BR112016020492B1 - PROTEIN COMPRISING TYPE III FIBRONECTIN DOMAIN - Google Patents

Abstract

STABILIZED FIBRONECTIN BASED ON MOLECULE STRUCTURES. The present invention relates to proteins comprising a fibronectin-based scaffold (FBS) domain, e.g., 10Fn3 molecules, which specifically bind to a target, and wherein the FBS domain is linked to its C- terminal in a region consisting of PmXn, where P is proline, X is any amino acid, and where n is 0 or an integer that is at least 1, and m is an integer that is at least 1, and where the PmXn portion provides an improved property to the FBS domain, for example, greater stability, relative to the protein that is not linked to the PmXn portion.

Description

CROSS REFERENCE TO RELATED PATENT APPLICATIONS

[001] This Patent application claims the benefit of U.S. Provisional Application No. 61 / 955,975, filed on March 20, 2014, and U.S. Provisional Patent Application No. 62 / 084,270, filed on November 25, 2014, the contents of which are specifically incorporated into the present invention by reference.

BACKGROUND

[002] Adnectins are a class of therapeutic proteins with high affinity and specific target binding properties that are derived from the tenth domain of human type III fibronectin (10Fn3). Whereas wild-type 10Fn3 is extremely stable and soluble, 10Fn3 variants, which contain on the order of 4 to 31 target-binding mutations of the wild-type sequence, vary widely in terms of stability and solubility. In other words, any mutations from the wild-type 10Fn3 sequence, even if necessary for target binding, carry a risk of reducing the stability of the protein. Consequently, it would be desirable to identify modifications that can be made to the wild-type 10Fn3 sequence that would stabilize it, preferably independently of the identity of the residues that mediate the binding of Adnectin to its therapeutic targets.

SUMMARY

[003] Provided in the present invention are stabilized fibronectin-based structures (FBS) of proteins, e.g., Fn3, such as 10Fn3 molecules (e.g., human 10Fn3 molecules) that are linked at their C-terminal end to a moiety that consists of the amino acid sequence PmXn, where P is proline, X is any amino acid, the symbol m represents an integer that is at least 1 and n is 0 or an integer that is at least 1, and in which the portion PmXn increases at least one characteristic, for example thermostability, of FBS proteins.

BRIEF DESCRIPTION OF FIGURES

[004] Figure 1: Representation of a crystal structure of the human 10Fn3 domain (PDB ID: 1FNA), and the protein sequence of the polypeptide visible in the structure. The last two residues defined in the structure, the "EI" in the sequence of interest, are shown as black spheres, immediately downstream of the C-terminal beta strand, G.

DETAILED DESCRIPTION Definitions

[005] An "amino acid residue" represents the remaining part of an amino acid after a water molecule has been lost (an H+ from the nitrogen side and an OH- from the carboxylic side) in the formation of a bond peptide.

[006] As used in the present invention, a "10Fn3 domain" or "10Fn3 portion" or "10Fn3 molecule" refers to wild-type 10Fn3 and biologically active variants, for example, biologically active variants that specifically bind to a target, such as a target protein. A wild-type human 10Fn3 domain may comprise one of the amino acid sequences set forth in SEQ ID NO: 1-8. Biologically active variants of a wild-type human 10Fn3 domain include 10Fn3 domains comprising at least, at most, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40 or 45 amino acid changes, i.e., substitutions, additions or deletions, in with respect to a 10Fn3 domain comprising any one of SEQ ID NOs: 1-8. A biologically active variant of a wild-type 10Fn3 domain may also comprise, or comprise at most, changes of 13, 1-5, 1-10, 1-15, 1-10, 1-25, 1-30, 1 - 35, 1-40 or 1-45 amino acids relative to a 10Fn3 domain comprising any of SEQ ID NOs: 1-8. In certain embodiments, a biologically active variant of a wild-type 10Fn3 domain comprises no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40 or 45 amino acid changes, i.e., substitutions, additions or deletions, relating to a domain of 10Fn3 comprising any of SEQ ID NOs: 1-8. Amino acid changes can be in a loop region, in a chain, or in the N-terminal or C-terminal region. Exemplary 10Fn3degenerate amino acid sequences allowing amino acid changes in the loop regions are provided in the present invention as SEQ ID NOs: 9-16.

[007] By "polypeptide" is meant any sequence of two or more amino acids, regardless of length, post-translational modification, or function. Polypeptides may include natural amino acids and non-natural amino acids, such as those described in U.S. Patent No. 6,559,126, incorporated herein by reference. Polypeptides can also be modified in any of a variety of conventional chemical ways (e.g., an amino acid can be modified with a protecting group; the carboxy-terminal amino acid can be made into a terminal amide group; the amino-terminal residue can be be modified with groups, for example, increasing lipophilicity, or the polypeptide may be glycosylated or otherwise chemically modified to increase in vivo stability or half-life). Modifications of the polypeptide may include attaching another structure, such as a cyclic compound or other molecule, to the polypeptide and may also include polypeptides that contain one or more amino acids in an altered configuration (i.e., R or S; or, L or D).

[008] A "region" of a 10Fn3 domain (or portion or molecule) as used in the present invention refers to a loop (AB, BC, CD, DE, EF and FG), a β-strand (A, B , C, D, E , F and G), N-terminal (corresponding to amino acid residues 1-7 of SEQ ID NO: 1), or C-terminal (corresponding to amino acid residues 93-94 of SEQ ID NO: 1).

[009] A "north pole loop" of a 10Fn3 domain (or portion) refers to any of the BC, DE and FG loops of a 10Fn3 domain.

[0010] A "south pole loop" of a 10Fn3 domain (or portion) refers to any of the AB, CD and EF loops of a 10Fn3 domain.

[0011] A "framework region" refers to any non-loop region of a human 10Fn3 domain. The framework region includes the beta-strands A, B, C, D, E, F and G, as well as the N-terminal region (amino acids corresponding to residues 1-7 of SEQ ID NO: 1) and the C-terminal region (amino acids corresponding to residues 93-94 of SEQ ID NO: 1).

[0012] "Percentage (%) of amino acid sequence identity" in the present invention is defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in a selected sequence, after sequence alignment and introduction of gaps, if necessary, to achieve maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be accomplished in a number of ways that are within the skill in the art, for example, using publicly available computer software such as BLASTSM, BLASTSM-2, ALIGN, ALIGN software. -2 or Megaalign (DNASTAR ®). Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms necessary to achieve maximum alignment over the full length of the sequences being compared.

[0013] For present purposes, the % amino acid sequence identity of a given amino acid sequence A, with, or against a given amino acid sequence B (which may alternatively be phrased as a given amino acid sequence A that has or comprises a given % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows: 100 times portion X/Y where X is the number of amino acid residues scored as identical matches by the sequence alignment program, such as BLASTSM, BLASTSM-2, ALIGN, ALIGN-2 or Megalign (DNASTAR®), in the program alignment of A and B, and where Y is the total number of amino acid residues in B. It will be It is appreciated that when the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not be equal to the % amino acid sequence identity of B to A .

[0014] As used in the present invention, an amino acid residue in a polypeptide is considered to "contribute to the binding" of a target if (1) any of the non-hydrogen atoms of the residue's side chain or main chain are found to be within five Angstroms of any atom of the binding target based on an experimentally determined three-dimensional structure of the complex, and/or (2) a mutation of the residue to its equivalent in wild-type 10Fn3 (e.g., SEQ ID NO: 1), for alanine, or to a residue having a side chain of similar or smaller size than the residue in question, leads to a measured increase in the equilibrium dissociation constant for the target (e.g., an increase in kon) .

[0015] The serum or plasma "half-life" of a polypeptide can generally be defined as the time required for the serum concentration of the polypeptide to be reduced by 50%, in vivo, for example, due to degradation of the polypeptide and/or clearance or sequestration of the polypeptide through natural mechanisms. The half-life can be determined in any manner known per se, such as by pharmacokinetic analysis. Suitable techniques will be apparent to the person skilled in the art, and may, for example, generally involve the steps of administering a suitable dose of a polypeptide to a primate; collecting blood or other samples from said primate at regular intervals; determining the level or concentration of the polypeptide in said blood sample; and calculating, from (a batch of) the data thus obtained, the time until the level or concentration of the polypeptide was reduced by 50% compared to the initial level after dosing. Methods for determining half-life can be found, for example, in Kenneth et al, Chemical Stability of Pharmaceuticals: A Handbook for Pharmacists (1986); . Peters et al, Pharmacokinetics Analysis: A Practical Approach (1996); and Gibaldi, M. et al., Pharmacokinetics, Second Edition Rev., Mareei Dekker (1982).

[0016] Serum half-life can be expressed using parameters such as t1/2-alpha, t1/2-beta and the area under the curve (AUC). An "increase in half-life" refers to an increase in any one of these parameters, any two of these parameters, or all three of these parameters. In certain embodiments, an increase in half-life refers to an increase in t1/2-beta, with or without an increase in t1/2-alpha and/or the AUC or both.

[0017] "Shelf life" of a pharmaceutical product, for example, a protein comprising a portion of FBS and a portion of HSA, is the period of time the product is stored before decomposition occurs. For example, shelf life can be defined as the time for 0.1%, 0.5%, 1%, 5%, or 10% of the product to decompose.

Overview

[0018] Provided in the present invention are proteins comprising a fibronectin-based scaffold (FBS) domain, e.g., Fn3, such as 10Fn3 molecules, which specifically bind to a target, and in which the FBS domain is linked. at its C-terminal end a region consisting of PmXn, characterized P is proline, X is any amino acid and where n is 0 or an integer that is at least 1 and m is an integer that is at least , 1. The Patent application is based, at least in part, on the discovery that the addition of a proline and optionally one or more amino acids to the C-terminal end of a 10Fn3 molecule increases at least one characteristic of 10Fn3 molecule, for example, its thermal stability or solubility, in relation to the unmodified 10Fn3 molecule.

[0019] The 10Fn3 molecules described in the present invention can be designed to bind to any target of interest. In exemplary embodiments, the target is a target antigen, polypeptide, or therapeutic protein of interest. Examples of therapeutically desirable targets include, for example, tumor necrosis factor alpha (TNF-alpha), VEGFR2, PCSK9, IL-23, EGFR and IGF1R.

Structure based on Fibronectin

[0020] As used in the present invention, a protein with "fibronectin-based structure" or "FBS" refers to the portion or proteins or portions that are based on a fibronectin type III ("Fn3") repeat . Fn3 is a small domain (about 10 kDa) that has the immunoglobulin (Ig) folding structure (i.e., a β-sandwich Ig structure consisting of seven β-strands and six loops). Fibronectin has 18 Fn3 repeats, and while sequence homology between the repeats is low, they all share a high similarity in tertiary structure. Fn3 domains are also present in many proteins other than fibronectin, such as adhesion molecules, cell surface molecules, e.g., cytokine receptors, and carbohydrate-binding domains. For reviews, see Bork et al., Proc. Natl. Academic. Sci. USA, 89 (19): 8990 to 8994 (1992); Bork et al., J. Mol. Biol., 242 (4): 309 to 320 (1994); . Campbell et al, Structure, 2 (5): 333 to 337 (1994); Harpez et al., J. Mol. Biol., 238 (4): 528 to 539 (1994)). The term "FBS" protein or moiety is intended to include structures based on Fn3 domains from these other proteins (i.e., non-fibronectin molecules).

[0021] An Fn3 domain is small, monomeric, soluble and stable. It has no disulfide bonds and is therefore stable under reducing conditions. The fn3 domains comprise, in order from N-terminal to C-terminal, a beta or a Beta-type filament, A; a loop, AB; a beta version or a strand of type Beta, B; a loop, BC; a beta version or a strand of type Beta, C; a loop, CD; a beta version or a beta-type strand, D; a loop, DE; a beta version or a beta-type strand, E; a loop, EF; a beta version or a beta-type strand, F; a loop, FG; and a beta or a beta-type filament, G. The seven anti-parallel B-filaments are arranged as two beta sheets that form a stable core, while creating two "faces" composed of the loops connecting the beta or a Beta type filament. Loops AB, CD and EF are located on one face ("the south pole") and loops BC, DE and FG are located on the opposite face ("the north pole").

[0022] The loops in FN3 molecules are structurally similar to the complementarity determining regions (CDRs) of antibodies and, when altered, can be involved in binding the Fn3 molecule to a target, for example, a target protein. Other regions of Fn3 molecules, such as the beta or beta-like filaments and N-terminal or C-terminal regions, when altered, may also be involved in binding to a target. Any or all of the AB, BC, CD, DE, EF, and FG loops can participate in binding to a target. Any of the beta or beta-like filaments can be involved in binding to a target. The fn3 domains can also bind to a target through one or more loops and one or more beta or beta-like filaments. Binding may also require N-terminal or C-terminal regions. An FBS domain for use in a protein may comprise all loops, all beta or beta-type filaments, or only a portion thereof, in which some loops and/or beta or beta-type filaments and/or the N-regions or C-terminus are modified (or altered) since the FBS domain preferentially binds specifically to a target. For example, an FBS domain may comprise 1, 2, 3, 4, 5, or 6 loops, 1, 2, 3, 4, 5, 6, 7, or 8 beta strands, and, optionally, an N-terminal region. and/or C-terminal, in which one or more loops, one or more beta strands, the N-terminal region and/or the C-terminal region are modified relative to the wild-type FBS domain.

[0023] In exemplary embodiments, the ligand (or target) of the FBS binding moieties described in the present invention are based on the tenth domain of type III fibronectin, that is, the tenth module of Fn3 (10Fn3). The amino acid sequence of a wild-type human 10Fn3 portion is as follows: VSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQE FTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRT (SEQ ID NO: 1) (loops AB, CD, and EF are underlined; loops BC, FG, and DE are emphasized in bold; the B -filaments are located between or adjacent to each of the loop regions, and the N-terminal region is shown in italics). The last two amino acid residues of SEQ ID NO: 1 are a portion of a C-terminal region.

