Fibronectin type iii domain binding serum albumin and uses thereof

Abstract

Fibronectin type III domain (FN3) molecules that bind serum albumin, related polynucleotides that encode FN3 domains that bind serum albumin, cells that express the FN3 domains, and related vectors, detectably labeled FN3 domains, and FN3 domains fused to heterologous moieties, are useful for extending the half-life of molecules in diagnostic and therapeutic applications.

Description

Technical Field

[0001] This embodiment relates to a fibronectin type III (FN3) domain that binds to serum albumin. Such an FN3 domain can be used, for example, to prolong the in vivo serum half-life of a drug or a protein conjugated thereto. Methods for manufacturing such molecules and pharmaceutical compositions comprising them are also provided. Background Technology

[0002] Rapid removal of drugs or biotherapeutic molecules from the bloodstream can limit their clinical effectiveness or lead to more frequent dosing to patients. A common clearance method is renal clearance via glomerular filtration. This pathway is most relevant to smaller biotherapeutic agents, as the rate of renal filtration is significantly reduced for molecules with molecular weights greater than 50,000 Daltons (Kontermann, Curr Opin Biotechnol 2011). Several approved biotherapeutic agents contain active moieties that are themselves below the filtration limit and are therefore rapidly cleared. To overcome this limitation, numerous techniques have been introduced to efficiently increase the size of therapeutic molecules to reduce renal filtration. There remains a need for compounds or methods that increase the half-life of drugs or therapeutic agents. This embodiment addresses these and other needs. Summary of the Invention

[0003] This embodiment provides a fibronectin type III (FN3) domain that binds to serum albumin. Related polynucleotides encoding the provided FN3 domain, cells expressing the provided FN3 domain, and related vectors are also described. Furthermore, methods for using the provided FN3 domain are described. For example, given the prolonged serum half-life of albumin, albumin-binding peptides can be used as fusion partners to produce therapeutic proteins or drugs with a longer or extended serum half-life.

[0004] In addition to the albumin-binding FN3 domain, a polynucleotide sequence encoding the protein is provided. A vector containing the polynucleotide, such as those expressing the albumin-binding FN3 domain described herein, is also provided. Cells capable of expressing the disclosed vector are also described. These cells may be mammalian cells (such as 293F cells, CHO cells), insect cells (such as Sf7 cells), yeast cells, plant cells, or bacterial cells (such as Escherichia coli). A process for producing the FN3 domain (protein) is also provided.

[0005] This embodiment also provides a method for fusing or otherwise linking the provided albumin-binding FN3 domain to various molecules to prolong the half-life of these molecules. Therefore, the albumin-binding FN3 domain can be used, for example, to prolong the half-life of therapeutic drugs.

[0006] In some embodiments, administration of a pharmaceutical composition comprising an FN3 domain that binds albumin is used to improve the in vivo half-life of a treatment partner. This half-life extension can be determined by a variety of methods known in the art, including, for example, by monitoring the pharmacokinetics of the FN3 domain that binds albumin. Non-limiting examples of such determinations of these methods are provided in the embodiments provided herein.

[0007] A kit comprising the provided albumin-binding FN3 domain is also provided. This kit can be used to perform methods using the albumin-binding FN3 domain provided herein, or other methods known to those skilled in the art. In some embodiments, the kit may include the FN3 domain described herein and reagents for detecting the presence of human serum albumin in biological samples. The kit may include one or more FN3 domains described herein and a container for containing the FN3 domain when not in use, instructions for use of the FN3 domain fixed to a solid support, and / or a detectable labeling form of the FN3 domain, as described herein. Attached Figure Description

[0008] Figure 1 The indirect pull-down of endogenous albumin from cynomolgus monkey and human serum using the albumin-binding FN3 domain is shown. The FN3 domain binding to albumin domain 1 is enclosed in dashed lines; the FN3 domain binding to albumin domain 3 is enclosed in solid lines. All constructs were prepared as bispecific gene fusions with one null FN3 domain (TC25) and one albumin-binding FN3 domain. *ABD refers to the albumin-binding domain (see J.T. Andersen, R. Pehrson, V. Tolmachev, MBDaba, L. Abrahmsen, C. Ekblad, J. Biol. Chem. 286:5234-5241 2011).

[0009] Figure 2 The pharmacokinetic properties of the FN3 domain of albumin-binding macaques were shown after a single IV dose of 5 mg / kg (H9) or 5 mg / kg (ALB40 (B7)). Detailed Implementation

[0010] definition

[0011] Various terms related to the described aspects are used throughout the specification and claims. Unless otherwise stated, these terms will be given their ordinary meaning in the art. Other specifically defined terms will be interpreted in a manner consistent with the definitions provided herein.

[0012] As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural indicators unless the content expressly indicates otherwise. Thus, for example, reference to “a cell” includes a combination of two or more cells, etc.

[0013] As used herein, the term "about," when referring to measurable values ​​such as amount, duration, etc., is intended to cover variations from the specified value up to ±10%, as such variations are suitable for performing the disclosed methods. Unless otherwise stated, all figures indicating amounts of components, properties such as molecular weight, reaction conditions, etc., used in the specification and claims should be understood to be modified by the term "about" in all cases. Therefore, unless indicated to the contrary, the numerical parameters set forth in the following specification and appended claims are approximate values ​​that may vary according to the desired properties sought to be obtained according to the invention. At least, and not in an attempt to limit the application of the doctrine of equivalence to the claims, each numerical parameter should be interpreted at least according to the number of significant figures reported and by applying ordinary rounding techniques.

[0014] Although the numerical ranges and parameters described in this invention are approximate, the values ​​described in specific examples are reported as precisely as possible. However, any numerical value inherently contains some errors, which necessarily arise from the standard deviations found in their respective test measurements.

[0015] "Isolated" means that a biological component (such as a nucleic acid, peptide, or protein) has been substantially isolated, additionally produced, or purified from other biological components of the organism in which the component is naturally present, namely other chromosomes and extrachromosomal DNA and RNA, and proteins. Therefore, "isolated" nucleic acids, peptides, and proteins include nucleic acids and proteins purified using standard purification methods. "Isolated" nucleic acids, peptides, and proteins can be part of a composition, and are still isolated if such a composition is not part of the native environment of the nucleic acid, peptide, or protein. The term also covers nucleic acids, peptides, and proteins prepared through recombinant expression in host cells, as well as chemically synthesized nucleic acids. As used herein, "isolated" FN3 domains are intended to refer to FN3 domains that substantially do not contain other FN3 domains with different antigen specificities (e.g., isolated FN3 domains that specifically bind human serum albumin substantially do not contain FN3 domains that specifically bind antigens other than human serum albumin). However, isolated FN3 domains that specifically bind to epitopes, allotypes, or variants of human serum albumin may be cross-reactive with other related antigens, such as antigens from other species (e.g., serum albumin species homologs).

[0016] As used herein, the term "fibronectin type III (FN3) domain" (FN3 domain) refers to a domain that frequently occurs in proteins including fibronectin, tendinin, intracellular cytoskeletal proteins, cytokine receptors, and prokaryotic enzymes (Bork and Doolittle, Proc Nat Acad Sci USA 89:8990-8994, 1992; Meinke et al., J Bacteriol 175:1910-1918, 1993; Watanabe et al., J Biol Chem 265:15659-15665, 1990). Exemplary FN3 domains include 15 different FN3 domains present in human tendinin C, 15 different FN3 domains present in human fibronectin (FN), and non-naturally synthesized FN3 domains, as described, for example, in U.S. Patent No. 8,278,419. Individual FN3 domains are represented by domain number and protein name, for example, the third FN3 domain (TN3) of tendinin, or the tenth FN3 domain (FN10) of fibronectin.

[0017] As used herein, the term "specific binding" or "specific binding" refers to the FN3 domain of the present invention binding at approximately 1 x 10-1 1 ... -6 M or smaller, for example, about 1x10 -7 M or smaller, approximately 1x10 -8 M or smaller, approximately 1x10 -9 M or smaller, approximately 1x10 -10 M or smaller, approximately 1x10 -11 M or smaller, approximately 1x10 -12 M or smaller, or about 1x10 -13 M or a smaller dissociation constant (K) D The ability to bind to a predetermined antigen. Typically, the described FN3 domain binds to a predetermined antigen (i.e., human serum albumin), and its K... D Compared to its K-type antigens for non-specific antigens (such as casein) D The difference is at least 10-fold lower, as measured using, for example, a Proteon Instrument (BioRad) via surface plasmon resonance. However, the described FN3 domain, which specifically binds to human serum albumin, may be cross-reactive with other relevant antigens, such as the same pre-determined antigens (homologs) from other species, such as the cynomolgus macaque (Macaca fascicularis) or the chimpanzee (Pan troglodytes).

