inc., New York; Ausubel, et al. (2001) Current Protocols in Molecular Biology, Vol. 3, John Wiley and Sons, Inc., NY, NY, pp. 16.0.5-16.22.17; Sigma-Aldrich, Co. (2001) Products for Life Science Research, St. Louis, Mo.; pp. 45-89; Amersham Pharmacia Biotech (2001) BioDirectory, Piscataway, N.J., pp. 384-391). 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, N.Y.; Harlow and Lane, supra). Standard techniques for characterizing ligand/receptor interactions are available (see, e.g., Coligan, et al. (2001) Current Protocols in Immunology, Vol. 4, John Wiley, Inc., New York).
All references cited herein are incorporated by reference to the same extent as if each individual publication, database entry (e.g. Genbank sequences or GeneID entries), patent application, or patent, was specifically and individually indicated to be incorporated by reference. This statement of incorporation by reference is intended by Applicants, pursuant to 37 C.F.R. § 1.57(b)(1), to relate to each and every individual publication, database entry (e.g. Genbank sequences or GeneID entries), patent application, or patent, each of which is clearly identified in compliance with 37 C.F.R. § 1.57(b)(2), even if such citation is not immediately adjacent to a dedicated statement of incorporation by reference. The inclusion of dedicated statements of incorporation by reference, if any, within the specification does not in any way weaken this general statement of incorporation by reference. Citation of the references herein is not intended as an admission that the reference is pertinent prior art, nor does it constitute any admission as to the contents or date of these publications or documents.
The present embodiments are not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the embodiments in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims.
The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice the embodiments. Various modifications of the embodiments in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims.

## Claims

1. A composition comprising a siRNA molecule comprising a sense strand and antisense strand, wherein:
the sense strand comprises a sequence of SEQ ID NO: 12, 18, 24, 30, 34, 152, 154, 164, 170, 182, 212, 214, or 216; and
the anti-sense strand comprises a sequence of SEQ ID NO: 13, 19, 25, 31, 35, 153, 155, 165, 171, 183, 213, 215, or 217.

2. The composition of claim 1, wherein the siRNA further comprises a linker, wherein the linker is covalently attached to the sense strand or the anti-sense strand of the siRNA.

3. The composition of claim 2, wherein the siRNA is linked to one or more FN3 domains.

4. The composition of claim 2, wherein the linker is covalently attached to the 3′ end of the sense strand.

5. The composition of claim 4, wherein the siRNA is linked to one or more FN3 domains.

6. The composition of claim 2, wherein the linker comprises a compound having the formula of:

7. The composition of claim 6, wherein the siRNA is linked to one or more FN3 domains.

8. The composition of claim 1, wherein the siRNA further comprises a vinyl phosphonate, wherein the vinyl phosphonate is covalently attached to the sense strand or the anti-sense strand of the siRNA.

9. The composition of claim 8, wherein the siRNA is linked to one or more FN3 domains.

10. The composition of claim 8, wherein the vinyl phosphonate is covalently attached to the 5′ end of the antisense strand.

11. The composition of claim 10, wherein the siRNA is linked to one or more FN3 domains.

12. The composition of claim 1, wherein the siRNA further comprises a linker covalently attached to the 3′ end of the sense strand and a vinyl phosphonate covalently attached to the 5′ end of the antisense strand.

13. The composition of claim 12, wherein the siRNA is linked to one or more FN3 domains.

14. The composition of claim 12, wherein the siRNA molecule comprises:
a sense strand of SEQ ID NO: 188 and an antisense strand of SEQ ID NO: 189;
a sense strand of SEQ ID NO: 190 and an antisense strand of SEQ ID NO: 191;
a sense strand of SEQ ID NO: 192 and an antisense strand of SEQ ID NO: 193;
a sense strand of SEQ ID NO: 196 and an antisense strand of SEQ ID NO: 197;
a sense strand of SEQ ID NO: 206 and an antisense strand of SEQ ID NO: 207; or
a sense strand of SEQ ID NO: 210 and an antisense strand of SEQ ID NO: 211.

15. The composition of claim 12, wherein
the sense strand comprises a sequence of SEQ ID NO: 188, 190, 192, 196, 206, or 210; and
the anti-sense strand comprises a sequence of SEQ ID NO: 189, 191, 193, 197, 207, or 211.