[0024] Wild-type human 10Fn3 molecules also include those that lack the N-terminal region or a portion thereof. For example, a wild-type 10Fn3 molecule may comprise SEQ ID NO: 1, wherein amino acid residues 1, 1-2, 1-3, 1-4, 1-5, 1-6 or 1-7 are excluded (SEQ ID NOs: 2-8, respectively). Table 1 shows the amino acid sequence of these wild-type human 10Fn3 portions: Table 1: Amino acid sequences of wild-type human 10Fn3 molecules with various N-terminal regions

[0025] In some embodiments, the AB loop corresponds to residues 14-17, the BC loop corresponds to residues 23-31, the CD loop corresponds to residues 37-47, the DE loop corresponds to residues 51-56, the EF loop corresponds to residues 63-67, and the FG loop corresponds to residues 75-87 of SEQ ID #: 1. The BC, DE, and FG loops align along the faces of the molecule, i.e., the "north pole", and the Loops AB, CD, and EF line up along the opposite face of the molecule, that is, the "south pole." In SEQ ID NO: 1, β-strand A corresponds to residues 8-13, β-strand B corresponds to residues 18-22, β-strand C corresponds to residues 32-36, beta-strand D corresponds to residues 48-50 , β-Filament e corresponds to residues 57-62, β-Filament F corresponds to residues 68-74, and C L corresponds to residues 88-92. The β-filaments are linked to each other through the corresponding loop, for example, filaments A and B are linked through AB loop in the formation β-filament A, AB loop, β-filament B, etc.

[0026] An example of FBS proteins that are based on human 10Fn3 domains are Adnectins (Adnexus, a subsidiary of Bristol-Myers Squibb). 10Fn3 adnectins are molecules in which CDR loop regions such as, β-filaments, the N-terminal and/or C-terminal regions of a 10Fn3 domain have been modified to evolve a protein capable of binding a compound of interest. For example, U.S. Patent No. 7,115,396 describes 10Fn3 domain proteins in which changes in the BC, DE, and FG loops result in high affinity of TNFα ligands. U.S. Patent No. 7,858,739 describes FN3 domain proteins in which changes in the BC, DE, and FG loops result in high-affinity VEGFR2 ligands.

[0027] In certain embodiments, the FBS portion comprises a 10Fn3 domain that is generally defined through the following degenerate sequence:

[0028] VSDVPRDLEVVAA(X)uLLISW(X)vYRITY(X)wFTV(X)xATISG L(X)yYTITVYA(X)zISINYRT (SEQ ID NO: 9),

[0029] or by means of a sequence selected from the group of SEQ ID NO: 10-16, which sequences are identical to SEQ ID NO: 9, except that 1, 2, 3, 4, 5, 6 or 7 N-terminal amino acids, respectively. Table 2 shows the amino acid sequences of these degenerate human 10Fn3 molecules. Table 2: Amino acid sequences of degenerate wild-type human 10Fn3 molecules with multiple N-terminal regions

[0030] In SEQ ID NOs: 25-32 and 50, the AB loop is represented by (X)u, the BC loop is represented by (X) v, the CD loop is represented by (X), W, the loop DE is represented by (X) x, the EF loop is represented by (X) y and the FG loop is represented by Xz. The symbol X represents any amino acid and the subscript after the symbol X represents an integer of the number of amino acids. In particular, U, V, W, X, Y, and Z can each be independently anywhere from 2-20, 2-15, 2-10, 2-8, 5-20, 5-15, 5-10 , 5-8, 6-20, 615, 6-10, 6-8, 2-7, 5-7, or 6-7 amino acids. The beta filament sequences (underlined in SEQ ID NO: 9) can have any number from 0 to 10, from 0 to 8, 0-6, 0-5, 0-4, 0-3, from 0 to 2 , or 0 to 1 substitutions, deletions or additions of all framework regions relative to the corresponding amino acids shown in SEQ ID NOs: 9-16. In some embodiments, the beta strand sequences may have any number from 0 to 10, from 0 to 8, 0-6, 05, 0-4, 0-3, 0-2, or from 0 to 1 substitutions, for example, conservative substitutions in all framework regions relative to the corresponding amino acids shown in SEQ ID NO: 9-16.

[0031] In certain embodiments, the amino acid residues (hydrophobic core residues in bold in SEQ ID NO: 9, supra) are fixed, and any substitutions, conservative substitutions, deletions or additions occur at other than the amino acid residues hydrophobic core residues. Thus, in some embodiments, the hydrophobic core residues of the polypeptides provided in the present invention have not been modified relative to the wild-type human 10Fn3 domain (e.g., SEQ ID NO: 1).

[0032] In some embodiments, a portion of FBS comprises a 10Fn3 domain, wherein the 10Fn3 domain comprises a loop, AB; a loop, BC; a loop, CD; a loop, DE; a loop, EF; and a loop, FG; and has at least one loop selected from the AB, BC, CD, DE, FG EF loop and with an amino acid sequence change relative to the corresponding loop sequence of the wild-type human 10Fn3 domain. In some embodiments, a single loop is changed. In some embodiments, a maximum of 2 loops are changed. In some embodiments, a maximum of 3 loops are changed. In some embodiments, the BC, DE and/or FG loops are changed. In certain embodiments, the AB, CD and EF loops are changed. In certain embodiments, the FG loop is the only loop that is changed. In certain embodiments, the CD loop is the only loop that is changed. In other embodiments, the CD and FG loops are both changed, and optionally, any other loops are changed. In certain embodiments, the CD and EF loops are both changed, and optionally, any other loops are changed. In some embodiments, one or more specific structure changes are combined with one or more loop changes. By "altered" is meant one or more amino acid sequence changes relative to a template sequence (i.e., the corresponding wild-type human fibronectin domain) and includes amino acid additions, deletions, and substitutions. Examples of 10Fn3 molecules comprising specific combinations of altered loops and/or framework regions (e.g., beta strands, N-terminal region and C-terminal region) are further described in the present invention.

[0033] It should be understood that not all residues within a loop region need to be modified in order to achieve a 10Fn3 binding domain having strong affinity for a desired target. Furthermore, insertions and deletions in the loop regions can also be made while still producing the 10Fn3 high-affinity binding domains.

[0034] In some embodiments, one or more loops selected from AB, BC, CD, DE, FG and EF can be extended or shortened in length relative to the corresponding loop in wild-type human 10Fn3. In any given polypeptide, one or more loops may be extended in length, one or more loops may be reduced in length, or combinations thereof. In some embodiments, the length of a given cycle may be extended by 2-25, 2-20, 2-15, 2-10, 2-5, 5-25, 5-20, 5-15, 510, 10- 25, 10-20, or 10-15 amino acids. In some embodiments, the length of a given cycle can be reduced by 1-15, 1-11, 1-10, 1-5, 1-3, 1-2, 2-10, or 2-5 amino acids. In particular, the FG loop of 10Fn3 is 13 residues long, whereas the corresponding loop in antibody heavy chains ranges from 4-28 residues. To optimize the binding of polypeptides that depend on FG for target binding antigen, therefore, the length of the FG loop of 10Fn3 can be altered in length as well as sequence to obtain the greatest flexibility and target binding affinity.

[0035] In some embodiments, the FBS portion comprises a 10Fn3 domain in which the non-loop regions comprise an amino acid sequence that is at least 80, 85, 90, 95, 98, or 100% identical to the non-loop regions of SEQ ID NO: 1, in which at least one loop selected from AB, BC, CD, dE, EF and FG is changed. For example, in certain embodiments, the AB loop may have up to four amino acid substitutions, insertions of up to 10 amino acids, deletions of up to 3 amino acids, or a combination thereof; the BC loop can have up to 10 amino acid substitutions, deletions of up to four amino acids, insertions of up to 10 amino acids, or a combination thereof; the CD loop can have up to 6 amino acid substitutions, insertions of up to 10 amino acids, deletions of up to 4 amino acids, or a combination thereof; the DE loop can have up to 6 amino acid substitutions, deletions of up to four amino acids, up to 13 amino acid insertions, or a combination thereof; the EF loop can have up to 5 amino acid substitutions, insertions of up to 10 amino acids, deletions of up to 3 amino acids, or a combination thereof; and/or the FG loop may have up to 12 amino acid substitutions, deletions of up to 11 amino acids, up to 25 amino acid insertions, or a combination thereof.

[0036] In some embodiments, a portion of FBS comprises a 10Fn3 domain having at least 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or the identity of 90% for a human 10Fn3 domain having an amino acid sequence selected from the sequence group comprising SEQ ID NOs: 1-16. In certain embodiments, the FBS portion provided in the present invention has at least 50% identity to an amino acid sequence selected from the group of amino acid sequences comprising SEQ ID NO: 1-16. In other embodiments, the FBS portion has at least 65% identity with an amino acid sequence selected from the group of amino acid sequences comprising SEQ ID NO: 1-16. In certain embodiments, one or more of the loops will not be modified relative to the sequence of the corresponding loop of the wild-type sequence and/or one or more of the β-strands will not be modified relative to the sequence of the corresponding B-strands of the wild-type sequence. wild-type and/or the N-terminal or C-terminal regions will not be modified. In certain embodiments, each of the beta or beta-like strands of a 10Fn3 domain in an FBS portion may comprise, consist essentially of, or consist of an amino acid sequence that is at least 80%, 85%, 90%, 95 % or 100% identical to the sequence of a corresponding beta or similar beta strand of SEQ ID NO: 1. Preferably, variations in the B-strand regions do not disturb the stability of the polypeptide under physiological conditions.

[0037] In some embodiments, the non-loop region of a 10Fn3 domain can be modified through one or more conservative substitutions. Up to 5%, 10%, 20% or even 30% or more of the amino acids in the 10Fn3 domain can be changed through a conservative substitution, without substantially altering the affinity of the ligand for a 10Fn3. In certain embodiments, the non-loop regions, e.g., the β-strands may comprise anywhere from 015, 0-10, 0-8, 0-6, 0-5, 0-4, 0-3, 1-15 , 1-10, 1-8, 1-6, 1-5, 1-4, 1-3, 215, 2-10, 2-8, 2-6, 2-5, 2-4, 5-15 , or 5-10 conservative amino acid substitutions. In exemplary embodiments, the structure modification can reduce the ligand binding affinity for the 10Fn3 of a ligand by at least 100-fold, 50-fold, 25-fold, 10-fold, 5-fold, or 2-fold. It may be that such changes may alter the immunogenicity of 10Fn3 in vivo, and where immunogenicity is reduced, these changes may be desirable. As used in the present invention, "conserving substitutions" are residues that are physically or functionally similar to the corresponding reference residues. That is, a conservative substitution and its reference residue have similar size, shape, electrical charge, chemical properties including the ability to form covalent or hydrogen bonds, or the like. Exemplary conservative substitutions include those that meet the criteria defined for an accepted point mutation in Dayhoff et al, Atlas of Protein Sequence and Structure, 5:. 345 to 352 (1978 and Suppl.). Examples of conservative substitutions include substitutions within the following groups: (a) valine, glycine; (B) glycine, alanine; (C) valine, isoleucine, leucine; (D) aspartic acid, glutamic acid; (E) asparagine, glutamine; (F) serine, threonine; (G) lysine, arginine, methionine; and (h) phenylalanine, tyrosine.

[0038] Also available in the present invention is the 10Fn3s domain with combinations of loop and structure modifications. The conjugates may comprise a 10Fn3 domain comprising (i) a modification in the amino acid sequence of at least one of the AB, BC, CD, DE, EF, or FG loops, and (ii) a modification in the amino acid sequence of at least a region of structure (i.e., a change in at least one β-strand, the N-terminal region, and or the C-terminal region), in which the modified loop(s) and the Structure-modified region(s) both contribute to binding of the same target. In exemplary embodiments, framework region modifications are located adjacent to modifications in a loop region, e.g., if the AB loop is modified, framework mutations may tend to be located in β-strand A and/or β -filament B, which are adjacent to the AB loop in the linear sequence of the 10Fn3 domain. In other embodiments, a set of modifications may be found together in loop and framework regions that are adjacent to each other in the linear sequence of the Fn3 domain. For example, FN3 ligands having both loop and backbone modifications may have groups of amino acid changes in the following combinations of loop and backbone regions that are adjacent to each other in the linear sequence of the Fn3 domain: β - Filament / loop/β-Filament, loop/β-filament/loop, loop/β-filament/β-filament, terminal region/β-filament/loop, or loop/β-filament/terminal region, etc. For example, fN3 domains having novel combinations of loop and framework modifications may have groups of modifications such that over a stretch of 20 contiguous amino acids, at least 15 of the amino acids are modified relative to wild type. In other embodiments, at least 17 out of 20, 18 out of 20, 17 out of 25, 20 out of 25, or 25 out of 30 residues in a contiguous stretch are modified from the wild-type Fn3 domain sequence through the corresponding amino acid segment. In certain embodiments, a given Fn3 domain may have two or three sets of modifications separated by stretches of (i.e., wild-type) unmodified sequence. For any given region (i.e., a loop, β-strand or terminal region) that is modified, all or only a portion of the region may be modified relative to the wild-type sequence. When a β-strand region is modified, preferably the hydrophobic core residues remain unchanged (i.e., wild-type) and one or more of the non-essential residues in the β-strand are modified.