[0018] As used herein, “serum half-life” can generally be defined as the time it takes for the serum concentration of an amino acid sequence, compound, or polypeptide to decrease by 50% in vivo, for example, due to degradation of the sequence or compound by natural mechanisms and / or clearance or isolation of the sequence or compound. The in vivo half-life of the amino acid sequence, compound, or polypeptide of the present invention can be determined in any manner known per se, such as by pharmacokinetic analysis. Suitable techniques will be apparent to those skilled in the art and may, for example, typically involve administering a suitable dose of the amino acid sequence, compound, or polypeptide of this disclosure to a warm-blooded animal (i.e., a human or another suitable mammal, such as a mouse, rabbit, rat, pig, dog, or primate, such as monkeys from the genus Macaca (e.g., and particularly cynomolgus monkeys (Macaca fascicularis) and / or rhesus monkeys (Macaca mulatta)) and baboons (Papio ursinus)); collecting a blood sample or other sample from said animal; determining the level or concentration of the amino acid sequence, compound, or polypeptide of the present invention in said blood sample; and calculating from the data thus obtained (figures) the time until the level or concentration of the amino acid sequence, compound, or polypeptide of the present invention is reduced by 50% compared to the initial level after administration. Reference, for example, is made to the experimental section below, and to standard manuals such as Kenneth, A et al: Chemical Stability of Pharmaceuticals: A Handbook for Pharmacists and Peters et al, Pharmacokinete analysis: A Practical Approach (1996). It also references Marcel Dekker's "Pharmacokinetics", M Gibaldi & D Perron, 2nd Rev.edition (1982).

[0019] Those skilled in the art will also understand (see, for example, pages 6 and 7 of WO 04 / 003019 and other references cited therein) that half-life can be expressed using parameters such as t1 / 2-α, t1 / 2-β, and area under the curve (AUC). In this specification, "half-life" means any one of these parameters, such as any two of these parameters, or substantially all three of these parameters.

[0020] The term "pharmacokinetics" is used according to its field-recognized meaning and refers to the study of how drugs work in the body, such as the effects and duration of drug action, the rates at which they are absorbed, distributed, metabolized, and eliminated from the body.

[0021] As used herein, the terms “substituting” or “substituted” or “mutating” or “mutated” refer to one or more amino acids or nucleotides that are altered, deleted, or inserted in a polypeptide or polynucleotide sequence to produce a variant of that sequence.

[0022] As used herein, the terms “randomizing” or “randomized” or “diversified” or “diversifying” refer to at least one substitution, insertion, or deletion in a polynucleotide or polypeptide sequence.

[0023] As used herein, “variant” refers to a polypeptide or polynucleotide that differs from a reference polypeptide or reference polynucleotide through one or more modifications, such as substitution, insertion, or deletion.

[0024] The term "library" refers to a collection of variants. This library can consist of peptide or polynucleotide variants.

[0025] As used herein, “Tencon” refers to the synthetic fibronectin type III (FN3) domain having the sequence shown in SEQ ID NO:1 and described in U.S. Publication No. US2010 / 0216708.

[0026] "Polynucleotide," synonymous with "nucleic acid molecule," "nucleotide," or "nucleic acid," refers to any polynucleotide or polydeoxynucleotide, which can be unmodified RNA or DNA or modified RNA or DNA. "Polynucleotide" includes, but is not limited to, single-stranded and double-stranded DNA, DNA of mixtures of single-stranded and double-stranded regions, single-stranded and double-stranded RNA, and RNA of mixtures of single-stranded and double-stranded regions, and hybrid molecules comprising DNA and RNA, which can be single-stranded or more typically double-stranded, or a mixture of single-stranded and double-stranded regions. Additionally, "polynucleotide" refers to a triple-stranded region containing RNA or DNA, or both RNA and DNA. The term polynucleotide also includes DNA or RNA containing one or more modified bases, and DNA or RNA with a backbone modified for stability or other reasons. "Modified" bases include, for example, triphenylmethylated bases and less common bases such as inosine. DNA and RNA can be modified in many ways; therefore, "polynucleotide" includes polynucleotides in chemical, enzymatic, or metabolically modified forms commonly found in nature, as well as chemical forms of DNA and RNA characteristic of viruses and cells. "Polynucleotides" also include relatively short nucleic acid chains, often called oligonucleotides.

[0027] A vector is a replicon, such as a plasmid, bacteriophage, granule, or virus, in which another nucleic acid fragment can be operatively inserted to cause the fragment to be replicated or expressed.

[0028] As used herein, the term "host cell" can be any type of cell, such as primary cells, cultured cells, or cells derived from a cell line. In specific embodiments, the term "host cell" refers to a cell transfected with a nucleic acid molecule and its offspring or potential offspring. Such offspring may differ from the parent cell transfected with the nucleic acid molecule, for example, due to mutations or environmental influences that may occur in subsequent generations or the integration of the nucleic acid molecule into the host cell genome. The terms "expression" and "production" are used synonymously herein and refer to the biosynthesis of a gene product. These terms include the transcription of a gene into RNA. These terms also cover the translation of RNA into one or more polypeptides and further cover all naturally occurring post-transcriptional and post-translational modifications. The expression or production of an antibody or its antigen-binding fragment can occur in the cytoplasm of a cell or in an extracellular environment such as a growth medium for cell culture. The meaning of "substantially the same" may vary depending on the context in which the term is used. Because natural sequence variations can exist between the heavy and light chains and the genes encoding them, it is expected that some degree of variation will be found in the amino acid sequences described herein or in genes encoding antibody or antigen-binding fragments, with little or no impact on their unique binding properties, such as specificity and affinity. This expectation is partly due to the degeneracy of the genetic code and the evolutionary success of conserved amino acid sequence variations that do not significantly alter the properties of the encoded proteins.

[0029] Overview of the publicly available FN3 domain

[0030] Tencon (SEQ ID NO:1) is a non-naturally occurring fibronectin type III (FN3) domain designed from a concordant sequence of the 15th FN3 domain from human tendinin-C (Jacobs et al., Protein Engineering, Design, and Selection, 25:107-117, 2012; US Publication No. 2010 / 0216708). The crystal structure of Tencon shows six surface-exposed rings that connect to seven β chains characterizing the FN3 domain, designated A, B, C, D, E, F, and G, and the rings designated AB, BC, CD, DE, EF, and FG rings (Bork and Doolittle, Proc Natl Acad Sci USA 89:8990-8992, 1992; US Patent No. 6,673,901). These loops, or selected residues within each loop, can be randomized to construct a library of fibronectin type III (FN3) domains, which can be used to select novel molecules that bind serum albumin. Table 1 shows the location and sequence (SEQ ID NO: 1) of each loop and β chain in Tencon.

[0031] Table 1.

[0032]

[0033]

[0034] Therefore, libraries designed based on Tencon sequences may have randomized FG loops, or randomized BC and FG loops, such as libraries TCL1 or TCL2 as described below. The Tencon BC loop is 7 amino acids long, thus allowing for diversification at the BC loop and randomization of 1, 2, 3, 4, 5, 6, or 7 amino acids in Tencon sequence-designed libraries. The Tencon FG loop is also 7 amino acids long, allowing for diversification at the FG loop and randomization of 1, 2, 3, 4, 5, 6, or 7 amino acids in Tencon sequence-designed libraries. Further diversity at the loops in Tencon libraries can be achieved through the insertion and / or deletion of residues at the loops. For example, FG and / or BC loops can be lengthened by 1–22 amino acids or shortened by 1–3 amino acids. The FG loop in Tencon is 7 amino acids long, while the corresponding loop range in the antibody heavy chain is 4–28 residues. To provide maximum diversity, the FG loop can be diversified in both sequence and length to correspond to the 4–28 residue length range of antibody CDR3. For example, the length of the FG ring can be further diversified by extending the ring by an additional 1, 2, 3, 4, or 5 amino acids.

[0035] Tencon sequence-based libraries can also have random substitution surfaces formed on one side of the FN3 domain and comprising two or more β-chains and at least one loop. One such substitution surface is formed by amino acids in the C and Fβ-chains and the CD and FG loops (C-CD-F-FG surface). A library design based on a Tencon-substituted C-CD-F-FG surface is described in U.S. Patent No. US2013 / 0226834. Tencon sequence-based libraries also include libraries designed based on Tencon variants, such as Tencon variants with substitutions at residue positions 11, 14, 17, 37, 46, 73, or 86 (residue numbers corresponding to SEQ ID NO: 1), and variants exhibiting improved thermostability. Exemplary Tencon variants are described in U.S. Publication No. 2011 / 0274623 and include Tencon27 (SEQ ID NO:4) having substitutions of E11R, L17A, N46V, and E86I compared to Tencon of SEQ ID NO:1.