16. The composition of claim 15, wherein the siRNA is linked to one or more FN3 domains.

17. The composition of claim 1, wherein the siRNA molecule comprises:
a sense strand of SEQ ID NO: 12 and an antisense strand of SEQ ID NO: 13;
a sense strand of SEQ ID NO: 18 and an antisense strand of SEQ ID NO: 19;
a sense strand of SEQ ID NO: 24 and an antisense strand of SEQ ID NO: 25;
a sense strand of SEQ ID NO: 30 and an antisense strand of SEQ ID NO: 31;
a sense strand of SEQ ID NO: 34 and an antisense strand of SEQ ID NO: 35;
a sense strand of SEQ ID NO: 152 and an antisense strand of SEQ ID NO: 153;
a sense strand of SEQ ID NO: 154 and an antisense strand of SEQ ID NO: 155;
a sense strand of SEQ ID NO: 164 and an antisense strand of SEQ ID NO: 165;
a sense strand of SEQ ID NO: 170 and an antisense strand of SEQ ID NO: 171;
a sense strand of SEQ ID NO: 182 and an antisense strand of SEQ ID NO: 183;
a sense strand of SEQ ID NO: 212 and an antisense strand of SEQ ID NO: 213;
a sense strand of SEQ ID NO: 214 and an antisense strand of SEQ ID NO: 215; or
a sense strand of SEQ ID NO: 216 and an antisense strand of SEQ ID NO: 217.

18. The composition of claim 17, wherein the siRNA is linked to one or more FN3 domains.

19. The composition of claim 1, wherein the siRNA is linked to one or more FN3 domains.

20. A pharmaceutical composition comprising a composition of claim 19.

21. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject the composition of claim 19.

22. A method of reducing the expression of KRAS in a cell, the method comprising contacting the cell with the composition of claim 19.

23. A method of delivering a siRNA that targets KRAS to a cell in a subject, the method comprising administering to the subject a pharmaceutical composition comprising the composition of claim 19.

24. A composition having a formula of (X1)n-(X2)q-(X3)y-L-X4, wherein:
X1 is a first FN3 domain;
X2 is second FN3 domain;
X3 is a third FN3 domain or half-life extender molecule;
L is a linker; and
X4 is a nucleic acid molecule,
wherein n, q, and y are each independently 0 or 1, and
wherein the nucleic acid molecule comprises a siRNA molecule having:
a sense strand comprising a sequence of SEQ ID NO: 12, 18, 24, 30, 34, 152, 154, 164, 170, 182, 188, 190, 192, 196, 206, 210, 212, 214, or 216; and
an anti-sense strand comprising a sequence of SEQ ID NO: 13, 19, 25, 31, 35, 153, 155, 165, 171, 183, 189, 191, 193, 197, 207, 211, 213, 215, or 217.

25. The composition of claim 24, wherein L comprises a compound having the formula of:

26. The composition of claim 24, wherein each of X1, X2, or X3 is linked by a linker, wherein the linker comprises a sequences of SEQ ID NO: 369, 370, 371, 372, 373, 374, 375, or 376.

27. The composition of claim 24, wherein X1 comprises a sequence of SEQ ID NO: 300-335, 337-368, 377-392, or 395-623.

28. The composition of claim 24, wherein X2 comprises a sequence of SEQ ID NO: 300-335, 337-368, 377-392, or 395-623.

29. The composition of claim 24, wherein X3 comprises a sequence of SEQ ID NO: 300-335, 337-368, 377-392, or 395-623.

30. A composition having a formula A1-B1, wherein A1 has a formula of C1-L1-XS and B1 has a formula of XAS-L2-F1, wherein:
C1 is a polymer;
L1 and L2 are each, independently, a linker;
XS is a 5′ to 3′ oligonucleotide sense strand of a double stranded siRNA molecule comprising a sequence of SEQ ID NO: 12, 18, 24, 30, 34, 152, 154, 164, 170, 182, 188, 190, 192, 196, 206, 210, 212, 214, or 216;
XAS is a 3′ to 5′ oligonucleotide antisense strand of a double stranded siRNA molecule comprising a sequence of SEQ ID NO: 13, 19, 25, 31, 35, 153, 155, 165, 171, 183, 189, 191, 193, 197, 207, 211, 213, 215, or 217; and
F1 is a polypeptide comprising at least one FN3 domain.

31. The composition of claim 30, wherein A1-B1 has a formula of:

32. The composition of claim 30, wherein F1 comprises a polypeptide having a formula of (X1)n-(X2)q-(X3)y,
wherein X1 is a first FN3 domain; X2 is second FN3 domain; X3 is a third FN3 domain or half-life extender molecule; and
wherein n, q, and y are each independently 0 or 1, provided that at least one of n, q, and y is 1.

33. The composition of claim 32, wherein X1 comprises a sequence of SEQ ID NO: 300-335, 337-368, 377-392, or 395-623.

34. The composition of claim 32, wherein X2 comprises a sequence of SEQ ID NO: 300-335, 337-368, 377-392, or 395-623.

35. The composition of claim 32, wherein X3 comprises a sequence of SEQ ID NO: 300-335, 337-368, 377-392, or 395-623.

36. The composition of claim 30, wherein the linker comprises a sequence of SEQ ID NO: 369, 370, 371, 372, 373, 374, 375, or 376.