[0039] In some embodiments, the 10Fn3 domains comprise a binding face along the "north side" of the molecule ("north side secondary ligands" or "WS ligands"). The WS linkers may comprise a modified CD loop and a modified FG loop, as compared to the corresponding CD and FG loop sequences set forth in SEQ ID NO: 1. The CD loop and the FG loop both contribute to binding to the same target. In certain embodiments, the linkers may comprise additional WS modifications to one or more regions within the Fn3 domain. For example, WS linkers may comprise structure modifications in one or more of the B-Filament regions adjacent to the CD and/or FG loops. In particular, WS linkers may comprise sequence modifications to one or more of β-Filament C, β-Filament D, β-Filament F, and/or structure modifications of G. Examples of beta-filament include modifications at positions region one or more of framework corresponding to amino acid positions: 33, 35, 49, 69, 71, 73, 89 and/or 91 of SEQ ID NO: 1. WS binding agents may also comprise modifications to the BC loop, particularly in the C-terminal portion of the BC loop. In one embodiment, the last two residues of the BC loop (i.e., corresponding to amino acids 30 and 31 in the 10Fn3 wild-type domain) are modified relative to the wild-type sequence. All or a portion of the additional loop and structure modifications may contribute to target binding, in conjunction with the modified CD and FG loops. Preferably, the hydrophobic core residues are not modified relative to the wild-type sequence.

[0040] Exemplary WS linkers include those that have a wild-type or mutated amino acid at positions 30, 31, 33, 35, 37, 38, 46, 47, 49, 50, 67, 69, 71, 73, 75, 76, 84, 85, 86, 87, 89 or 91.

[0041] In some embodiments, a 10Fn3 domain comprises modifications to the CD, DE and, in some cases, EF loops, wherein the loop modifications all contribute to target binding. These polypeptides are referred to as "front linkers". The forward linkers may additionally comprise modifications of one or more framework regions, particularly in regions that flank or framework are adjacent to a modified loop region. For example, the forward linkers may comprise a structural modification in one or more of the β-strand C, β-strand D, and/or β-strand E relative to the sequences of the corresponding β-strands of the wild-type Fn3 domain, e.g. , the human 10Fn3 domain (SEQ ID NO: 1). Preferably, the hydrophobic core residues are not modified relative to the wild-type sequence. Exemplary structure modifications that may be present in the forward linkers include modifications at one or more positions corresponding to amino acid positions 36, 49, 58 and/or 50 of SEQ ID NO: 1. Such structure modifications may contribute to binding to the target in conjunction with the modified loops. In certain embodiments, the forward linkers may comprise sets of modifications, encompassing various loop regions and the Fn3 filament, e.g., 10Fn3 domain. In particular, the forward linkers may comprise modifications to at least 15, 20, 24, 25, or 27 of the 31 residues among the amino acids corresponding to residues 36 to 66 of wild-type Fn3, e.g., human 10Fn3 domain (SEQ ID NO: 1). Loop and/or strand modifications may include amino acid substitutions, deletions and/or insertions, or combinations thereof. In exemplary embodiments, the CD loop is extended in length or reduced in length relative to the CD loop of Fn3, e.g., wild-type human 10Fn3 domain (SEQ ID NO: 1).

[0042] In some embodiments, the 10Fn3 domains comprise modifications to the EF and FG loops, wherein the loop modifications contribute to binding of the same target. These polypeptides are referred to as "loop linkers" herein. Loop linkers may comprise additional modifications to other loop and/or framework regions. For example, a linker may contain modifications back to at least a portion of the AB loop, preferably the N-terminal portion of the AB loop. In an exemplary embodiment, the first two amino acids of the AB loop (i.e., corresponding to amino acid residues 14 and 15 of the 10Fn3 wild-type domain) are modified relative to the wild-type sequence. In certain embodiments, a loop linker may also contain one or more backbone modifications, especially modifications in one or more backbone regions that are adjacent to a modified loop region. For example, the loop linkers may contain one or more modifications in one or more of the A-strands, G-strands, the N-terminal region, and/or the C-terminal region. Preferably, the hydrophobic core residues are not modified relative to the wild-type sequence. The structure of exemplary modifications include modifications at one or more positions corresponding to amino acid positions 1-7, 9-13, 89, 91, 93 and/or 94 of SEQ ID NO: 1. One or more loop modifications and/or or additional structure may contribute to target binding, along with modified FE and FG loops. Suitable modifications of loop types and/or framework region include amino acid substitutions, deletions and/or insertions, or combinations thereof. In certain embodiments, the amino acid sequence of the FG loop is extended in length or reduced in length relative to the FG loop of the wild-type human 10Fn3 domain (SEQ ID NO: 1).

[0043] In certain embodiments, a back linker may comprise a cluster of modified amino acid residues across a contiguous stretch of several regions in the 10Fn3 domain. For example, at least 14 of the first 15 amino acid residues of Fn3, e.g., the 10Fn3 domain can be modified relative to the corresponding residues in wild-type Fn3, e.g., human 10Fn3 domain (SEQ ID NO: 1), and / or at least 15 of the 18 residues among the amino acids corresponding to residues 80 to 97 (or 94) of wild-type Fn3, e.g., human 10Fn3 domain (SEQ ID no: 1 or 23) can be modified with respect to residues corresponding to the wild-type sequence. When reference is made to the amino acids at the additional 94 C-terminal positions in a 10Fn3 molecule, which is in the context of a 10Fn3 molecule comprising the flexible linker between the 10th and 11th repeat of the Fn3 domain, i.e., EIDKPSQ, forming thus a protein length of 101 amino acids (thus, SEQ ID NO: 1 linked to EIDKPSQ at its C-terminal end is represented by SEQ ID NO: 23).

[0044] VSDVPRD LEVVAATPTSLLISWDAPAVTVRYYRITYGETG GNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPI SINYRT EIDKPSQ (SEQ ID NO: 23)

[0045] In certain embodiments, a 10Fn3 domain comprises modifications in the amino acid sequences of β-strand A, AB loop, β-strand B, loop CD, β-Filament E, loop EF, and β-Filament F, in relation to to the sequences of the corresponding regions of the wild-type sequence. These polypeptides are referred to as "south pole ligands" or "SP ligands" described in the present invention. The modified loops and filaments contribute to binding to the same target. The amino acid sequence of the CD loop can be extended in length or reduced in length relative to the CD loop of wild-type Fn3, for example, the human 10Fn3 domain (SEQ ID NO: 1 or 23). South pole linkers may comprise additional modifications to the β-G strand and/or the C-terminal region relative to the sequence of the corresponding region of the wild-type sequence. In exemplary embodiments, the south pole linkers may include one or more amino acid modifications corresponding to positions 11, 12, 19, 60, 61, 69, 91, 93 and 95-97 of the wild-type sequence.

[0046] In some embodiments, a 10Fn3 domain comprises modified BC, DE and FG loops, as compared to the corresponding BC, DE and FG loop sequences set forth in SEQ ID NO: 1 or 23, as well as additional modifications in one or more of the residues of β-strand C, β-strand D, β-strand F and β-strand G. The modifications of β-strand and loop region together contribute to target binding. These proteins are referred to as "Northwest ligands", or "NW ligands", herein. In exemplary embodiments, the NW linkers comprise one or more framework modifications at any one or combination of amino acid positions corresponding to framework region positions R33, T49, Y73 and S89 of SEQ ID NO: 1 or 23. Suitable modifications to the loop and framework regions include amino acid substitutions, deletions and/or insertions, or combinations thereof. In certain embodiments, one or more of the BC, DE, and FG loops are extended in length or reduced in length, or combinations thereof, relative to the wild-type sequence. In one embodiment, each of the BC, DE, and FG loops are extended in length or reduced in length, or combinations thereof, relative to the wild-type sequence (e.g., SEQ ID NO: 1 or 23). In certain embodiments, only a portion of the BC loop is modified, particularly the C-terminal portion, relative to the wild-type sequence. For example, the BC loop can only be modified at amino acid residues corresponding to amino acids 27-31 of the wild-type BC loop, whereas the remainder of the BC loop (i.e., corresponding to residues 23-26 of the wild-type loop ) is left unchanged.

[0047] In some embodiments, a 10Fn3 domain comprises a modified BC, DE and FG loop, as well as one or more modifications in any of, or combination of, the N-terminal region, β-strand A, β-strand B and/or or β-Filament E. These proteins are referred to as "Northeast ligands", or "NE ligands", herein. In exemplary embodiments, the NE linkers are modified in any one or combination of the amino acids corresponding to the framework region of positions 1-7, E9, L19, S21 and/or T58 of the wild-type sequence (SEQ ID NO: 1 or 23). The combination of modified loop and framework regions contributes to target binding.

[0048] In some embodiments, a 10Fn3 domain comprises modifications in one or more loops of the AB, CD, DE and EF, as well as additional modifications in one or more of the β-filament B, β-Filament D and/or β -Filament E. These proteins are referred to as "South Front ligands" herein. The combination of modified loop and strand residues contributes to target binding. In exemplary embodiments, a South Front linker may be modified at one or more amino acid positions corresponding to framework region positions L19, T49, T58, S60, and/or G61 of SEQ ID NO: 1 or 23 and/or to a or more amino acid positions corresponding to loop region positions T14-S17, P51, T56, G40-E47, and/or K63-G65 of SEQ ID NO: 1 or 23. In exemplary embodiments, a South Front linker may be extended into length or reduced in length in the AB loop, between the amino acids corresponding to residues 18 and 20 of the wild-type sequence, and/or in the CD loop.

[0049] In some embodiments, a 10Fn3 domain comprises a β-strand A and a modified β-strand G, when compared to the corresponding strand of SEQ ID NO: 1 or 23. These proteins are referred to as "AG ligands" or ligands of "AG Filament" described in the present invention. In certain embodiments, AG filament linkers comprise clusters of modifications to the N-terminal and C-terminal portions of Fn3, e.g., 10Fn3 domain, while the middle portion of Fn3 remains unchanged. For example, an AG strand linker may comprise modifications to 16 of 19 of the first 19 amino acids in the 10Fn3 domain (i.e., corresponding to amino acid positions 1-19 of SEQ ID NO: 1 or 23) and changes to 13- 17 of 18 of the last 18 amino acids in the 10Fn3 domain (i.e., corresponding to amino acid positions 84-101 of SEQ ID NO: 9) or in 14-18 of 22 of the last 22 amino acids in the 10Fn3 domain (i.e., corresponding to amino acid positions 80-101 of SEQ ID NO: 9). In exemplary embodiments, an AG linker may comprise modifications at one or more positions corresponding to positions 1-7, 9, 11-17, 19, 84-89 and 91-97 of SEQ ID NO: 9. Preferably, the regions modified into an AG ligand contribute to binding to the same target.

[0050] In some embodiments, a 10Fn3 domain comprises a modified CD and EF loop, as well as additional modifications in any of, or the combination of, residues corresponding to positions 69 or 91-97 of SEQ ID NO: 1 or 23. These proteins are referred to as "Southwest ligands", or "SW ligands", described in the present invention. The modified loop and framework regions contribute to target binding.

[0051] In certain embodiments, the proteins comprise a 10Fn3 domain having reduced immunogenicity, in which a portion of the BC loop is left as wild type. Preferably, such polypeptides have lower immunogenicity relative to a polypeptide with equivalent modifications to a larger portion of the BC loop. In exemplary embodiments, the N-terminal portion of the BC loop is left as wild type. For example, the first 1, 2, 3, 4, 5, or 5 residues of the BC loop can be left as wild type, while the remaining C-terminal residues of the BC loop can be modified. In Fn3 designs having at least a portion of the N-terminal region of the BC loop as wild type, it may be desirable to leave all or a portion of β-strand B and/or β-strand C relatively unmodified to the Fn3 sequence. wild type, as well as in particular the portions of β-strand B and/or C-strand β that are adjacent to the BC loop (i.e., the C-terminal portion of the β-strand B and/or the N-terminal portion of β-strand C). In exemplary embodiments, FN3 domains having the wild-type sequence in an N-terminal portion of the BC loop and reduced immunogenicity may not have any modifications in the N-terminal region, loop A, β-Filament AB, and β-strand B. . in Fn3 designs with a portion of the BC loop as wild type, the modified portion of the BC loop may contribute to achieving binding along with changes to other regions of the 10Fn3 domain.

[0052] In certain embodiments, the proteins comprise a 10Fn3 domain having reduced immunogenicity, in which the strong anchorage of HLA in the β-filament B/BC loop/C β-Filament region (the "BC anchor") has been removed or destroyed (e.g., modified relative to the wild-type sequence in a manner that reduces binding affinity for one or more HLA receptors). For example, the BC anchor can be removed or destroyed by modifying the Fn3, e.g., 10Fn3 domain at one or more positions corresponding to positions L19, S21, R33 and/or T35 of SEQ ID NO: 1. When the anchor AC has been removed or destroyed, it is possible to modify the BC loop sequence without significantly increasing the immunogenic potential of the BC region. Therefore, many of these FN3 models have modifications in the BC loop, in addition to modifications in the β-B strand and/or β-C strand. The BC loop can contribute to target binding, optionally in combination with changes to other regions. of the Fn3 domain. Modifications to β-strand B and/or β-strand C may or may not contribute to the binding objective.

[0053] In exemplary embodiments, an FBS, for example, a 10Fn3 domain, binds to a desired target, with a Kd of less than 500 nM, 100 nM, 50 nM, 10 nM, 5 nM, 1 nM, 500 pM , 100 pM or less. In some embodiments, the FBS, e.g., the 10Fn3 domain, binds to a desired target with a Kd between 1 pM and 1 µM, between 100 pM and 500 nM, between 1 nM and 500 nM, or between 1 nM and 100 nM. nM. In exemplary embodiments, the 10Fn3 portion specifically binds to a target that is not bound via a wild-type 10Fn3 domain, particularly the human wild-type 10Fn3 domain having, for example, SEQ ID NO: 1-8.