[0036] Libraries based on Tencon and other FN3 sequences can be randomized at selected residue positions using random or restricted amino acid sets. For example, variants with random substitutions in the library can be generated using the NNK codon, which encodes all 20 naturally occurring amino acids. In other diverse schemes, the DVK codon can be used to encode the amino acids Ala, Trp, Tyr, Lys, Thr, Asn, Lys, Ser, Arg, Asp, Glu, Gly, and Cys. Optionally, the NNS codon can be used to generate all 20 amino acid residues while simultaneously reducing the frequency of stop codons. For example, variants can be generated using... This technology (http: / / www.sloning.com) synthesizes libraries with FN3 domains exhibiting a biased amino acid distribution at desired locations. The technology utilizes pre-fabricated double-stranded triplet libraries, which serve as universal building blocks sufficient for the synthesis of thousands of genes. The triplet libraries represent all possible sequence combinations necessary to construct any desired DNA molecule. Codon names are based on well-known IUB codes.

[0037] The FN3 domains that bind or specifically bind to human serum albumin as described herein can be isolated by generating an FN3 library such as the Tencon library, which uses cis-display to link a DNA fragment encoding a scaffold protein to a DNA fragment encoding RepA to generate a pool of protein-DNA complexes formed post-translational in vitro, wherein each protein is stably bound to the DNA encoding it (US Patent No. 7,842,476; Odegrip et al., Proc Natl Acad Sci US A101,2806-2810,2004), and the specific binding of the library to human serum albumin can be determined by any method known in the art and described in the examples. Exemplary and well-known methods that can be used include ELISA, sandwich immunoassays, and competitive and non-competitive assays (see, for example, Ausubel et al., eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York). The identified FN3 domains that specifically bind to human serum albumin can be further characterized according to desired features.

[0038] The FN3 domains that specifically bind human serum albumin described herein can be generated using any FN3 domain as a template to create a library and screen for molecules that specifically bind human serum albumin in the library using the methods provided herein. Exemplary FN3 domains that can be used are the third FN3 domain of tendinin C (TN3) (SEQ ID NO:81), the third FN3 domain of Fibcon (SEQ ID NO:82), and the tenth FN3 domain of fibronectin (FN10) (SEQ ID NO:83). Standard cloning and expression techniques are used to clone the library into a vector or to synthesize a double-stranded cDNA cassette for in vitro expression or translation of the library. For example, ribosome display (Hanes and Pluckthun, Proc Natl Acad Sci USA, 94,4937-4942, 1997), mRNA display (Roberts and Szostak, Proc Natl Acad Sci USA, 94,12297-12302, 1997), or other cell-free systems can be used (US Patent No. 5,643,768). Libraries of FN3 domain variants can be expressed as fusion proteins displayed on, for example, any suitable phage surface. Methods for displaying fusion peptides on phage surfaces are well known (US Publication No. 2011 / 0118144; International Publication No. WO2009 / 085462; US Patent No. 6,969,108; US Patent No. 6,172,197; US Patent No. 5,223,409; US Patent No. 6,582,915; US Patent No. 6,472,147).

[0039] In some embodiments described herein, the FN3 domain that specifically binds to human serum albumin is based on the Tencon sequence of SEQ ID NO:1 or the Tencon27 sequence of SEQ ID NO:4, wherein SEQ ID NO:1 or SEQ ID NO:4 optionally has substitutions at residue positions 11, 14, 17, 37, 46, 73 and / or 86.

[0040] The FN3 domain of the present disclosure, which specifically binds to human serum albumin, can be modified to improve their properties, such as improving thermal stability and the reversibility of thermal folding and unfolding. Several methods have been applied to increase the apparent thermal stability of proteins and enzymes, including rational design based on comparisons with highly similar thermally stable sequences, design for stable disulfide bonds, mutations that increase α-helix tendency, modification of salt bridges, alteration of protein surface charge, directed evolution, and composition of common sequences (Lehmann and Wyss, Curr Opin Biotechnol, 12, 371-375, 2001). High thermal stability can increase the yield of expressed proteins, improve solubility or activity, reduce immunogenicity, and minimize the need for a cold chain during manufacturing. Residues that can be substituted to improve the thermal stability of Tencon (SEQ ID NO:1) are residues at positions 11, 14, 17, 37, 46, 73, or 86, and are described in U.S. Publication No. 2011 / 0274623. The substitutions corresponding to these residues can be incorporated into the molecules containing the FN3 domain of the present invention.

[0041] Measurements of protein stability and protein instability can be viewed as similar or dissimilar aspects of protein integrity. Proteins are sensitive to, or “unstable” by, denaturation caused by heat, ultraviolet radiation, or ionizing radiation; changes in osmotic pressure and pH in liquid solutions; mechanical shear forces exerted by small-pore filtration; ultraviolet radiation; ionizing radiation such as gamma radiation; chemical or thermal dehydration; or any other action or force that may cause protein structural damage. Molecular stability can be determined using standard methods. For example, molecular stability can be measured by measuring thermal melting (“T”) using standard methods. m Temperature, measured in degrees Celsius (°C), is the temperature at which half of the molecules unfold. Typically, T... m The higher the temperature, the more stable the molecule. Besides heat, the chemical environment can also alter a protein's ability to maintain a specific three-dimensional structure.

[0042] In one embodiment, the FN3 domain of this disclosure that specifically binds to human serum albumin can exhibit behavior similar to that via T m Compared to the same structural domain before the modification, the stability is increased by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more.

[0043] Chemical denaturation can also be measured using a variety of methods. Chemical denaturants include guanidine hydrochloride, guanidine thiocyanate, urea, acetone, organic solvents (DMF, benzene, acetonitrile), salts (ammonium sulfate, lithium bromide, lithium chloride, sodium bromide, calcium chloride, sodium chloride); reducing agents (e.g., dithiothreitol, β-mercaptoethanol, dinitrobenzene, and hydrides such as sodium borohydride), nonionic and ionic detergents, acids (e.g., hydrochloric acid (HCl), acetic acid (CH3COOH), haloacetic acids), hydrophobic molecules (e.g., phospholipids), and targeted denaturants. The quantification of the degree of denaturation can depend on the loss of functional properties, such as the ability to bind target molecules, or on physicochemical properties, such as aggregation tendency, exposure of residues previously inaccessible to the solvent, or the breaking or formation of disulfide bonds.

[0044] The FN3 domain of this disclosure can be generated as a monomer, dimer, or polymer, for example, as a way to increase the valence of target molecule binding and thus increase affinity, or to generate a bispecific or multispecific scaffold that simultaneously binds two or more different target molecules. Dimers and polymers can be generated by linking monospecific, bispecific, or multispecific protein scaffolds, for example, by using amino acid linkers, such as linkers containing polyglycine, glycine, and serine, or alanine and proline. Exemplary linkers include (GS)2 (SEQ ID NO:71), (GGGS)2 (SEQ ID NO:72), (GGGGS)5 (SEQ ID NO:73), (AP)2 (SEQ ID NO:74), (AP)5 (SEQ ID NO:75), and (AP)2 (SEQ ID NO:74). 10 (SEQ ID NO:76), (AP) 20 (SEQ ID NO:77) and A(EAAAK)5AAA (SEQ ID NO:78). Dimers and polymers can be linked to each other in the N-to-C direction. The use of naturally occurring and artificial peptide linkers to ligate peptides into novel linker-fusion peptides is well known in the literature (Hallewell et al., J Biol Chem 264, 5260-5268, 1989; Alfthan et al., Protein Eng. 8, 725-731, 1995; Robinson & Sauer, Biochemistry 35, 109-116, 1996; U.S. Patent No. 5,856,456).

[0045] Human serum albumin binder

[0046] The FN3 domain is rapidly cleared from circulation via renal filtration and degradation due to its small size of ~10 kDa. In some aspects, this disclosure provides for the specific binding of the FN3 domain of serum albumin, such as human serum albumin (HSA), to prolong the half-life of the FN3 domain or another therapeutic agent associated with or linked to the albumin-binding FN3 domain.

[0047] In some embodiments, the FN3 domain that binds to human serum albumin contains a starting methionine (Met) linked to the N-terminus of the molecule.

[0048] In some embodiments, the human serum-bound albumin FN3 domain contains a cysteine ​​residue (Cys) linked to the C-terminus or N-terminus of the FN3 domain.

[0049] The addition of N-terminal Met and / or C-terminal Cys can promote expression and / or conjugate to another molecule, such as another half-life-extending molecule, such as PEG, Fc region, another FN3 domain, etc.