[0054] In certain embodiments, a portion of FBS comprises an amino acid sequence that is at least 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to an amino acid sequence selected from the group of sequences consisting of SEQ ID Nos: 1-16, and the FBS specifically binds to a target, for example, with a Kd of less than 500 nM, 100 nM, 50 nM, 10 nM, 5 nM, 1 nM, 500 pM, 100 pM or less. The FBS portion may comprise amino acid changes (or changes) in one or more loops and one or more framework regions.

[0055] In some embodiments, one or more residues of the "arginine-glycine-aspartic acid" (RGD) integrin binding motif (amino acids 78-80 of SEQ ID NO: 1) may be substituted, so as to disrupt the integrin binding. In some embodiments, the FG loop of the polypeptides provided in the present invention does not contain an RGD integrin binding site. In one embodiment, the RGD sequence is substituted by means of a polar neutral amino-acidic amino acid sequence through the amino acid acid (at the N-terminus to C-terminus). In certain embodiments, the RGD sequence is replaced with SGE or RGE.

[0056] In some embodiments, the amino acid sequences of the N-terminal and/or C-terminal regions of a portion of FBS are modified through deletion, substitution or insertion relative to the amino acid sequences of the corresponding regions of the 10Fn3 domains that comprise, for example, SEQ ID NO: 1.

[0057] In certain embodiments, the amino acid sequence of the first 1, 2, 3, 4, 5, 6, 7, 8 or 9 residues of SEQ ID NO: 1 may be modified or deleted in the polypeptides presented in the present invention in relation to to the sequence of the corresponding amino acids in the wild-type human 10Fn3 domain of SEQ ID NO: 1. In exemplary embodiments, the amino acids corresponding to amino acids 1-7, 8 or 9, of any of SEQ ID NOs: 1-16 are substituted with an alternative N-terminal region that is between 1-20, 1-15, 1-10, 1-8, 1-5, 1-4, 1-3, 1-2, or 1 amino acids in length. Exemplary alternative N-terminal regions include (represented by the single amino acid code letter) H, MG, L, MGVSDVPRDL (SEQ ID NO: 24) and GVSDVPRDL (SEQ ID NO: 25), or N-terminal truncations of any of SEQ ID NOs: 24 or 25. Other suitable alternative N-terminal regions include, for example, XnSDVPRDL (SEQ ID NO: 26), XnDVPRDL (SEQ ID NO: 27), XnVPRDL (SEQ ID NO: 28), XnPRDL (SEQ ID NO: 29), XnRDL (SEQ ID NO: 30), xndl (SEQ ID NO: 31), or Met or Gly, and when n = 2, X is Met-Gly. When a Met-Gly sequence is added to the N-terminus of a 10Fn3 domain, the M will normally be removed by scission, leaving a G at the N-terminus. In other embodiments, the alternative N-terminal region comprises the amino acid sequence MASTSG (SEQ ID NO: 32).

[0058] As further described in the present invention, in some embodiments, the first seven or eight residues (i.e., residues 1-7 or 1-8) of SEQ ID NO: 1, are deleted, generating a 10Fn3 domain having the sequence of, for example, SEQ ID NO: 8. Additional sequences may also be added to the N- or C-terminus of a 10Fn3 domain having the amino acid sequence of any of SEQ ID NOs: 1-16. For example, in some embodiments, the N-terminal extension consists of an amino acid sequence selected from the group consisting of: H, MG, and G. For example, any of SEQ ID NO: 1-16 may be preceded by M, MG, or G.

[0059] In certain embodiments, a portion of FBS is based on an Fn3 repeat other than the 10th repeat domain of fibronectin type III, e.g., human fibronectin. For example, an FBS moiety may be similar to any other type of fibronectin III repeat, e.g., the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 11th, 12th, 13th, 14th, 15th. , 16, 17, and repeats fN3 18. In still other embodiments, a portion of FBS may be from a molecule other than fibronectin. Exemplary FBS portions may be derived from tenascin, a protein that is composed of 15 Fn3 domains with similar sequence similarities to one another as found in fibronectin. These repeats are described, for example, in Jacobs et al, Protein Engineering, Design, Selection, 25: 107 (2012). Based on the homology of the repeats in the fibronectin molecule and those in the tenascin molecule, artificial molecules based on these homologies were created. Proteins that comprise a consensus sequence of amino acids based on the homology of the domains in the fibronectin molecule are referred to as Fibcon and FibconB (WO 2010/093627 and Jacobs et al. (2012) supra.) and those that are based on the homology of the Tenascin domains in the molecule are referred to as Tencon. An exemplary Fibcon amino acid sequence comprises the following amino acid sequence: MPAPTDLRFTNETPSSLLISWTPPRVQITGYIIRYGPVGSDGRVKEFT VPPSVSSATITGLKPGTEYTISVIALKDNQESEPLRGRVTTGG (FibconB; SEQ ID NO: 33),

[0060] wherein the AB loop consists of amino acids 13-16 (TPSS; SEQ ID NO: 34), BC loop consists of amino acids 22-28 (TPPRVQI; SEQ ID NO: 35), CD loop consists of amino acids 38- 43 (VGSDGR; SEQ ID NO: 36), DE loop consists of amino acids 51-54 (PSV; SEQ ID NO: 37), EF loop consists of amino acids 60-64 (GLKPG; SEQ ID NO: 38) and FG loop consists of amino acids 75-81 (KDNQESEP; SEQ ID NO: 39). Another Fibcon amino acid sequence comprises the following amino acid sequence: LDAPTDLQVTNVTDTSITVSWTPPSATITGYRITYTPSNGPGEPKELTV PPSSTSVTITGITPGVEYVVSVYALKDNQESPPLVGTCTT (SEQ ID NO: 40; Jacobs et al., Supra).

[0061] FN3 proteins derived from Tenascin include Tencons (WO 2010/051274, WO 2010/051310 and WO 2011/137319, which are specifically incorporated into the present invention by reference). An exemplary Tencon protein has the following amino acid sequence: LPAPKNLVVSEVTEDSLRLSWTAPDAAFDSFLIQYQESEKVGEAINLT VPGSERSYDLTGLKPGTEYTVSIYGVKGGHRSNPLSAEFTT (SEQ ID NO: 41; Jacobs et al., supra, and WO 2011/137319),

[0062] wherein the AB loop consists of amino acids 13-16 (TEDS; SEQ ID NO: 42, BC loop consists of amino acids 22-28 (TAPDAAF; SEQ ID NO: 43), CD loop consists of amino acids 38-43 (SEKVGE; SEQ ID NO: 44), DE loop consists of amino acids 51-54 (GSER; SEQ ID NO: 45), EF loop consists of amino acids 60-64 (GLKPG; SEQ ID NO: 46) and FG loop consists at amino acids 75-81 (KGGHRSN; SEQ ID NO: 47).

[0063] The Fibcon, FibconB or Tencon moiety, or binding target variants thereof, either alone or linked to a heterologous moiety can be fused, as described in the present invention. The Fn3 domains of other proteins, for example, cell surface hormone and cytokine receptors, chaperonins, and carbohydrate binding domains, can be conjugated as described in the present invention.

[0064] FBS proteins or portions are described, for example, in WO 2010/093627, WO 2011/130324, WO 2009/083804, WO 2009/133208, WO 02/04523, WO 2012/016245, WO 2009/ 023184, WO 2010/051310, WO 2011/020033, WO 2011/051333, WO 2011/051466, WO 2011/092233, WO 2011/100700, WO 2011/130324, WO 2011/ 130328, WO 2011/137319, WO 2010/051274, WO 2009/086116, WO 09/058379, WO 2013/067029 WO 2012/016245, WO 2014/120891 and WO 2014/043344 (all of which are specifically incorporated into the present invention by reference) : any of the proteins or portions of FBS described in these publications can be used as described in the present invention.

[0065] In certain embodiments, a protein comprises at least 2 FBS moieties, for example, the protein comprises a multivalent FBS moiety. For example, a multivalent FBS may comprise 2, 3 or more FBS moieties, e.g., 10Fn3 domains, which are covalently associated. In exemplary embodiments, the FBS moiety is a dimeric or bispecific protein comprising two 10Fn3 domains.

[0066] The FBS portions, for example, the 10Fn3 domains, in a multivalent protein can be linked via a polypeptide linker. Exemplary polypeptide linkers include polypeptides having 1-20, 1-15, 1-10, 1-8, 1-5, 1-4, 1-3, or 1-2 amino acids. Suitable linkers to join the 10Fn3 domains are those that allow the separate domains to fold independently of each other forming a three-dimensional structure that allows binding to a high-affinity target molecule. Specific examples of suitable linkers include glycine-serine based linkers, glycine-proline based linkers, proline-alanine based linkers, as well as any other linkers described in the present invention. In some embodiments, the linker is a glycine-proline based linker. These linkers comprise glycine and proline residues and can be between 3 and 30, 10 and 30, and 3 and 20 amino acids in length. Examples of such linkers include GPG, GPGPGPG (SEQ ID NO: 48) and GPPGPGPGPG (SEQ ID NO: 49). In some embodiments, the linker is a proline-alanine based linker. These linkers comprise proline and alanine residues and can be between 3 and 30, 10 and 30, 3 and 20 and 6 and 18 amino acids in length. Examples of such linkers include PAPAPA (SEQ ID NO: 50), PAPAPAPAPAPA (SEQ ID NO: 51) and PAPAPAPAPAPAPAPAPA (SEQ ID NO: 52). In some embodiments, the linker is a glycine-serine based linker. These linkers comprise glycine and serine residues and can be between 8 and 50, 10 and 30, and 10 and 20 amino acids in length. Examples of such linkers include GSGSGSGSGS ((GS) 5; SEQ ID NO: 53), GSGSGSGSGSGS ((GS) 6; SEQ ID NO: 54), GSGSGSGSGSGSGSGSGSGS ((GS) 10; SEQ ID NO: 55), GGGGSGGGGSGGGGS (( G4S) 4; SEQ ID NO: 56), GGGGSGGGGSGGGGSGGGGSGGGGS ((G4S) 5; SEQ ID NO: 57), and GGGGSGGGGSGGGSG (SEQ ID NO: 58). In exemplary embodiments, the linker does not contain any Asp-Lys (NS) pairs. PmXn portions, e.g. stabilization portions

[0067] In certain embodiments, FBS, for example, a portion of 10Fn3, is linked at its C-terminal end to a portion consisting of PmXn, where P is a proline, X is any amino acid, the symbol m represents a integer that is at least 1 and n is 0 or an integer that is at least 1 and P is N-terminal of , its amino acid 94 (based on the amino acid numbering of SEQ ID NO: 1). The PmXn moiety can be linked via a peptide bond to amino acid 94 of a 10Fn3 moiety. A PmXn moiety may be linked to a 10Fn3 moiety having an amino acid sequence that is homologous to that of SEQ ID NO: 1 or comprises, consists essentially of, or consists of an amino acid sequence shown in Table 1 or 2. A single proline residue at the end of SEQ ID NO: 1 is referred to as "95Pro" or "Pro95" or "P95" or "95P".

[0068] Exemplary 10Fn3 moieties linked to a PmXn moiety include the following: LEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKS TATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRTPmXn (SEQ ID NO: 59) VSDVPRDLEVVAATPTSLLISWDAPAVTVRYRITYGETGGNSPVQE FTVPGSK STATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRTPm Xn (SEQ ID NO: 60)

[0069] In PmXn, m can be 1, 2, 3 or more. For example, m could be 1-3 or m could be 1-2. "N" can be 0, 1, 2, 3 or more, for example, n can be 1-3 or 1-2.

[0070] As further described in the present invention, these portions of 10Fn3 can be modified to bind to a target (and form the FBS portions) by modifying the amino acid sequence of one or more loop and/or one or more β-strands . The FBS moieties that are linked to PmXn are referred to in the present invention as "modified FBS moieties". Therefore, provided in the present invention are proteins comprising an FBS moiety comprising an amino acid sequence that is at least about 50%, 60%, 70%, 80%, 90%, or 95% identical to SEQ ID NO: 59 or 60, where the protein comprises PmXn, and where the FBS specifically binds to a target (other than through the RGD domain).

[0071] In PmXn, n may be 0, in which case, the C-terminal amino acid of the protein is Pm, for example, P. In certain embodiments, n is not 0, and may be, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more. For example, n can be 0-10, 0-5, 0-3, 1-10, 1-5, 1-3, or 1-2. However, more than 10 amino acids can be linked to proline. For example, in a set of FBS moiety or an FBS moiety fused to another polypeptide, the C-terminal amino acid of the FBS moiety may be linked to one or more prolines, and the last proline is linked to the second FBS moiety or to the heterologous portion. Therefore, in certain embodiments, n may be an integer that ranges from 0-100, 0-200, 0-300, 0-400, 0-500 or more.

[0072] In certain embodiments, PmXn comprises a cysteine. For example, the first amino acid after proline may be a cysteine, and the cysteine may be the last amino acid in the molecule or the cysteine may be followed by one or more amino acids. The presence of a cysteine allows the conjugation of heterologous moieties to an FBS moiety, e.g. chemical moieties, e.g. PEG. Examples of PmXn moieties comprising a cysteine include: PmCXn, wherein C is a cysteine. Another example is PmXn1CXn2, where n1 and n2 are independently 0 or an integer that is at least 1. For example, N1 may be and N2 may be 1, 2, 3, 4, or 5. PmXn Portions Examples include those listed in Table 3. Table 3: Examples of PmXn portions

[0073] Any of the PmXn moieties, for example those shown in Table 3, may be followed by a histidine tail, for example, 6xHis tag, or other tags. This does not exclude that a histidine tail may be included in PmXn.

[0074] The addition of a PmXn moiety to an FBS moiety improves one or more characteristics of the FBS moiety. For example, as shown in the Examples, it improves the thermal stability of a 10Fn3 moiety relative to the portion that is not linked to a PmXn moiety. Improving thermal stability is expected to improve other desirable properties such as solubility, correct folding and expression level. For example, as shown in the example, the presence of a PmXn moiety at the C-terminal end of an FBS increases the solubility of SBF relative to SBF, which is not linked to a PmXn moiety.

[0075] Thus, in certain embodiments, the Tm of an FBS, for example, a 10Fn3, portion is increased by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 , 11, 12, 13, 14, or 15 °C relative to the FBS portion that is not linked to a PmXn portion. For example, the Tm can be increased 1-30, 1-25, 1-20, 1-15, 1-10, or 1-5°C relative to the FBS portion that is not linked to a PmXn portion. TM can be measured by thermal scanning fluorescence (TSF), for example, as follows. Protein samples, e.g. HTPP samples, are normalized to 0.2 mg/ml in PBS. 1 ul of SYPRO® orange dye diluted 1:40 with PBS is added to 25 uL of each sample and the plate is sealed with a 96-well clear adhesive microplate seal. Samples are scanned using a BioRad RT-PCR machine by increasing the temperature from 25°C - 95°C at a rate of 2 degrees per minute. Data is analyzed using the BioRad CFX 2.0 software manager. Tm can also be measured by Differential Scanning Calorimetry (DSC) as follows. A 0.5 mg/ml solution is scanned in a VP-capillary differential scanning calorimeter (GE Microcal) by raising the temperature from 15°C to 110°C at a rate of 1 degree per minute under 70 p.s.i. pressure. Data is analyzed against an appropriate buffer series using fine-tuning control using Origin Software (OriginLab Corp).

[0076] In certain embodiments, the solubility of an FBS moiety is improved by binding it to a PmXn moiety. Such a molecule may exist in a concentration of at least 10 mg/ml, 20 mg/ml, 30 mg/ml, 40 mg/ml, 50 mg/ml, 60 mg/ml, 70 mg/ml, 80 mg/ml , 90 mg/ml or 100 mg/ml.

PKE Portions

[0077] FBS moieties can be linked to PKE moieties to extend the half-life of the FBS moieties. Exemplary PKE moieties include human serum albumin; proteins that bind to human serum albumin (e.g., binding to ASH from FBS or ABD); fc; or any part or variant thereof; and PEG. These moieties can be linked N-terminally or C-terminally to the PmXn moiety and/or the FBS.

Cysteine Conjugate Labels and Therapeutics

[0078] In certain embodiments, an FBS moiety linked to a PmXn moiety (and which refers to a "modified FBS moiety"), wherein at least one or more of the "X" amino acids is a cysteine residue, is linked through one or more cysteines, to a heterologous moiety, such as a labeling moiety, a biologically active moiety (e.g., a therapeutic agent) or a binding moiety.

[0079] The FBS moieties described in the present invention can be conjugated through a C-terminal cysteine to a therapeutic agent to form an immunoconjugate such as a drug-FBS conjugate (FBS-DC; also "Adnectinaa drug conjugate").

[0080] In an FBS-DC, the SBF is conjugated to a drug, with FBS functioning as a targeting agent to direct the SBF-DC to a target cell that expresses its antigen, such as a cancer cell. Preferably, the antigen is a tumor-associated antigen, that is, one that is exclusively expressed or overexpressed by the cancer cell. Once there, the drug is released, either within the target cell or in its vicinity, to act as a therapeutic agent. For a review of the mechanism of action and use of drug conjugates as used with antibodies, for example, in cancer therapy, see Schrama et al, Nature Rev. Drug Disc., 5: 147 (2006).

[0081] Suitable therapeutic agents for use in drug conjugates include antimetabolites, alkylating agents, minor DNA groove ligands, DNA intercalators, DNA cross-linking agents, histone deacetylase inhibitors, nuclear export inhibitors, proteasome, topoisomerase I or II inhibitors, heat shock protein inhibitors, tyrosine kinase inhibitors, antibiotics and antimitotic agents. In an FBS-DC, the FBS and the therapeutic agent are preferably conjugated through a cleavable linker, such as a peptidyl, disulfide, or hydrazone linker. More preferably, the linker is a peptidyl linker, such as Val-Cit, Ala-Val, Ala-ValVal, Lys-Lys, Pro-Val-Gly-Val-Val (SEQ ID NO: 89), Ala-Asn - Val, Val- Leu-Lys, Ala-Ala-Asn, Cit-Cit, Val-Lys, Lys, Cit, Ser or Glu. The FBS-DCs can be prepared according to methods similar to those described in U.S. Patent No. 7,087,600; 6,989,452; and 7,129,261; PCT Publication No. WO 02/096910.; WO 07/038658; WO 07/051081; WO 07/059404; WO 08/083312; and WO 08/103693; U.S. Patent Publication No. 2006/0024317.; 2006/0004081; and 2006/0247295; the descriptions of which are incorporated herein by reference. A linker itself may be linked, for example, covalently, for example using maleimide chemistry, to a cysteine of the PmXn moiety, wherein at least one X is a cysteine. For example, a linker can be covalently attached to an FBS PmXn, wherein at least one X is a cysteine. For example, a ligand can be attached to one of FBS-PmCn, where P is a proline, C is a cysteine, and m and n are integers that are at least 1, for example, 1-3. Binding to a cysteine can be accomplished as is known in the art using maleimide chemistry (e.g., Imperiali, B., et al, Protein Engineering: Nucleic Acids and Molecular Biology, Vol 22, pp 65 to 96, Gross, HJ, Ed . (2009)). For attaching a linker to a cysteine on an FBS, the linker may, for example, comprise a maleinimide moiety, which portion then reacts with the cysteine to form a covalent bond. In certain embodiments, the amino acids surrounding cysteine are optimized to facilitate the chemical reaction. For example, a cysteine residue can be surrounded by a negatively charged amino acid through a faster reaction compared to a cysteine that is surrounded by a stretch of positively charged amino acids (EP 1074563).

[0082] For the treatment of cancer, the drug is preferably a cytotoxic drug that causes the death of the target cancer cell. Cytotoxic drugs that can be used in FBS-DCs include the following types of compounds and their analogues and derivatives:

[0083] (a) enediines such as calicheamicin (see, for example, Lee et al, J. Am Chem Soe, 109: 3464, 3466 (1987)). E uncialamycin (see, for example, Davies et al, WO 2007/038868 A2 (2007) and Chowdari et al, US Patent No. 8709431 B2 (2012));

[0084] (b) tubulysins (see, for example, Domling et al, US Patent No. 7778814 B2 (2010); Cheng et al, US Patent No. 8394922 B2 (2013); E Cong et al, US Publication No. ° 2014 / 0227295 A1;

[0085] (c) CC-1065 and duocarmycin (see, for example, Boger, in US Patent No. 6,5458,530 B1 (2003); Sufi et al, US Patent No. 8461117 B2 (2013); E Zhang et al, US Publication No. 2012/0301490 A1 (2012));

[0086] (d) Epothilones (see, for example, Vite et al, US Publication No. 2007/0275904 A1 (2007) and US Patent No. RE42,930 E (2011).);

[0087] (e) Auristatins (see, for example, Senter et al, US Patent No. 6,844,869 B2 (2005) and Doronina et al, US Patent No. 7,498,298 B2 (2009)..);

[0088] (f) Pyrrolobenzodiazepine (PBD) dimers (see, for example, Howard et al, US Publication Nos. 2013/0059800 A1 (2013) and 2013/0028919 A1 (2013);.. and WO 2013/041606 A1 ( 2013)); It is

[0089] (g) Maytansinoids such as DM1 and DM4 (see, for example, Chari et al., US Patent No. 5,208,020 (1993) and Amphlett et al., US Patent No. 7,374,762 B2 (2008 )).

[0090] In certain embodiments, one of FBS-PmXn, in which at least one X is a cysteine, is linked to a label or detectable moiety for use, for example, in vitro or in vivo or image detection.

[0091] Detectable labels may be any of several types currently used in the field of in vitro diagnostics, including particle labels including metallic sols such as colloidal gold, isotopes such as I125 or Tc99 presented, for example, with a chelating agent. peptide of the N2S2, N3S or N4 type, chromophores, including fluorescent tags, biotin, luminescent tags, phosphorescent tags and the like, as well as enzymatic tags that convert a given substrate to a detectable tag, and polynucleotide tags which are described below to amplification, such as through the polymerase chain reaction. A biotinylated antibody is then detectable through binding to avidin or streptavidin. Suitable enzyme labels include horseradish peroxidase, alkaline phosphatase and the like. For example, the marker may be the enzyme alkaline phosphatase, detected by measuring the presence or formation of chemiluminescence upon conversion of 1,2-dioxetane substrates such as adamantyl methoxy phenyl phosphoryloxy dioxane (AMPPD), 3-(4-({1 methoxyspiro, 2-dioxetane-3,2'-(5'-chloro)tricycle 3.3.1.1{3,7}decan}-4-yl) disodium phenyl phosphate (CSPD), as well as CDP and CDP-STAR® substrates or other luminescents well known to those skilled in the art, for example, suitable lanthanide chelates such as terbium (III) and europium (III).

[0092] Detectable moieties that can be used include radioactive agents, such as: radioactive heavy metals such as iron chelates, radioactive gadolinium or manganese chelates, positron emitters of oxygen, nitrogen, iron, carbon, or gallium , 18F 60Cu, 61Cu, 62Cu, 64Cu , 124I, 86Y, 89Zr, 66Ga, 67Ga, 68Ga, 44Sc, 47Sc, 11C, 111In, 114min, 114In, 125I, 124I, 131I, 123I, 131I, 123I, 32Cl, 33cl, 34Cl, 74Br, 75Br, 76Br, 77Br, 78Br, 89Zr, 186Re, 188Re, 86Y, 90Y, 177Lu, 99Tc, 212Bi, 213Bi, 212Pb, 225Ac, or 153Sm.

[0093] The means of detection is determined by the chosen label. The appearance of the tag or its reaction products can be achieved using the naked eye, in the case where the tag is particulate and accumulates at appropriate levels, or by means of instruments such as a spectrophotometer, a luminometer, a fluorometer, and the like. all in accordance with standard practice.

[0094] A detectable moiety can be linked to a cysteine according to methods known in the art. When the detectable moiety is a radioactive agent, for example, those described in the present invention below, the detectable moiety is linked to an FBS through a chelating agent that is reactive with cysteine residues, such as a maleimide-containing chelating agent, such as such as maleimide-NODAGA or maleimide-DBCO. Maleimide-NODAGA or maleimide-DBCO can be reacted with a cysteine at the C-terminal end of an FBS (e.g., through the PmXn moiety, in which at least one X is a cysteine residue), to obtain FBS NODAGA or FBS DBCO, respectively. Any of the following chelating agents may be used, provided that it comprises, or can be modified to comprise, a reactive moiety that reacts with cysteines: DFO, DOTA and their derivatives (CB-DO2A, 3p-C-DEPA, TCMC, oxo -DO3A), TE2A, CB- TE2A, CB-TE1A1P, CB-TE2P, MM-TE2A, DM-TE2A, diamsar and derivatives, NODASA, NODAGA, NOTE, NETA, TACN-TM, DTPA, 1B4M-DTPA, CHX - A '' - DTPA, TRAP (PRP9), Nopo, AAZTA and derivatives (from data), H2dedpa, H4octapa, H2azapa, H5decapa, H6phospa, chelating agents based on HBED, SHBED, BPCA, CP256, PCTA, HEHA, PEPA , EDTA, TETA, and TRITA, and close analogues and derivatives thereof.

[0095] In certain embodiments, an FBS is labeled with a PET tag and used as an in vivo imaging agent. For example, an FBS can be labeled with the PET tracer 64Cu. 64Cu can be linked to an FBS with a C-terminal cysteine with a chelating agent such as maleimide-NODAGA.

Exemplary molecules

[0096] In certain embodiments, a protein comprises (i) an FBS portion comprising an amino acid sequence that is at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95 %, 97%, 98%, or 99% identical to any of SEQ ID NOs: 116; and (ii) PmXn, where P is a proline, X is any amino acid, the symbol m represents an integer of at least 1 and n is 0 or an integer of at least 1, wherein the protein specifically binds to a target (e.g., with a Kd of less than 500 nM, 100 nM, 50 nM, 10 nM, 5 nM, 1 nM, 500 pM, 100 pM or less, as determined, for example, by Surface Plasmon Resonance (SPR) , such as Biacore) and wherein the PmXn portion improves at least one property of the FBS unit relative to a protein consisting of the unmodified FBS portion.

[0097] In certain embodiments, an improved property conferred by a PmXn moiety is a protein of improved stability, e.g., an increase in melting temperature (Tm) of at least 1°C, 2°C, 3°C, 4 °C, 5 °C, 6 °C, 7 °C, 8 °C, 9 °C, 10 °C, 11 °C, 12 °C, 13 °C, 14 °C, 15 °C, 1-15 °C, 2-15 °C, 315 °C, 5-15 °C, 5-15 °C, 1-10 °C, 2-10 °C, 3-10 °C, 4 -10°C or 5-10°C. Tm can be determined, for example, by means of thermal scanning fluorescence (TSF) or differential scanning calorimetry (DSC). "Improved Tm" refers to a statistically significant improvement in Tm.

[0098] In certain embodiments, a protein comprising an FBS moiety and a PmXn moiety is present in a composition, e.g., a pharmaceutical composition, at a concentration of at least 10 mg/ml, 20 mg/ml, 30 mg /ml, 40 mg/ml, 50 mg/ml, 60 mg/ml, 70 mg/ml, 80 mg/ml, 90 mg/ml or 100 mg/ml.