[0050] In some embodiments, the FN3 domain comprises the amino acid sequence of SEQ ID No: 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, or 69. In some embodiments, the FN3 domain (protein) is isolated. In some embodiments, the FN3 protein comprises SEQ ID NO: 51 having at least one substitution compared to SEQ ID NO: 51. In some embodiments, the substitution is located at residue position 10 corresponding to SEQ ID NO: 51. In some embodiments, the substitution (mutation) is A10V. In some embodiments, the substitution is A10 to G, L, I, T, or S. In some embodiments, the substitution at position 10 is any naturally occurring amino acid.

[0051] In some embodiments, the amino acid sequence contained in the FN3 domain has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequences of SEQ ID NO: 51, 52, 53, 54, 55, 56, 57%, 98%, or 69. In some embodiments, the amino acid sequence comprising the FN3 domain has at least 85%, 86%, 87%, 88%, 89%, 90%, 90%, 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence of SEQ ID NO: 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, or 69, provided that the protein has a substitution at position 10 corresponding to SEQ ID NO: 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, or 69. In some embodiments, the substitution is A10V. In some embodiments, the substitution is A10G, A10L, A10I, A10T, or A10S. In some embodiments, the substitution at position 10 is any naturally occurring amino acid.

[0052] In some embodiments, when compared with the amino acid sequences of SEQ ID No: 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, or 69, the isolated FN3 domain contains amino acid sequences having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 substitutions. In some embodiments, the substitutions are located at position 10 corresponding to position 10 of SEQ ID No: 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, or 69.

[0053] In some embodiments, the provided FN3 domain contains a cysteine ​​residue at at least one of the residue positions corresponding to residue positions 6, 11, 22, 25, 26, 52, 53, 61, 88 or 6, 8, 10, 11, 14, 15, 16, 20, 30, 34, 38, 40, 41, 45, 47, 48, 53, 54, 59, 60, 62, 64, 70, 88, 89, 90, 91 or 93 of SEQ ID NO 1, or at the C-terminus. Although the positions are listed serially, each position may also be selected individually. In some embodiments, the cysteine ​​residue is located at the position corresponding to position 6, 53 or 88.

[0054] In some embodiments, the isolated FN3 domain as described herein may comprise sequences such as SEQ ID NO: 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, or 69, wherein the C chain, CD loop, F chain, and FG loop are respectively replaced by the C chain, CD loop, F chain, and FG loop of any of the described albumin-binding FN3 domain sequences (i.e., SEQ ID No: 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, or 69), or by sequences having at least 85%, 90%, 95%, 97%, 98%, or 99% identity with the C chain, CD loop, F chain, and FG loop sequences of the four core FN3 domain sequences.

[0055] In some embodiments, the isolated albumin-binding FN3 domain comprises sequences as shown in SEQ ID NO: 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68 or 69.

[0056] In some embodiments, the FN3 domain includes SEQ ID NO:51, which has the following substitutions: A10V, N33A, A35S, W37A, P39A, G40A, I41A, G42A, W47A, R49A, K69A, W71A, H73A, A79S, S80A, P82A, I85A, or R87A. In some embodiments, the FN3 domain includes SEQ ID NO:51 with substitutions at positions 10, 33, 25, 37, 39, 40, 41, 42, 47, 49, 69, 71, 73, 79, 80, 82, 85, or 87. In some embodiments, the FN3 domain includes SEQ ID NO:51 with substitutions at position 10. In some embodiments, the FN3 domain includes SEQ ID NO:51 at position 10 having a permutation and additional permutations A10V, A10G, A10L, A10I, A10T, or A10S.

[0057] In some embodiments, the FN3 domain contains an amino acid sequence that is 90% identical to that of SEQ ID NO:51, or has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 substituted amino acid sequences when compared with the amino acid sequence of SEQ ID NO:51.

[0058] In some embodiments, the substitution is an alanine substitution. In some embodiments, the substitution is G, L, I, T, or S. In some embodiments, the substitution or alteration is the substitution or alteration of any other naturally occurring amino acid residue. "Naturally occurring amino acid residue" refers to one of the 20 amino acid residues, such as alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine. In some embodiments, W37 or W47 is substituted with different hydrophobic residues, such as glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, or methionine. In some embodiments, W37 or W47 is substituted with phenylalanine or tyrosine.

[0059] Fusion product binding to the FN3 domain of human serum albumin

[0060] In some embodiments, this disclosure provides conjugates comprising an FN3 domain that binds to serum albumin and at least one additional portion. The additional portion can be used for any diagnostic, imaging, or therapeutic purpose. In some embodiments, the additional portion is an antisense oligonucleotide, siRNA, miRNA, antibody, another FN3 domain, etc. Other examples of FN3 domains include, but are not limited to, those provided in U.S. Patent Nos. 8,278,419, 9,200,059, 1,004,0842, 8,569,227, 9,234,029, 9,982,253, 9,200,273, 9,897,612, 10,196,446, 8,415,291, 8,617,894, 9,695,228, 9,725,497, 9,156,887, or 10,280,200, each of which is incorporated herein by reference in its entirety, including the specific FN3 domain provided therein. Or those provided in U.S. Patent Application Nos. 15 / 629090, 16 / 218990, 15 / 637276, 15 / 148312, 15 / 611296, 15 / 839915, 15 / 840281, 15 / 840303, 62 / 914643, 62 / 914654, or 62 / 914725, each of which is incorporated herein by reference in its entirety, including the specific FN3 domain provided therein.

[0061] In some embodiments, the serum half-life of the portion fused to the FN3 domain is increased relative to the serum half-life of the portion not fused to the FN3 domain. In some embodiments, the serum half-life of the FN3 domain fusion is at least 20%, 40%, 60%, 80%, 100%, 120%, 150%, 180%, 200%, 400%, 600%, 800%, 1000%, 1200%, 1500%, 1800%, 1900%, 2000%, 2500%, or 3000% longer than the serum half-life of the portion not fused to the FN3 domain. In other embodiments, the serum half-life of the FN3 domain fusion is at least 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 10, 12, 13, 15, 17, 20, 22, 25, 27, 30, 35, 40, or 50 times greater than the serum half-life of the portion not fused to the FN3 domain. In some embodiments, the serum half-life of the FN3 domain fusion in cynomolgus monkeys is at least 2 hours, 2.5 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 15 hours, 20 hours, 25 hours, 30 hours, 35 hours, or 40 hours.

[0062] Therefore, the FN3 domain fusion molecules described herein can be used to increase the half-life of a therapeutic portion by forming a fusion between the therapeutic portion and the FN3 domain. Such fusion molecules can be used to treat conditions that respond to the biological activity of the therapeutic portion contained in the fusion. This disclosure contemplates the use of the described FN3 domain fusion molecules in diseases caused by dysregulation of any of the following proteins or molecules.

[0063] Heterogeneous part

[0064] In some embodiments, the described FN3 domain is fused with a second portion, which is an organic small molecule, nucleic acid molecule, or protein. In some embodiments, the FN3 domain is fused with a therapeutic portion of a target receptor, receptor ligand, viral capsid protein, immune system protein, hormone, enzyme, antigen, or cell signaling protein. Fusion can be formed by attaching the second portion to either end of the FN3 domain, i.e., an FN3 domain-therapeutic molecule or a therapeutic molecule-FN3 domain arrangement.

[0065] In other exemplary embodiments, the FN3 domain is fused to one or more additional FN3 domains. For example, the FN3 domain may be fused to one, two, three, four, or more additional FN3 domains. The additional FN3 domains may bind to the same or different targets other than serum albumin. Examples of FN3 domains are provided herein and are incorporated herein by reference.

[0066] In some embodiments, the FN3 domain is linked to another molecule. In some embodiments, the other molecule is a drug or therapeutic agent, a protein, an antibody, a polymer, or a toxin. In some embodiments, the other molecule is an FN3 domain that binds to a molecule other than human albumin. In some embodiments, other FN3 domains bind to CD71. In some embodiments, the FN3 domain is directly linked to another molecule. In some embodiments, the FN3 domain is linked to another molecule via a linker. In some embodiments, the linker is a peptide linker. In some embodiments, the peptide linker includes the sequences (GS)2 (SEQ ID NO:71), (GGGS)2 (SEQ ID NO:72), (GGGGS)5 (SEQ ID NO:73), (AP)2 (SEQ ID NO:74), (AP)5 (SEQ ID NO:75), (AP)10 (SEQ ID NO:76), (AP)20 (SEQ ID NO:77), and A(EAAAK)5AAA (SEQ ID NO:78).

[0067] In some embodiments, this application provides an FN3-Y fusion that can be represented by the following formula: FN3-XY or Y-XFN3, wherein FN3 is an FN3 domain as described herein (including any N-terminal and / or C-terminal extensions), X is a peptide linker (suitable linkers include, for example, any of SEQ ID NOs: 71-78), and Y is a therapeutic portion as described herein.

[0068] In some embodiments, this application provides FN3-Y fusions that can be represented by the following formula: FN3-Xi-Cys-X2-Y or Y-Xi-Cys-X2-FN3, wherein FN3 is an FN3 domain as described herein (including any N-terminal and / or C-terminal extensions), Xi is an optional peptide linker (suitable linkers include, for example, any of SEQ ID NOs: 71-78), Cys is a cysteine ​​residue, X2 is a chemically derived spacer, and Y is a therapeutic moiety as described herein. In exemplary embodiments, the chemically derived spacer comprises a maleimide moiety that can be used to conjugate a therapeutic moiety to the C-terminal Cys of the FN3 domain, or to conjugate the FN3 domain to the C-terminal Cys of a therapeutic moiety, via Michael addition as further described herein. In other aspects, the described FN3 domain can be combined with two or more therapeutic moieties. For example, the two portions can be combined into the FN3 domain in various arrangements, such as, for example, the N-to-C-terminus of a fusion sequence, as follows: XY-FN3, X-FN3-Y, or FN3-XY, where X and Y represent two distinct therapeutic portions. The two distinct therapeutic portions can be selected from any of the portions disclosed herein.

[0069] In some embodiments, the bispecific FN3 molecule comprises a first FN3 domain and a second FN3 domain, wherein the first FN3 domain comprises the serum albumin-binding FN3 domain described herein, and the second FN3 domain binds to target proteins other than human serum albumin.

[0070] Deimmunization by binding peptides

[0071] The amino acid sequence of serum albumin binders and their fusions can be altered to eliminate one or more B or T cell epitopes. Proteins comprising the FN3 domain fusions described herein can be deimmunized to render them non-immunogenic or less immunogenic to a given substance. Deimmunization can be achieved by altering the structure of the protein. Any deimmunization technique known to those skilled in the art can be employed, see, for example, WO00 / 34317, the disclosure of which is incorporated herein by reference in its entirety.

[0072] In one embodiment, the presence of MHC class II binding motifs in the sequences of serum albumin binders and their fusions can be analyzed. For example, comparisons can be made with databases of MHC-binding motifs, such as by searching the “motifs” database on sitewehil.wehi.edu.au. Alternatively, computational threading methods can be used to identify MHC class II binding peptides, such as those designed by Altuvia et al. (J.Mol.Biol.249 244-250 (1995)), thereby testing the binding energies of consecutive overlapping peptides from polypeptides to MHC class II proteins. Computational binding prediction algorithms include iTopeTM, Tepitope, SYFPEITHI, EpiMatrix (EpiVax), and MHCpred. To aid in the identification of MHC class II binding peptides, sequence features associated with successfully presented peptides, such as amphiphilic and Rothbard motifs, and cleavage sites of cathepsin B and other processing enzymes, can be searched.

[0073] Once potential (e.g., human) T-cell epitopes are identified, these epitopes are then eliminated as needed by altering one or more amino acids. Typically, this involves a change in one or more amino acids within the T-cell epitope itself. This might involve altering amino acids adjacent to the epitope in terms of the protein's primary structure, or amino acids that are not adjacent in the primary structure but are adjacent in the molecule's secondary structure. The most common alteration considered would be amino acid substitution, but in some cases, amino acid addition or deletion may be appropriate. All alterations can be accomplished using recombinant DNA technology, so the final molecule can be prepared by expression from a recombinant host, for example, through maturation methods, but also using protein chemistry or any other molecular alteration approach.

[0074] Once the identified T-cell epitopes are removed, the deimmunized sequences can be analyzed again to ensure that no new T-cell epitopes are generated, and if they are, the epitopes can be deleted.

[0075] Not all computationally identified T-cell epitopes need to be removed. Those skilled in the art will understand the importance of the “strength” of a particular epitope, or more precisely, its potential immunogenicity. Various computational methods generate scores for potential epitopes. Those skilled in the art will recognize that it may only be necessary to remove epitopes with high scores. They will also recognize that there is a balance between removing potential epitopes and maintaining protein binding affinity or other biological activities. Therefore, one strategy is to sequentially introduce the substitution into the described FN3 domain or an FN3 domain fusion protein, and then test target binding or other biological activities and immunogenicity.

[0076] Other modifications

[0077] In some embodiments, serum albumin binders and their fusions may further comprise post-translational modifications. Exemplary post-translational protein modifications include phosphorylation, acetylation, methylation, ADP ribosylation, ubiquitination, glycosylation, carbonylation, sumoylation, biotinylation, or the addition of peptide side chains or hydrophobic groups. Thus, modified serum albumin binders and their fusions may contain non-amino acid elements, such as lipids, polysaccharides, or monosaccharides, as well as phosphates. A preferred form of glycosylation is sialylation, which conjugates one or more sialic acid moieties to a peptide. The sialic acid moieties improve solubility and serum half-life while reducing the protein's potential immunogenicity. See, for example, Raju et al. Biochemistry. 2001 Jul 31; 40(30):8868-76. The effects of these non-amino acid elements on the function of serum albumin binders or their fusions can be tested to understand their ability to bind specific serum albumins (e.g., HSA or RhSA) and / or the functional roles conferred by specific non-FN3 moieties in the context of the fusion (e.g., the effect of FGF21 on glucose uptake).

[0078] Vector & Polynucleotide Implementation Methods

[0079] This disclosure also includes nucleic acid sequences encoding any of the proteins described herein. As those skilled in the art will understand, due to the degeneracy of the third base, almost every amino acid can be represented by more than one triplet codon in the encoding nucleotide sequence. Furthermore, small base pair changes may result in conserved substitutions in the encoded amino acid sequence, but are not expected to significantly alter the biological activity of the gene product. Therefore, nucleic acid sequences encoding the proteins described herein may be slightly modified in sequence while still encoding their respective gene products.

[0080] Nucleic acids encoding any of the various proteins or polypeptides disclosed herein can be chemically synthesized. Codon usage can be selected to improve expression in the cell. This codon usage will depend on the cell type selected. Specific codon usage patterns have been developed for Escherichia coli and other bacteria, as well as mammalian cells, plant cells, yeast cells, and insect cells. See, for example: Mayfield et al, Proc Natl Acad Sci US A. 2003 100(2):438-42; Sinclair et al. Protein Expr Purif. 2002(1):96-105; Connell ND. Curr Opin Biotechnol. 2001(5):446-9; Makrides et al. Microbiol Rev. 1996 60(3):512-38; and Sharp et al. Yeast. 1991 7(7):657-78.

[0081] General techniques for nucleic acid manipulation are within the scope of those skilled in the art and are also described, for example, in Sambrook et al., *Molecular Cloning: A Laboratory Manual*, Vols. 1-3, Cold Spring Harbor Laboratory Press, 2nd ed. 1989, or F. Ausubel et al., *Current Protocols in Molecular Biology* (Green Publishing and Wiley-Interscience: New York, 1987), and in periodic updates, which are incorporated herein by reference. Protein-encoding DNA is operatively linked to suitable transcriptional or translational regulatory elements derived from mammalian, viral, or insect genes. Such regulatory elements include transcription promoters, optional operon sequences controlling transcription, sequences encoding suitable mRNA ribosome binding sites, and sequences controlling the termination of transcription and translation. Also incorporated is the ability to replicate in the host, typically conferred by the origin of replication, and selection genes that facilitate the recognition of transformants. Suitable regulatory elements are well known in the art.

[0082] The proteins and fusion proteins described herein can be generated as fusion proteins with heterologous peptides, which are preferably signal sequences or other peptides having a specific cleavage site at the N-terminus of a mature protein or peptide. The selected heterologous signal sequences are preferably those recognized and processed by host cells (i.e., cleaved by signal peptidases). For prokaryotic host cells that do not recognize and process the native signal sequence, the signal sequence is replaced by a prokaryotic signal sequence selected from, for example, the group consisting of alkaline phosphatase, penicillinase, LPP, or heat-stable enterotoxin II leader sequences. For yeast secretion, the native signal sequence can be replaced by, for example, yeast invertase leader sequences, factor leader sequences (including *Saccharomyces* and *Kluyveromyces* α-factor leader sequences), or acid phosphatase leader sequences, *Candida albicans* glucosylamylase leader sequences, or the signal described in PCT Publication No. WO 90 / 13646. In mammalian cell expression, mammalian signal sequences and viral secretion leader sequences, such as the herpes simplex gD signal, can be used. The DNA of this precursor region can be ligated within the reading frame to the DNA encoding the protein.