[0099] In certain embodiments, a protein comprising an FBS portion and a PmXn portion is present in a composition, for example, a pharmaceutical composition, primarily as a monomer, for example, at least 80%, 85%, 90% , 95%, 98%, or 99% of the protein in the composition is in monomeric form. The monomeric degree of a protein solution can be determined using Size Exclusion Chromatography (SEC), for example, using a Superdex column (GE Healthcare) on an Agilent 1100 or 1200 HPLC system, with UV detection at nm. A214 and A280 nm and with fluorescence detection (excitation = 280 nm, emission = 350 nm). A buffer of 100 mM sodium sulfate, 100 mM sodium phosphate, 150 mM sodium chloride, (e.g., pH 6.8) at a suitable SEC column flow rate may be employed. Gel filtration standards (Bio-Rad Laboratories, Hercules, CA) are used for molecular weight calibration.

[00100] In certain embodiments, a protein comprising an FBS portion and a PmXn portion has a biological activity that is at least as strong as that of the unmodified FBS portion. Biological activity can be binding affinity to a target or a biological activity in an assay, for example, the ability to destroy tumor cells. In certain embodiments, the biological activity of the protein is within 5%, 10%, 25%, 50%, 100%, 2 times or more of the activity of the unmodified portion of FBS.

[00101] A protein comprising an FBS moiety and a PmXn may also comprise a combination of the aforementioned characteristics. For example, a protein may be soluble in concentrations up to 50 mg/ml, be present in at least 90% monomeric form, and/or have a biological activity that is at least as potent as that of unmodified FBS. .

[00102] When referring to an improved property, reinforcement is a statistically significant improvement.

Nucleic Acid-Protein Technology

[00103] One way to quickly make and test FBS domains with specific binding properties is the protein-Adnexus nucleic acid technology, a Bristol-Myers Squibb Company. Such in vitro expression and tagging technology, termed profusion, which exploits nucleic acid protein fusions (RNA-DNA protein fusions) can be used to identify novel polypeptides and amino acid motifs that are important for binding to proteins. Protein-nucleic acid technology is a technology that covalently couples a protein that encodes its genetic information. For a detailed description of the RNA-protein technology and screening methods of fibronectin-based protein structure libraries, see Szostak et al, US Patent No. 6,258,558; 6,261,804; 6,214,553; 6,281,344; 6,207,446; 6,518,018; PCT Publication No. WO 00/34784.; WO 01/64942; WO 02/032925; and Roberts et al., Proc Natl. Academic. Sci., 94: 12297-12302 (1997), incorporated into the present invention by reference.

Modalities of Vectors and Polynucleotides

[00104] Nucleic acids encoding any of the various proteins comprising an FBS portion and a PmXn portion described in the present invention can be synthesized chemically, enzymatically or recombinantly. Codon usage can be selected to improve expression in a cell. Such codon usage will depend on the cell type selected. Specialized codon usage patterns, which were developed for E. coli and other bacteria, as well as mammalian cells, plant cells, yeast cells, and insect cells. See, for example: Mayfield et al., Proc. Natl. Academic. Sci. USA, 100 (2): 438 to 442 (21 January 2003); Sinclair et al., Protein Expr. Purif, 26 (1): 96 to 105 (Oct. 2002); Connell, N.D., Curr. Opinion. Biotechnol., 12 (5): 446 to 449 (Oct. 2001); Makrides et al., Microbiol. Rev., 60 (3): 512 to 538 (September 1996); and Sharp et al, Yeast, 7 (7): 657 to 678 (Oct. 1991).

[00105] General techniques for manipulating nucleic acids are described, for example, in Sambrook et al, Molecular Cloning:. A Laboratory Manual, Second Edition, Vols. 1-3, Cold Spring Harbor Laboratory Press (1989), or Ausubel, F. et al., Current Protocols in Molecular Biology, Greeb Publishing and Wiley-Interscience, New York (1987) and periodic updates, incorporated into the present invention by of reference. The DNA encoding the polypeptide is operably linked to suitable transcription or translational regulatory elements derived from mammalian, viral or insect genes. Such regulatory elements include a transcription promoter, an optional operator sequence to control transcription, a sequence encoding appropriate mRNA ribosome binding sites, and sequences that control transcription and translation termination. The ability to replicate in a host, normally conferred by an origin of replication, and a selection gene to facilitate recognition of transformants are additionally incorporated.

[00106] The proteins described in the present invention can be produced in recombinant form not only directly, but also as a polypeptide with a heterologous polypeptide, which is preferably a signal sequence or another polypeptide having a specific cleavage site at the N-terminus of the protein mature or polypeptide. The preferred heterologous signal sequence selected is one that is recognized and processed (i.e., cleaved by a signal peptidase) by the host cell. For prokaryotic host cells that do not recognize and process the native signal sequence, the signal sequence is replaced with a prokaryotic signal sequence selected from, for example, the group of alkaline phosphatase, penicillinase, lpp, or leader and stable commands. heat enterotoxin II. For secretion in yeast, the native signal sequence may be replaced by, for example, the yeast invertase command, a factor command (including Saccharomyces and Kluyveromyces alpha factor leaders), or acid phosphatase command, the command of C. albicans glucoamylase, or the signal described in PCT publication No. WO 90/13646. In expression in mammalian cells, mammalian signal sequences as well as viral secretion leaders, for example, the herpes simplex gD signal, are available. The DNA for this precursor region can be linked in reading frame to the DNA encoding the protein.

[00107] Both expression and cloning vectors contain a nucleic acid sequence that allows the vector to replicate in one or more selected host cells. Generally, in cloning vectors this sequence is one that allows the vector to replicate independently of the host's chromosomal DNA, and includes origins of replication or autonomous replication sequences. Such sequences are well known for a variety of bacteria, yeasts and viruses. The pBR322 plasmid origin of replication is suitable for most Gram-negative bacteria, the 2μ plasmid origin is suitable for yeast, and various viral origins (SV40, polyoma, adenovirus, VSV, or BPV) are useful for cloning vectors in mammalian cells. Generally, the origin of replication component is not required for mammalian expression vectors (the SV40 origin can typically be used just because it contains the early promoter).

[00108] Expression and cloning vectors may contain a selection gene, also called a selectable tag. Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate, or tetracycline, (b) complement auxotrophic deficiencies, or (c) provide critical nutrients not available from of complex means, for example, the gene encoding D-alanine racemase for Bacilli.

[00109] A suitable selection gene for use in yeast is the trp1 gene present on the yeast plasmid YRp7 (Stinchcomb et al, Nature, 282: 39 (1979)). The trp1 gene provides a selection tag for a mutant strain of yeast lacking the ability to grow on tryptophan, e.g., ATCC No. 44076 or PEP4-1. Jones, Genetics, 85:12 (1977). The presence of the trp1 lesion in the yeast host cell genome then provides an effective environment to detect transformation through growth in the absence of tryptophan. Similarly, Leu2-deficient yeast strains (ATCC® 20622 or 38626) are complemented by known plasmids carrying the Leu2 gene.

[00110] Expression and cloning vectors usually contain a promoter that is recognized by the host organism and is operably linked to the nucleic acid that encodes the protein. Suitable promoters for use with prokaryotic hosts include the phoA, beta-lactamase and lactose promoter systems, alkaline phosphatase promoters, a tryptophan (trp) promoter system, and hybrid promoters such as the tac promoter. However, other known bacterial promoters are suitable. Promoters for use in bacterial systems will also contain a Shine-Dalgarno (S.D.) sequence operatively linked to the DNA encoding the protein.

[00111] Promoter sequences for eukaryotes are known. Virtually all eukaryotic genes have an AT-rich region located approximately 25 to 30 bases upstream of the site where transcription is initiated. Another sequence found 70 to 80 bases upstream of the start of transcription of many genes is a CNCAAT region (SEQ ID NO: 109) in which N can be any nucleotide. At the 3' end of most eukaryotic genes is an AATAAA (SEQ ID NO: 110) sequence that may be the signal for adding the poly A tail to the 3' end of the coding sequence. All of these sequences are suitably inserted into eukaryotic expression vectors.

[00112] Examples of promoter sequences suitable for use with yeast hosts include promoters for 3-phosphoglycerate kinase or other glycolytic enzymes, such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose isomerase -6- phosphate, 3- phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase and glucokinase.

[00113] Other yeast promoters, which are inducible promoters having the additional advantage of transcription controlled by growth conditions, are the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, metallothionein , glyceraldehyde-3-phosphate, and enzymes responsible for the use of maltose and galactose. Suitable vectors and promoters for use in yeast expression are further described in EP Patent Publication No. 73657 and PCT Publication No. s. WO 2011/124718 and WO 2012/059486. Yeast stimulators are also advantageously used with yeast promoters.

[00114] Transcription from vectors in mammalian host cells can be controlled, for example, by means of promoters obtained from the genomes of viruses such as polyoma virus, avian poxvirus, adenovirus (such as Adenovirus 2), bovine papilloma, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and most preferably Simian virus 40 (SV40), heterologous mammalian promoters, e.g. the ACTIN® promoter or an immunoglobulin promoter, shock promoters thermal, provided that such promoters are compatible with the host cell systems.

[00115] The early and late promoters of the SV40 virus are conveniently obtained as an SV40 restriction fragment that also contains the SV40 viral origin of replication. The human cytomegalovirus immediate early promoter is conveniently obtained as a HindIII E restriction fragment. A system for expressing DNA in mammalian hosts using bovine papillomavirus as a vector is described in US Patent No. 4,419,446. A modification of this system is described in U.S. Patent No. 4,601,978. See also Reyes et al, Nature, 297: 598 to 601 (1982) on the expression of β-human interferon cDNA in mouse cells under the control of a herpes simplex virus thymidine kinase promoter. Alternatively, long terminal repeat Rous sarcoma virus can be used as the promoter.

[00116] Transcription of a DNA encoding a protein by higher eukaryotes is often increased by inserting an enhancer sequence into the vector. Many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, α-fetoprotein, and insulin). Typically, however, an enhancer from a virus of eukaryotic cells is used. Examples include the SV40 enhancer on the late side of the replication origin (bp 100270), the cytomegalovirus early promoter, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers. See also Yaniv, Nature, 297: 17-18 (1982) on enhancer elements for activation of eukaryotic promoters. The enhancer may be spliced into the vector at a position 5' or 3' of the polypeptide coding sequence, but is preferably located at a location at the 5' end of the promoter.

[00117] Expression vectors used in eukaryotic host cells (e.g., yeast, fungi, insects, plants, animals, human, or nucleated cells of other multicellular organisms) will also contain the sequences necessary for transcription termination and stabilization of the mRNA. Such sequences are commonly available from 5' and, occasionally 3', untranslated regions of eukaryotic or viral DNA or cDNA. These regions contain segments of nucleotides transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding the polypeptide. A useful transcription termination component is the polyadenylation region of bovine growth hormone. See, WO 94/11026 and the expression vector described therein.

[00118] Recombinant DNA can also include any type of protein tag sequence that can be useful for purifying proteins. Examples of protein tags include, but are not limited to, a histidine tag, a FLAG® tag, a myc tag, an HA tag, or a GST tag. Suitable cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cellular hosts can be found in Cloning Vectors: A Laboratory Manual, Elsevier, New York (1985), the relevant description of which is incorporated into the present invention. through reference.

[00119] The expression construct can be introduced into the host cell using a method appropriate to the host cell, as will be evident to a person skilled in the art. A variety of methods for introducing nucleic acids into host cells are known in the art, including, but not limited to, electroporation; transfection using calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran, or other substances; microprojectile bombardment; lipofection; and infection (when the vector is an infectious agent).

[00120] Suitable host cells include prokaryotes, yeast or mammalian cells, bacterial cells. Suitable bacteria include Gram-negative or Gram-positive organisms, for example E. coli or Bacillus spp. Yeast, preferably Saccharomyces species, such as S. cerevisiae, can also be used for the production of polypeptides. Various mammalian or insect cell culture systems can also be employed to express the recombinant proteins. Baculovirus systems for producing heterologous proteins in insect cells are reviewed by Luckow et al. (Bio/Technology 6:47 (1988)). Examples of suitable mammalian host cell lines include endothelial cells, monkey kidney COS-7 cells, CV-1, Chinese hamster ovary (CHO) L, C127, 3T3 cells, human embryonic kidney cells, HeLa , 293, 293T and the BHK cell lines. Purified proteins are prepared by culturing suitable vector/host systems to express the recombinant proteins. The FBS protein is then purified from culture media or cell extracts.

Protein Production

[00121] Host cells are transformed with the expression described in the present invention or the cloning vectors for the production of proteins and cultivated in conventional nutrient media modified as appropriate to induce promoters, select transformants or amplify the genes encoding the desired sequences.

[00122] The host cells used to produce the proteins can be cultured in a variety of media. Commercially available media such as Ham's F10 (Sigma), Minimum Essential Medium ((MEM), (Sigma)), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), (Sigma)) are suitable. for culturing host cells. Furthermore, any of the media described in Ham et al., Meth. Enzymol., 58:44 (1979), Barnes et al., Anal. Biochem., 102:255 (1980), US Patent Nos. 4,767,704; 4,657,866; 4927762; 4560655; or 5,122,469; PCT Publication No. WO 90/03430.; WO 87/00195; or U.S. Patent RE30,985 can be used as culture media for host cells. Any of these media can be supplemented as needed with hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers ( such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as the drug Gentamycin), trace elements (defined as inorganic compounds usually present in final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements may also be included in appropriate concentrations which will be known to those skilled in the art. Culture conditions, such as temperature, pH and the like, are those previously used with the host cell selected for expression, and will be apparent to the person skilled in the art.