[0083] Expression vectors used in eukaryotic host cells (e.g., yeast, fungi, insects, plants, animals, humans, or nucleated cells from other multicellular organisms) will also contain sequences necessary for terminating transcription and stabilizing mRNA. Such sequences are typically obtained from the 5' end and occasionally the 3' untranslated region of eukaryotic or viral DNA or cDNA. These regions contain nucleotide fragments transcribed into polyadenylated fragments within the untranslated portion of mRNA encoding multivalent antibodies. A useful transcription termination component is the bovine growth hormone polyadenylated region. See PCT Publication No. WO 94 / 11026 and the expression vectors disclosed therein.

[0084] Recombinant DNA may also include any type of protein tag sequence that can be used to purify proteins. Examples of protein tags include, but are not limited to, histidine tags, FLAG tags, myc tags, HA tags, or GST tags. Cloning and expression vectors suitable for bacterial, fungal, yeast, and mammalian cell hosts can be found in Cloning Vectors: A Laboratory Manual (Elsevier, New York, 1985), the relevant disclosure of which is incorporated herein by reference.

[0085] It will be apparent to those skilled in the art that the expression construct is introduced into a host cell using a method suitable for the host cell. Various methods for introducing nucleic acids into host cells are known in the art, including but not limited to electroporation; transfection with calcium chloride, rubidium chloride, calcium phosphate, DEAE-glucan, or other substances; particle bombardment; lipid transfection; and infection (where the vector is the infectious agent).

[0086] Suitable host cells include prokaryotes, yeast, mammalian cells, or bacterial cells. Suitable bacteria include Gram-negative or Gram-positive organisms such as *Escherichia coli* or *Bacillus spp.* yeast, preferably from yeast species such as *Saccharomyces cerevisiae*, which can also be used to produce peptides. Various mammalian or insect cell culture systems can also be used to express recombinant proteins. Luckow and Summers (Bio / Technology, 6:47, 1988) reviewed baculovirus systems for producing heterologous proteins in insect cells. In some cases, it is necessary to produce proteins in vertebrate cells, such as for glycosylation, and the proliferation of vertebrate cells in culture (tissue culture) has become a routine procedure. Examples of suitable mammalian host cell lines include endothelial cells, COS-7 monkey kidney cells, CV-1, L cells, C127, 3T3, Chinese hamster ovary (CHO), human embryonic kidney cells, HeLa, 293, 293T, and BHK cell lines. For many applications, the smaller size of the protein polymers described herein will make E. coli a preferred expression method.

[0087] Protein production

[0088] The host cells are transformed with the expression or cloning vectors for protein production described herein and cultured in a conventional nutrient medium that is appropriately modified to induce promoters, select transformants, or amplify genes encoding desired sequences.

[0089] The host cells used to produce the proteins of this invention can be cultured in a variety of culture media. Commercially available media such as Ham's F10 (Sigma), Minimal Essential Medium ((MEM), Sigma), RPMI-1640 (Sigma), and Dulbecco Modified Eagle Medium ((DMEM), Sigma) are suitable for culturing host cells. Additionally, any culture medium described in Ham et al, Meth. Enz. 58:44 (1979), Barnes et al, Anal. Biochem. 102:255 (1980), U.S. Patent Nos. 4,767,704, 4,657,866, 4,927,762, 4,560,655, or 5,122,469; WO 90 / 03430; WO 87 / 00195; or U.S. Patent No. Re. 30,985 can be used as the culture medium for host cells. Any of these culture 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), and antibiotics (such as GENTAMYCIN). TM The formula may include drugs, trace elements (defined as inorganic compounds typically present in the micromolar range at final concentrations), and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations known to those skilled in the art. Culture conditions, such as temperature, pH, etc., are those previously used with the host cells selected for expression and will be obvious to those skilled in the art.

[0090] The proteins disclosed herein can also be generated using a cell translation system. For this purpose, the nucleic acid encoding the protein must be modified to allow in vitro transcription to produce mRNA and to allow cell-free translation of the mRNA in the specific cell-free system used. Exemplary eukaryotic cell-free translation systems include, for example, translation systems without mammalian or yeast cells, and exemplary prokaryotic cell-free translation systems include, for example, bacterial cell-free translation systems.

[0091] The proteins disclosed herein can also be produced by chemical synthesis (e.g., by the method described in Solid Phase Peptide Synthesis, 2nd ed. 1984, The Pierce Chemical Co., Rockford, IL). Modifications to the proteins can also be produced by chemical synthesis.

[0092] The proteins disclosed herein can be purified using methods commonly known in the field of protein chemistry for the separation / purification of proteins. Non-limiting examples include extraction, recrystallization, salting out (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 combination thereof. After purification, the protein can be exchanged for different buffers and / or concentrated using any of a variety of methods known in the art, including but not limited to filtration and dialysis.

[0093] The purified protein is preferably at least 85% pure, more preferably at least 95% pure, and most preferably at least 98% pure. Regardless of the exact purity value, the protein is pure enough to be used as a pharmaceutical product.

[0094] Imaging, Diagnostics and Other References

[0095] Based on the properties of the heterologous molecules fused with the FN3 domain, the FN3 domain fusions provided herein can be used to treat a variety of diseases and disorders. The application of the FN3 domain fusions can be determined by those skilled in the art based on their knowledge and the information provided herein. This document describes in detail the uses of various FN3 domain fusion proteins. FN3 domain fusions can be administered to any mammalian subject or patient, including humans and non-human objects.

[0096] The serum albumin binders and fusion molecules described herein can be detectably labeled and used to contact cells expressing proteins, for example, those bound by fusion molecules for imaging or diagnostic applications. Any method known in the art for conjugating proteins to detectable moieties can be employed, including those described by Hunter, et al., Nature 144:945 (1962); David, et al., Biochemistry 13:1014 (1974); Pain, et al., J. Immunol. Meth. 40:219 (1981); and Nygren, J. Histochem. and Cytochem. 30:407 (1982).

[0097] In some embodiments, the serum albumin binder and fusion molecule described herein are further attached to a detectable marker (e.g., the marker may be a radioactive isotope, a fluorescent compound, an enzyme, or an enzyme cofactor). The marker may be a radioactive reagent, such as: radioactive heavy metals such as iron chelates, radioactive chelates of gadolinium or manganese, positron emitters of oxygen, nitrogen, iron, carbon, or gallium. 43 K, 52 Fe、 57 Co、 67 Cu、67 Ga、 68 Ga、 123 I, 125 I, 13 T, 132 I or 99 Tc. Serum albumin binders or fusion molecules attached to this portion can be used as imaging agents and administered in amounts effective for diagnostic purposes in mammals such as humans, followed by detection of the localization and accumulation of the imaging agent. The localization and accumulation of the imaging agent can be detected by radioscintigraphy, magnetic resonance imaging, computed tomography, or positron emission tomography. As will be apparent to those skilled in the art, the amount of radioisotope to be administered depends on the radioisotope. Those skilled in the art can readily determine the amount of imaging agent to be administered based on the specific activity and energy of a given radionuclide used as the active portion.

[0098] Serum albumin binders and fusion molecules can also be used as affinity purification agents. In this process, the protein is immobilized on a suitable support, such as Sephadex resin or filter paper, using methods well-known in the art. The protein can be used with any known assay, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays (Zola, Monoclonal Antibodies: A Manual of Techniques, pp. 147-158 (CRC Press, Inc., 1987)). Exemplary therapeutic formulations and administration methods are also described.

[0099] This invention provides a method for administering a therapeutic portion fused with a described FN3 domain, wherein the half-life of the therapeutic portion is prolonged upon fusion with the described FN3 domain. The techniques and dosages used for administering the fusion construct will vary depending on the type of therapeutic portion fused with the described FN3 domain and the specific condition being treated, but can be readily determined by a person skilled in the art. Typically, regulatory agencies require that protein reagents used as therapeutic agents be formulated to have acceptablely low levels of pyrogens. Therefore, therapeutic formulations are generally distinguished from other formulations in that they are substantially pyrogen-free, or contain at least no more than an acceptable level of pyrogens as determined by an appropriate regulatory agency (e.g., the FDA). In some embodiments, pharmaceutical formulations of the described FN3 domain and its fusion molecule comprise, for example, 1-20 mM succinic acid at pH 4.0-7.0, 2-10% sorbitol, and 1-10% glycine. In an exemplary embodiment, pharmaceutical formulations of the FN3 domain and its fusion molecule comprise, for example, 10 mM succinic acid at pH 6.0, 8% sorbitol, and 5% glycine.

[0100] In some embodiments, the described FN3 domains and their fusions are pharmaceutically acceptable for mammals, particularly humans. A “pharmaceutically acceptable” polypeptide is a polypeptide administered to animals without significant adverse medical consequences. Examples of pharmaceutically acceptable FN3 domains and their fusions disclosed herein include FN3 domains lacking an integrin-binding domain (RGD) and compositions that are substantially free of endotoxins or have very low endotoxin levels.

[0101] The therapeutic composition may be administered in a unit dosage form with a pharmaceutically acceptable diluent, carrier, or excipient. As a non-limiting example, administration may be parenteral (e.g., intravenous, subcutaneous), oral, or topical. The composition may be in the form of pills, tablets, capsules, liquids, or sustained-release tablets for oral administration; liquids for intravenous, subcutaneous, or parenteral administration; or gels, lotions, ointments, creams, or polymers or other sustained-release loading agents for topical administration.

[0102] Methods well-known in the art for preparing formulations can be found, for example, in "Remington: The Science and Practice of Pharmacy" (20th ed., ed. ARGennaro AR., 2000, Lippincott Williams & Wilkins, Philadelphia, PA). Formulations for parenteral administration may, for example, contain excipients, sterile water, saline, polyalkylene glycols such as polyethylene glycol, plant-derived oils, or hydrogenated naphthalene. Biocompatible, biodegradable lactide polymers, lactide / glycolic acid copolymers, or polyoxyethylene-polyoxypropylene copolymers can be used to control the release of the compound. Nanoparticle formulations (e.g., biodegradable nanoparticles, solid lipid nanoparticles, liposomes) can be used to control the biodistribution of the compound. Other potentially useful parenteral delivery systems include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes. The concentration of the compound in the formulation varies depending on many factors, including the dosage of the drug to be administered and the route of administration.

[0103] Peptides can optionally be administered as pharmaceutically acceptable salts, such as non-toxic acid addition salts or metal complexes commonly used in the pharmaceutical industry. Examples of acid addition salts include organic acids such as acetic acid, lactic acid, dihydroxynaphthyl acid, maleic acid, citric acid, malic acid, ascorbic acid, succinic acid, benzoic acid, palmitic acid, succinic acid, salicylic acid, tartaric acid, methanesulfonic acid, toluenesulfonic acid, or trifluoroacetic acid; polymeric acids such as tannic acid and carboxymethyl cellulose; and inorganic acids such as hydrochloric acid, hydrobromic acid, and phosphoric acid sulfate. Metal complexes include zinc and iron. In one example, peptides are formulated in the presence of sodium acetate to increase thermal stability.

[0104] Preparations intended for oral use include tablets containing the active ingredient in a mixture with non-toxic, pharmaceutically acceptable excipients. These excipients may be, for example, inert diluents or fillers (e.g., sucrose and sorbitol), lubricants, flow aids, and anti-adhesion agents (e.g., magnesium stearate, zinc stearate, stearic acid, silica, hydrogenated vegetable oil, or talc).

[0105] Formulations intended for oral use may also be provided as chewable tablets, or as hard gelatin capsules in which the active ingredient is mixed with an inert solid diluent, or as soft gelatin capsules in which the active ingredient is mixed with a water or oil medium.

[0106] Therapeutic effective dose refers to the dose that produces the therapeutic effect of administration. The exact dose will depend on the disease to be treated and can be determined by those skilled in the art using known techniques. Typically, FN3 domain fusions are administered at doses of about 0.01 μg / kg to about 50 mg / kg daily, preferably 0.01 mg / kg to about 30 mg / kg daily, and most preferably 0.1 mg / kg to about 20 mg / kg daily. The peptide can be administered daily (e.g., once, twice, three or four times daily) or less frequently (e.g., every other day, once or twice a week, or monthly). Additionally, as is known in the art, adjustments may need to be made for age and weight, general health condition, sex, diet, timing of administration, drug interactions, and disease severity, and these can be determined by those skilled in the art using routine experiments.

[0107] Kit for detecting human serum albumin

[0108] This article provides kits for detecting human serum albumin in biological samples. These kits include one or more FN3 domains that bind serum albumin as described herein, along with kit usage instructions.

[0109] The provided FN3 domain for binding serum albumin can be in solution; lyophilized; immobilized on a substrate, carrier, or plate; or detectably labeled.

[0110] The described kit may also include additional components for performing the methods described herein. For example, the kit may include methods for obtaining samples from subjects, obtaining control or reference samples (e.g., samples from subjects with slowly progressive cancer and / or subjects without cancer), one or more sample compartments, and / or guidance material describing the performance and tissue-specific controls or standards of the methods of the present invention.

[0111] The method for determining serum albumin levels may further include, for example, buffer solutions or other reagents used in the assay to determine serum albumin levels. Instructions may be, for example, printed instructions for performing the assay and / or instructions for assessing serum albumin levels.

[0112] The described kit may also include methods for separating samples from a subject. These methods may include one or more devices or reagents capable of obtaining fluids or tissues from a subject. Methods for obtaining samples from a subject may also include methods for separating blood components, such as serum, from blood samples. Preferably, the kit is designed for human subjects.

[0113] The embodiments described herein relate to a method for detecting the presence of human serum albumin in a biological sample, including contacting the biological sample with the FN3 domain of serum albumin as described herein and evaluating the binding of the biological sample to the protein.

[0114] The embodiments described herein relate to a method for prolonging the half-life of a target molecule in human subjects, the method comprising conjugating an FN3 domain that binds to serum albumin as described herein to a target molecule, thereby prolonging the half-life of the target molecule in human subjects. In some embodiments, the method further includes administering the conjugated molecule to a human. In some embodiments, the target molecule is a drug, an antibody, an FN3 domain that binds to a molecule other than human albumin, or a toxin. In some embodiments, the conjugate is a peptide linker. In some embodiments, the peptide linker includes the sequences (GS)2 (SEQ ID NO:71), (GGGS)2 (SEQ ID NO:72), (GGGGS)5 (SEQ ID NO:73), (AP)2 (SEQ ID NO:74), (AP)5 (SEQ ID NO:75), (AP)10 (SEQ ID NO:76), (AP)20 (SEQ ID NO:77), and A(EAAAK)5AAA (SEQ ID NO:78).

[0115] Example

[0116] The following examples are provided to supplement the existing disclosure and to provide a better understanding of the subject matter described herein. These examples should not be considered as limiting the subject matter. It should be understood that the examples and implementations described herein are for illustrative purposes only, and various modifications or changes thereto will be apparent to those skilled in the art and will be included therein, and can be made without departing from the true scope of the invention.

[0117] Example 1: Constructing a TENCON library using random rings

[0118] Tencon (SEQ ID NO:1) is an immunoglobulin-like scaffold with a fibronectin type III (FN3) domain, designed from a shared sequence of 15 FN3 domains derived from human tendinin-C (Jacobs et al., Protein Engineering, Design, and Selection, 25:107-117, 2012; US Patent No. 8,278,419). The crystal structure of Tencon reveals six surface-exposed loops connecting seven β chains. These loops, or selected residues within each loop, can be randomized to construct libraries of fibronectin type III (FN3) domains, which can be used to selectively bind novel molecules to specific targets.

[0119] Tencon:

[0120] LPAPKNLVVSEVTEDSLRLSWTAPDAAFDSFLIQYQESEKVGEAINLTVPGSERSYDLTGLKPGTEYTVSIYGVKGGHRSNPLSAEFTT(SEQ ID NO 1):

[0121] Various libraries were generated using the Tencon scaffold and various design strategies. Typically, libraries TCL1 and TCL2 produced a good bond. The generation of TCL1 and TCL2 libraries is described in detail in International Publication No. WO 2014081944A2.

[0122] Building the TCL1 Library

[0123] A library, TCL1, designed to randomize only the FG loop (SEQ ID NO:1) of Tencon, was constructed for use in a cis-display system (Jacobs et al., Protein Engineering, Design, and Selection, 25:107-117, 2012). In this system, a double-stranded DNA containing the Tac promoter sequence, the Tencon library coding sequence, the RepA coding sequence, cis-elements, and ori-elements was generated. Expression in an in vitro transcription / translation system produced a complex of the Tencon-RepA fusion protein cis-binding to the DNA encoding it. The complex binding to the target molecule was then isolated and amplified by polymerase chain reaction (PCR), as described below.

[0124] The TCL1 library for cis-display was constructed via successive rounds of PCR to produce two halves of the final linear double-stranded DNA molecule; the 5' fragment contained the promoter and Tencon sequence, while the 3' fragment contained the repA gene and cis and ori elements. These two halves were combined by restriction digestion to produce the complete construct. The TCL1 library was designed to incorporate only the random amino acid KGGHRSN (SEQ ID NO:32) into the FG loop of the Tencon. The NNS codon was used to construct the library, potentially incorporating all 20 amino acids and a stop codon into the FG loop. The TCL1 library contains six separate sub-libraries, each with a different random FG loop length, ranging from 7 to 12 residues, to further increase diversity.