[00123] The proteins described in the present invention can also be produced using cell-free translation systems. For such purposes, the nucleic acids encoding the protein must be modified to permit in vitro transcription to produce mRNA and to permit cell-free translation of the mRNA in the particular cell-free system being used (eukaryotic, such as a mammal or yeast cell-free or prokaryotic translation system, such as a bacterial cell-free translation system).

[00124] Proteins can also be produced through chemical synthesis (for example, by the methods described in Solid Phase Peptide Synthesis, Second Edition, Pierce Chemical Co., Rockford, IL (1984)). Modifications to the protein can also be produced through chemical synthesis.

[00125] The proteins described in the present invention can be purified using isolation/purification methods for proteins generally known in the area of protein chemistry. Non-limiting examples include extraction, recrystallization, salt exchange (e.g., with ammonium sulfate or sodium sulfate), centrifugation, dialysis, ultrafiltration, adsorption chromatography, ion exchange chromatography, hydrophobic chromatography, normal phase chromatography, reversed phase chromatography, gel filtration, gel permeation chromatography, affinity chromatography, electrophoresis, countercurrent distribution or any combinations thereof. After purification, proteins can be exchanged into different buffers and/or concentrated by any of a variety of methods known in the art, including, but not limited to, filtration and dialysis.

[00126] The purified protein is preferably at least 85% pure, more preferably at least 95% pure, and most preferably at least 98% or 99% pure. Regardless of the exact numerical purity value, the protein is pure enough for use as a pharmaceutical product.

Exemplary uses

[00127] The modified FBS proteins can be used for any purpose for which FBS proteins that are not modified by the addition of a PmXn moiety can be used.

[00128] In one aspect, the application provides proteins comprising a portion of modified FBS that are useful in treating disorders. The diseases or disorders that can be treated will be dictated by the binding specificity of the FBS moiety. As described in the present invention, modified FBS moieties can be designed to bind to any target of interest. Exemplary targets include, for example, TNF-alpha, VEGFR2, PCSK9, IL-23, EGFR and IGF1R. As just one example, modified FBS portions that bind to TNF-alpha can be used to treat autoimmune diseases such as rheumatoid arthritis, inflammatory bowel disease, psoriasis and asthma. The modified FBS proteins described in the present invention can also be used to treat cancer.

[00129] In certain embodiments, a method for treating an individual who has a disease, for example, cancer, comprises administering to the individual a modified FBS-drug conjugate.

[00130] Methods for administering proteins to an individual are provided in the present invention. In some embodiments, the individual is a human being. In some embodiments, the proteins are pharmaceutically acceptable to a mammal, in particular a human. A "pharmaceutically acceptable" composition refers to a composition that is administered to an animal without significant adverse medical consequences. Examples of pharmaceutically acceptable compositions include compositions that comprise portions of FBS that lack the integrin-binding domain (RGD) and compositions that are essentially free of pyrogens or endotoxin or have very low levels of endotoxins or pyrogens.

[00131] Other uses of the modified FBS proteins described in the present invention include their use in in vitro or in vivo detection assays. For example, they can be used for the detection of a target molecule in a sample. A method may comprise contacting the sample with a modified FBS described in the present invention, wherein said contact is carried out under conditions that allow the formation of the FBS-target complex; and detecting said complex, thereby detecting said target in said sample. Detection can be performed using any technique recognized in the art, such as, for example, radiography, immunoassay, fluorescence detection, mass spectroscopy, or surface plasmon resonance. The sample can be from a human or another mammal. For diagnostic purposes, appropriate agents are detectable markers that include radioisotopes, for whole-body imaging, and radioisotopes, enzymes, fluorescent markers, and other brands of antibodies suitable for sampling testing.

[00132] In certain embodiments, the modified FBS described in the present invention is useful in a variety of diagnostic and imaging applications. In certain embodiments, a modified FBS is labeled with a moiety that is detectable in vivo and such labeled FBS can be used as in vivo imaging agents, e.g., for whole-body imaging. For example, in one embodiment, a method for detecting a tumor comprising a given antigen in an individual comprising administering to the individual a modified FBS linked to a detectable tag, and after a suitable time, detecting the tag in the individual.

[00133] An FBS imaging agent can be used to diagnose a disorder or disease associated with increased levels of a given antigen, for example, a cancer in which a tumor selectively overexpresses the antigen. In a similar way, a modified FBS that specifically binds to a particular antigen can be used to monitor antigen levels in an individual being treated for a condition associated with the antigen. Modified FBS can be used with or without modification, and can be labeled by covalently or non-covalently attaching a detectable moiety.

Formulation and Administration

[00134] The Patent application additionally provides pharmaceutically acceptable compositions comprising the proteins described in the present invention, wherein the composition is essentially endotoxin and/or pyrogen-free.

[00135] Therapeutic formulations comprising proteins are prepared for storage by mixing the described proteins having the desired degree of purity with optional physiologically acceptable excipients or stabilizers, (Osol, A., Ed., Remington's Pharmaceutical Sciences, 16th Edition (1980)), in the form of aqueous, lyophilized solutions or other dry formulations. Acceptable carriers, excipients or stabilizers are non-toxic to receptors at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecydimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkylparabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m -cresol); low molecular weight (less than about 10 residues); protein polypeptides, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose or dextrans; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; counterion-forming salt such as sodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionic surfactants such as Tween, PLURONIC® or polyethylene glycol (PEG).

[00136] The formulations of the present invention may also contain more than one active compound, as necessary for the particular indication to be treated, preferably those with complementary activities that do not adversely affect each other. Such molecules are suitably present in combination in amounts that are effective for the intended purpose.

[00137] Proteins can also be trapped in a microcapsule prepared, for example, through coacervation techniques or through interfacial polymerization, for example, hydroxymethyl cellulose or gelatin microcapsule and poly (methyl methacrylate) microcapsule, respectively, in microcapsule systems. colloidal drug delivery (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or in macroemulsions. Such techniques are described in Osol, A., Ed., Remington's Pharmaceutical Sciences, 16th Edition (1980).

[00138] The formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes.

[00139] Sustained release preparations can be prepared. Suitable examples of sustained release preparations include semipermeable matrices of solid hydrophobic polymers containing the proteins described in the present invention, which are in the form of shaped articles, for example, films, or microcapsule. Examples of sustained release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl methacrylate), or poly(vinyl alcohol)), polylactides (US Patent No. 3,773,919), L-glutamic acid copolymers and y -ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON Depot® (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate) and polyacid D-(-)-3 hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid allow the release of molecules over more than 100 days, certain hydrogels release proteins over shorter periods of time. When encapsulated proteins remain in the body for a long time, they can denature or aggregate as a result of exposure to humidity at 37°C, resulting in a loss of biological activity and possible changes in immunogenicity. Rational strategies can be devised for stabilization depending on the mechanism involved. For example, if the aggregation mechanism is to be formation of intermolecular S--S bonds through thio-disulfide exchange, stabilization can be achieved by modifying the sulfhydryl residues, lyophilizing from acidic solutions, controlling the moisture content , use of appropriate additives and development of specific polymer matrix compositions.

[00140] Although the person skilled in the art will understand that the dosage of each protein will be dependent on the identity of the protein, preferred dosages may range from about 10 mg/square meter to about 2000 mg/square meter, more than preferably from about 50 mg/square meter to about 1000 mg/square meter.

[00141] For therapeutic applications, proteins are administered to an individual in a pharmaceutically acceptable dosage form. They can be administered intravenously as a bolus or over a continuous period of time, via intramuscular, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, topical, or inhalation routes. The protein can also be administered by intratumoral, peritumoral, intralesional or perilesional routes, to exert local as well as systemic therapeutic effects. Suitable pharmaceutically acceptable carriers, diluents and excipients are well known and can be determined by those skilled in the art and the clinical situation. Examples of suitable carriers, diluents and/or excipients include: (1) Dulbecco's phosphate saline buffer, pH about 7.4, containing about 1 mg/ml to 25 mg/ml human serum albumin, ( 2) 0.9% saline (0.9% w/v NaCl), and (3) 5% (w/v) dextrose. The methods of the present invention can be practiced in vitro, in vivo or ex vivo.

[00142] Administration of proteins, and one or more additional therapeutic agents, whether co-administered or administered sequentially, can occur as described above for therapeutic applications. Suitable pharmaceutically acceptable carriers, diluents and excipients for co-administration will be understood by those skilled in the art and will depend on the identity of the particular therapeutic agent to be co-administered.

[00143] When present in an aqueous dosage form, rather than being lyophilized, the protein will typically be formulated at a concentration of about 0.1 mg/ml to 100 mg/ml, although wide variation outside these ranges will be permitted. . For the treatment of disease, the appropriate dosage of proteins will depend on the type of disease being treated, the severity and course of the disease, whether the proteins are administered for preventive or therapeutic purposes, the course of prior therapy, the patient's medical history and response to fusion and, the discretion of the attending physician. The protein is appropriately administered to the patient all at once or over a series of treatments.

Exemplary modalities

[00144] 1. An isolated fibronectin-based scaffold protein (FBS) that specifically binds to a target, wherein the FBS is a non-naturally occurring FBS, and wherein the FBS is linked at its C-terminal end to a portion consisting of the amino acid sequence PmXn, where P is proline, X is any amino acid, the symbol m represents an integer that is at least 1 and n is 0 or an integer that is at least 1, and in that the PmXn moiety provides an improved property for the FBS parent protein to the FBS protein that is not bound to the PmXn moiety.

[00145] 2. The FBS protein isolated according to the embodiment, wherein the portion consists of P (m is 1 and n is 0).

[00146] 3. The isolated FBS protein according to the embodiment, wherein the portion consists of PP (the symbol m represents 2 and the symbol n represents 0).

[00147] 4. The FBS protein isolated according to the embodiment, wherein the portion consists of PmXn, where n is 1-150.

[00148] 5. The FBS protein isolated according to the embodiment, wherein the portion consists of PmXn, where n is 1-10.

[00149] 6. The FBS protein isolated according to the embodiment, wherein the portion consists of PmXn, where n is 1-5.

[00150] 7. The protein isolated from FBS according to the embodiment, wherein the portion consists of PI, PC, PID, PIE, PIDK (SEQ ID NO: 61), PIEK (SEQ ID NO: 63), PIDKP ( SEQ ID NO: 65), PIEKP (SEQ ID NO: 67), PIDKPS (SEQ ID NO: 69), PIEKPS (SEQ ID NO: 71), PIDKPC (SEQ ID NO: 73), PIEKPC (SEQ ID NO: 75 ), PIDKPSQ (SEQ ID NO: 77), PIEKPSQ (SEQ ID NO: 79), PIDKPCQ (SEQ ID NO: 81), PIEKPCQ (SEQ ID NO: 83), PHHHHHH (SEQ ID NO: 87) or PCHHHHHH (SEQ ID NO: 86).

[00151] 8. The FBS protein isolated according to any one of embodiments 1-7, wherein the protein is an FBS Fn3 protein.

[00152] 9. The FBS protein isolated according to embodiment 8, wherein the Fn3 protein is a 10Fn3 protein.

[00153] 10. The FBS protein isolated according to embodiment 9, wherein the 10Fn3 protein is a human 10Fn3 protein.

[00154] 11. The FBS protein isolated according to any one of embodiments 1-10, comprising an amino acid sequence that is at least 50% identical to SEQ ID NO: 1.

[00155] 12. The FBS protein isolated according to any one of embodiments 1-11, which comprises mutating at least one amino acid in at least one loop or framework region.

[00156] 13. The FBS protein isolated according to any one of embodiments 1-12, which comprises the amino acid sequence VSDVPRDLEVVAA (X) uLLISW (X) vYRITY (X) WFTV (X) xATISGL (X) yYTITVYA (X ) zISINYRT (SEQ ID NO: 9), where (X) L, (X) v, (X) w, (X) X, (X), Y and (X) Z consists of the wild-type amino acid sequence ( SEQ ID NO: 1) or comprises at least one amino acid difference with the corresponding wild-type sequence, and the sequence optionally comprises from 1-10 backbone, N-terminal and/or C-terminal mutations.

[00157] 14. The FBS protein isolated according to any one of embodiments 1-13, comprising an amino acid sequence that is at least 50% identical to SEQ ID NO: 1, and in which the C-terminal amino acid residue of the FBS protein is fused to a portion consisting of PmXn, wherein the FBS protein specifically binds to a target with a Kd of less than 500 nm, which target is not bound by a wild-type 10Fn3 molecule comprising SEQ ID NO: 1.

[00158] 15. The FBS protein isolated according to embodiment 14, comprising an amino acid sequence that is at least 70% identical to SEQ ID NO: 1.

[00159] 16. The FBS protein isolated according to any one of embodiments 1-15, wherein the portion consisting of PmXn is linked to the C-terminal amino acid residue of the FBS protein through a peptide bond.

[00160] 17. The FBS protein isolated according to any one of embodiments 1-16, wherein PmXn is P or PC.

[00161] 18. The FBS protein isolated according to any one of embodiments 1-17, comprising an amino acid sequence that is at least 60% identical to SEQ ID NO: 1, and in which the C-terminal amino acid residue of the FBS protein is linked to a proline via a peptide bond, and wherein the FBS protein specifically binds to a target with a Kd of less than 500 nm, which target is not bound by a 10Fn3-type molecule wild type comprising SEQ ID NO: 1.

[00162] 19. The FBS protein isolated according to any one of embodiments 1-18, wherein at least one X of the PmXn is a cysteine and wherein the cysteine.

[00163] 20. The FBS protein isolated according to modality 19, in which cysteine is conjugated with a heterologous moiety.

[00164] 21. The FBS protein isolated according to embodiment 20, in which the heterologous molecule is a detectable portion.

[00165] 22. The FBS protein isolated according to embodiment 21, or 21, wherein the heterologous molecule is a drug molecule and the drug and FBS portion form an FBS-drug conjugate.

[00166] 23. The FBS protein isolated according to any one of embodiments 1-22, wherein the improved property conferred by the PmXn moiety is improved stability.

[00167] 24. The FBS protein isolated according to embodiment 23, wherein the improved stability is an increase in Tm of at least 1 °C, 2 °C, 3 °C, 4 °C, 5 °C or more.

[00168] The following representative examples contain important additional information, exemplification and guidance, which can be adapted for the practice of the present invention in its various embodiments and their equivalents. These examples are intended to help illustrate the present invention and are not intended to, nor should they be construed as, limiting its scope.

EXAMPLES Example 1: Improved thermostability through addition of a proline at the C-terminal end of 10Fn3 Molecules

[00169] In nature, 10Fn3 is a part of a long sequence of type III fibronectin domains; the domain immediately downstream of 10Fn3 is called 11Fn3. The following sequence shows the wild-type sequences of 10Fn3 (in italics) and 11Fn3 (in bold) and junction domains between them (underlined).

[00170] Q ID NO: 90)

[00171] Based on NMR and 10Fn3 crystal structures and sequence alignment between 10Fn3 and 11Fn3 (Dickinson et al, "The type III crystal structure of the tenth cell adhesion module of human fibronectin", J. Mol Biol , 36: 1079 to 1092 (1994)), the first two residues of this sequence, RT, are part of the final beta strand of 10Fn3 ("L strand"), and the remainder of the sequence, EIDKPSQ, is a flexible linker between the tenth and eleventh structured type III fibronectin domains.

[00172] The C terminus of 10Fn3-based protein engineering domains is often modified from the wild-type sequence to facilitate their cloning, expression and purification. During a study of different engineered C termini of adnectins it was found that a mutation from RTE to RTP led to increased thermal stability of several different adnectins. Table 4 lists different C terminals used in this study. Table 4: Wild-type linker between 10Fn3 and 11Fn3 and the modified Adnectin C-terminal sequences that were compared in this study. A "short engineered C-terminus with P and His tag" and "short engineered C-terminus with PC" contain the RTE to RTP mutation.

[00173] The effect of mutating RTE to RTP on the thermostability of Adnectins was tested by comparing several pairs of Adnectins that differ in their C termini. Table 5 describes the pairs of Adnectins used in this study, including their names, targets and thermodynamic properties. Adnectin 1 is an Adnectin that binds to target local. Total protein sequences are shown below. Table 5: Clone name, C-terminus, target, and melting temperature (as determined using differential scanning calorimetry at 0.5 mg/ml in PBS, pH 7.4). Adnectins PRD-1414 and PRD-1417 were conjugated to 40 kDa, 2-branched PEG (NOF, Cat. # GL2- 400MA01)

[00174] The amino acid sequences of the proteins used in this example:

[00175] ADX_2382_D09:

[00176] MGVSDVPRDLEVVAATPTSLLISWDAPAEGYGYYRITYGE TGGNSPVQEFTVPVSKGTATISGLKPGVDYTITVYAVEFDFPGAGYY HRPISINYRTEIDKPSQHHHHHH* (SEQ ID NO: 96)

[00177] ADX_5484_A03:

[00178] MGVSDVPRDLEVVAATPTSLLISWDAPAEGYGYYRITYGE TGGNSPVQEFTVPVSKGTATISGLKPGVDYTITVYAVEFDFPGAGYY HRPISINYRTPHHHHHH* (SEQ ID NO: 97)

[00179] ADX_2987_H07:

[00180] MGVSDVPRDLEVVAATPTSLLISWTLPHAGRAHYYRITYG ETGGNSPVQEFTVPGRGVTATISGLKPGVDYTITVYAVTVTTTKVIHY KPISINYRTEIDKPSQHHHHHH* (SEQ ID NO: 97)

[00181] ADX_5484_A04:

[00182] MGVSDVPRDLEVVAATPTSLLISWTLPHAGRAHYYRITYG ETGGNSPVQEFTVPGRGVTATISGLKPGVDYTITVYAVTVTTTKVIHY KPISINYRTPHHHHHH* (SEQ ID NO: 98)

[00183] PRD-1414:

[00184] MGVSDVPRDLEVVAATPTSLLISWDAPAEGYGYYRITYGE TGGNSPVQEFTVPVSKGTATISGLKPGVDYTITVYAVEFDFPGAGYY HRPISINYRTEC * (SEQ ID NO: 99)

[00185] PRD-1417:

[00186] MGVSDVPRDLEVVAATPTSLLISWDAPAEGYGYYRITYGE TGGNSPVQEFTVPVSKGTATISGLKPGVDYTITVYAVEFDFPGAGYY HRPISINYRTPC * (SEQ ID NO: 100)

[00187] The "*" in each sequence above indicates the stop codon, and the C-terminus of each Adnectin.

[00188] The N-terminus of M was removed during bacterial expression of each protein. C residues in purified PRD-1417 and PRD-1414 protein were conjugated to PEG prior to characterization of each protein.

[00189] As shown in Table 5, Adnectins with C-terminals that contain a proline instead of the wild-type glutamic acid at the C-terminal position (RTE to RTP mutation) show higher thermal stability. This increased stability was observed for several examples in which an additional modeled C-terminus on the natural linker between human 10Fn3 and 11Fn3 was replaced with a shorter, engineered C-terminus containing only the proline and a hexahistidine purification tag. Improved thermostability was also observed in a 10Fn3 protein having a short engineered C-terminal end and a polyethylene glycol (PEG) with the addition of a C-terminal proline. The increased stability can be attributed to the presence of a proline at this specific position.

Example 2: Stabilization of a Second PCSK9 Adnectin with a C-terminal Proline

[00190] This Example shows that the thermostability of another PCSK9 Adnectin molecule is also improved through the addition of a C-terminal proline.

[00191] In this example, the C-terminus of pegylated Adnectin PCSK9 (branched PEG 40 kDa) ADX_2013_E01 was modified from NYRTEIEKPCQ (SEQ ID NO: 101) to NYRTPC (SEQ ID NO: 102) and thermostability measured by TSF. The amino acid sequence of this Adnectin is provided in WO 2011/130354. The results indicate that Adnectin pegylated with the C-terminal NYRTEIEKPCQ (SEQ ID NO: 101) has a Tm of 70°C, whereas Adnectin pegylated with the C-terminal NYRTPC (SEQ ID NO: 102) has a Tm of 76°C.

[00192] In this way, the thermostability of the present Adnectin PCSK9 is reinforced at 6°C by the presence of a C-terminal proline.

Example 3: Comparison of Multiple C Terminals on Thermostability

[00193] This example shows a comparison between the thermal stability of Adnectins with or without a proline at their C-terminal end, as well as Adnectins having 2 prolines at their C-terminal end.

[00194] Two of the Adnectins described in Example 1 (ADX_2392_D09 binds to PCSK9 and ADX_2987_H07 binds to myostatin) and an additional target binding other than Adnectin (Adnectin 1, binding to target X sequences) were modified at their C-terminal end , as indicated in Table 6 and its thermal stability and % monomer were determined. An Adnectin ("Adnectin 1") to a different target (X) was also modified with the same C-terminal sequences. Thermostability was determined by TSF and the % monomer was determined by SEC. Table 6: Percentage of monomer and thermostability of Adnectins with various C termini

[00195] The results, which are presented in Table 6, show that the identity of the C terminus does not have any significant effect on the % monomer, however, it does have an effect on the melting temperature (Tm). As described in Example 1, NYRTPH6 (SEQ ID NO: 105) increases thermal stability relative to NYRTH6 (SEQ ID NO: 104), both for Adnectins: by 4 °C for Adnectin PCSK9 and by 6 °C for adnectin myostatin. Furthermore, the presence of a second proline provides a stabilizing effect that is similar to that provided by a single proline.

[00196] In this way, the presence of one or two prolines at the C-terminus increases the thermostability of Adnectins relative to the same molecule without the C-terminal proline(s).

Example 4: Addition of a proline improves the solubility of the C-terminus

[00197] This Example demonstrates that the presence of a C-terminal proline improves the solubility of a set of Adnectin compared to the same tandem Adnectin without the C-terminal proline.

[00198] A tandem Adnectin, that is, two Adnectins each binds to a different target, linked by a linker, and containing either a NYRTE (SEQ ID NO: 107), C-terminal or a NYRTP (SEQ ID NO: 108) C terminus were expressed in E. coli BLR cells and fermented at 30 °C. The titer of the pool without the proline was 1.2 g of soluble protein/L and 2.8 g/L of total protein , indicating that the expressed protein was 43% soluble. The pool titer with proline was 2.56 g/L soluble and 2.61 g/L total. Even after renaturating the insoluble portion of the pool without the proline, and obtaining a solution that was 98% pure and monomeric at 2.5 mg/mL, this protein composition did not show significant binding to its target, which suggests that may not be refolded correctly.

[00199] Thus, considering that the only difference between the two proteins was the presence of a P at the C-terminal end, the results suggest that the presence of a proline provides enhanced solubility of the set.

[00200] The complete description of all cited documents (including patents, patent applications, scientific articles, abstracts, laboratory manuals, books, GenBank® accession numbers, SWISS-PROT® accession numbers, or other descriptions) second Plan, detailed description, a brief description of the drawings, and examples are incorporated into the present invention by reference in their entirety.

[00201] The present invention is not to be limited in its scope by means of the embodiments described in the present invention, which are intended as illustrations of individual aspects of the present invention, and any that are functionally equivalent are within the scope of the present invention. Various modifications to the models and methods of the present invention, in addition to those described in the present invention, will be apparent to persons skilled in the art from the foregoing description and teachings, and are similarly intended to fall within the scope of the present invention. invention. Such modifications or other embodiments may be practiced without departing from the true scope and spirit of the present invention.

Claims (18)

1. Isolated protein, characterized by the fact that it comprises a tenth type III fibronectin domain (10Fn3), in which (i) The amino acid of the 10Fn3 domain that is located in the position corresponding to position 94 of the wild-type 10Fn3 domain presented in SEQ ID NO: 1 is directly linked to a moiety consisting of the amino acid sequence PmXn, where P is proline, X is any amino acid, m is an integer that is at least 1 and n is 0 or an integer that is at least 1, and (ii) wherein the PmXn moiety provides an improved property for the protein relative to the protein that is not bound to the PmXn moiety. 2. Isolated protein according to claim 1, characterized by the fact that the portion linked to the 10Fn3 domain consists of P (m is 1 and n is 0). 3. Isolated protein according to claim 1, characterized by the fact that the portion linked to the 10Fn3 domain consists of PP (m is 2 and n is 0). 4. Isolated protein according to claim 1, characterized by the fact that the portion linked to the 10Fn3 domain consists of PmXn, where n is 1-150. 5. Isolated protein according to claim 1, characterized by the fact that the portion linked to the 10Fn3 domain consists of PmXn, where n is 1-10. 6. Isolated protein according to claim 1, characterized by the fact that the portion linked to the 10Fn3 domain consists of PmXn, where n is 1-5. 7. Isolated protein according to claim 1, characterized by the fact that the portion linked to the 10Fn3 domain consists of PI, PC, PID, PIE, PIDK (SEQ ID NO: 61), PIEK (SEQ ID NO: 63), PIDKP (SEQ ID NO: 65), PIEKP (SEQ ID NO: 67), PIDKPS (SEQ ID NO: 69), PIEKPS (SEQ ID NO: 71), PIDKPC (SEQ ID NO: 73), PIEKPC (SEQ ID NO : 75), PIDKPSQ (SEQ ID NO: 77), PIEKPSQ (SEQ ID NO: 79), PIDKPCQ (SEQ ID NO: 81), PIEKPCQ (SEQ ID NO: 83), PHHHHHH (SEQ ID NO: 87) or PCHHHHHH (SEQ ID NO: 86). 8. Isolated protein according to any one of claims 1 to 7, characterized in that it comprises the amino acid sequence LEVVAA(X)uLLISW(X)vYRITY(X)w FTV(X)xATISGL(X)yYTITVY A(X )ZISINYRT (SEQ ID NO: 16), wherein (X)u, (X)v, (X)w, (X)x, (X)y and (X)z consist of the wild-type amino acid sequence (SEQ ID NO:1) or comprises at least one difference in amino acids compared to the corresponding loop of the wild-type sequence. 9. Isolated protein according to claim 1, characterized by the fact that the 10Fn3 domain is defined by the amino acid sequence defined in SEQ ID NO: 100. 10. Isolated protein according to claim 9, characterized by removing the N-terminal methionine residue (M) from the 10Fn3 domain. 11. Isolated protein according to any one of claims 1 and 4 to 10, characterized in that the PmXn portion is defined by at least one X being a cysteine. 12. Isolated protein according to any one of claims 1 to 11, characterized by the fact that the PmXn portion is defined as consisting of PmCXn or PmXn1CXn2, wherein C is cysteine and n1 and n2 are independently 0 or an integer that is at least minus 1. 13. Isolated protein according to claim 11 or 12, characterized by the conjugation of cysteine in the PmXn portion to a heterologous molecule. 14. Isolated protein according to claim 13, characterized in that the heterologous molecule is defined by a PKE moiety selected from human serum albumin, a protein that binds human serum albumin, an Fc and polyethylene glycol (PEG) . 15. Isolated protein according to claim 13, characterized in that the heterologous molecule is defined by a detectable portion. 16. Isolated protein according to claim 15 or 15, characterized in that the heterologous molecule is defined by a drug moiety and the drug moiety and the protein form a protein-drug conjugate. 17. Isolated protein according to any one of claims 1 to 16, characterized by the fact that the improved property conferred by the PmXn moiety is defined by improved stability. 18. Isolated protein according to claim 17, characterized in that the improved stability is an increase in Tm of at least 1°C, 2°C, 3°C, 4°C, 5°C or more.