[0125] TCL1 Document Library (SEQ ID NO:2)

[0126] LPAPKNLVVSEVTEDSLRLSWTAPDAAFDSFLIQYQESEKVGEAINLTVPGSERSYDLTGLKPGTEYTVSIYGVX 7-12 PLSAEFTT;

[0127] in

[0128] X1, X2, X3, X4, X5, X6, and X7 are any amino acids; and

[0129] X8, X9, X 10 X 11 and X 12 It can be any amino acid or a deletion.

[0130] Building the TCL2 Library

[0131] A TCL2 library was constructed in which the BC and FG loops of Tencon were randomized, and the amino acid distribution at each position was strictly controlled. Table 2 shows the amino acid distribution at the desired loop positions in the TCL2 library. The designed amino acid distribution served two purposes. First, based on the analysis of the Tencon crystal structure and / or homology modeling, the library was biased towards residues predicted to be structurally important for Tencon folding and stability. For example, position 29 was fixed as a subset of only hydrophobic amino acids because this residue is embedded in the hydrophobic core of the Tencon fold. The second layer of design involved biasing the amino acid distribution towards residues preferentially found in the antibody heavy chain HCDR3 to effectively generate a high-affinity binder (Birtalan et al., J Mol Biol 377:1518-28, 2008; Olson et al., Protein Sci 16:476-84, 2007). To achieve this goal, the “Designed Distribution” in Table 2 refers to the following distribution: 6% alanine, 6% arginine, 3.9% asparagine, 7.5% aspartic acid, 2.5% glutamic acid, 1.5% glutamine, 15% glycine, 2.3% histidine, 2.5% isoleucine, 5% leucine, 1.5% lysine, 2.5% phenylalanine, 4% proline, 10% serine, 4.5% threonine, 4% tryptophan, 17.3% tyrosine, and 4% valine. This distribution does not contain methionine, cysteine, or a stop codon.

[0132] TCL2 Document Library (SEQ ID NO:3)

[0133] LPAPKNLVVSEVTEDSLRLSWX1X2X3X4X5X6X7X8SFLIQYQESEKVGEAINLTVPGSERSYDLTGLKPGTEYTVSIYGVX9X 10 X 11 X 12 X 13 SX 14 X 15 LSAEFTT; where

[0134] X1 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr, or Val;

[0135] X2 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr, or Val;

[0136] X3Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val;

[0137] X4 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr, or Val;

[0138] X5 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr, or Val;

[0139] X6 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr, or Val;

[0140] X7 is Phe, Ile, Leu, Val, or Tyr;

[0141] X8 is Asp, Glu, or Thr;

[0142] X9 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr, or Val;

[0143] X 10 It is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr, or Val;

[0144] X 11 It is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr, or Val;

[0145] X 12 It is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr, or Val;

[0146] X 13It is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr, or Val;

[0147] X 14 It is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr, or Val; and

[0148] X 15 It is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr, or Val;

[0149] Table 2.

[0150]

[0151]

[0152] * Residue numbering is based on the Tencon sequence of SEQ ID NO:1

[0153] Subsequently, these libraries were improved in various ways, including constructing libraries on a stable Tencon framework (US Patent No. 8,569,227) that incorporated the substitution E11R / L17A / N46V / E86I (Tencon27; SEQ ID NO:4) compared to wild-type Tencon, and altering the randomization positions in the BC and FG rings. Tencon27 is described in International Patent Application No. WO 2013049275. This resulted in new libraries designed to randomize only the Tencon's FG ring (library TCL9) or a combination of the BC and FG rings (library TCL7). These libraries were constructed for use in cis-display systems (Odegripet al., Proc Natl Acad Sci USA 101:2806-2810, 2004). Details of this design are shown below:

[0154] Stable Tencon (Tencon27) (SEQ ID NO:4)

[0155] LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFLIQYQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVKGGHRSNPLSAIFTT

[0156] TCL7 (randomized FG and BC rings) (SEQ ID NO:5)

[0157] LPAPKNLVVSRVTEDSARLSWX1X2X3X4X5X6X7X8X9FDSFLIQYQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVX 10 X 11 X 12 X 13 X 14 X 15 X 16 X 17 X 18 X 19 SNPLSAIFTT;

[0158] in

[0159] X1, X2, X3, X4, X5, X6, X 10 X 11 X 12 X 13 X 14 X 15和 X 16 It is A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, or Y; and

[0160] X7, X8, X9, X 17 X 18 and X 19 It is A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, Y or missing.

[0161] TCL9 (randomized FG ring) (SEQ ID NO:6)

[0162] LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFLIQYQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIYGV X1X2X3X4X5X6X7X8X9X 10 X 11 X 12 SNPLSAIFTT;

[0163] X1, X2, X3, X4, X5, X6, and X7 are A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, or Y; and

[0164] X8, X9, X 10 X 11 and X12 It is A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, Y or missing.

[0165] For library construction, DNA fragments encoding random BC loops (6-9 positions in length) or FG loops (7-12 positions in length) are synthesized using Slonomics technology (Sloning Biotechnology GmbH) to control the amino acid distribution of the library and eliminate stop codons. Two different sets of DNA molecules with randomized BC or FG loops are synthesized independently and then combined using PCR to produce a complete library product.

[0166] Construction of FG Ring Library (TCL9)

[0167] A set of synthetic DNA molecules was generated, consisting of the complete gene sequence of the 5'Tac promoter followed by the Tencon, except for the random codons in the FG loop (SEQ ID NOs:26-31). For FG loop randomization, all amino acids except cysteine ​​and methionine were encoded in equal percentages. The lengths of the varied portions allowed them to encode 7, 8, 9, 10, 11, or 12 amino acids in the FG loop. Sublibraries of each length variation were synthesized individually at a scale of 2 μg and then amplified by PCR using oligonucleotides Sloning-FOR (SEQ ID NO:9) and Sloning-Rev (SEQ ID NO:10).

[0168] The 3' fragment of the library is a constant DNA sequence containing display elements, including a PspOMI restriction site, the coding region of the repA gene, and cis and ori elements. Using plasmid (pCR4Blunt) (Invitrogen) as a template, PCR was performed using M13 forward and M13 reverse primers to amplify the fragment. The resulting PCR product was digested with PspOMI overnight and purified by gel electrophoresis. To ligate the 5' portion of the library DNA to the 3' DNA containing the repA gene, 2 pmol (~540 ng to 560 ng) of 5' DNA was ligated overnight at 37°C with an equimolar (~1.25 μg) 3' repA DNA in the presence of NotI, PspOMI, and T4 ligase. The ligated library product was amplified using oligonucleotides POP2250 (SEQ ID NO: 11) and DigLigRev (SEQ ID NO: 12) after 12 cycles of PCR. For each sub-library, the DNA from 12 PCR reactions was pooled and purified by Qiagen column centrifugation. The yield of each sub-library of TCL9 ranged from 32 to 34 μg.

[0169] Construction of FG / BC ring library (TCL7)

[0170] The TCL7 library provides a library with randomized Tencon BC and FG loops. In this library, BC loops of 6-9 amino acids in length are combined with randomized FG loops of 7-12 amino acids in length. Synthesized Tencon fragments BC6, BC7, BC8, and BC9 (SEQ ID NO: 13-16) were generated to include the Tencon gene, which encodes the N-terminal portion of a protein up to and including residue VX, thereby replacing the BC loop with 6, 7, 8, or 9 randomized amino acids. These fragments were synthesized prior to the discovery of the L17A, N46V, and E83I mutations (CEN5243), but these mutations were introduced in the molecular biology steps described below. The following steps were performed to combine this fragment with the fragment encoding the randomized FG loop.

[0171] First, using oligonucleotides POP2222ext (SEQ ID NO:18) and LS1114 (SEQ ID NO:19), DNA fragments encoding the Tac promoter and the 5' sequence of Tencon up to the nucleotides encoding amino acid A17 (130mer-L17A, SEQ ID NO:17) were generated by PCR. This was done to include the L17A mutation in the library (CEN5243). Next, using BC6, BC7, BC8, or BC9 as templates and oligonucleotides LS1115 (SEQ ID NO:20) and LS1117 (SEQ ID NO:21), a DNA fragment encoding Tencon residue R18-V75, including a randomized BC loop, was amplified by PCR. This PCR step introduced a BsaI site at the 3' end. Subsequently, using oligonucleotides POP2222ext and LS1117 as primers, these DNA fragments were ligated by overlap PCR. The resulting 240 bp PCR products were pooled and purified using a Qiagen PCR purification kit. The purified DNA was digested with BsaI-HF and then purified by gel electrophoresis.

[0172] Using FG7 (SEQ ID NO:31), FG8 (SEQ ID NO:30), FG9 (SEQ ID NO:29), FG10 (SEQ ID NO:28), FG11 (SEQ ID NO:27), and FG12 (SEQ ID NO:26) as templates, fragments encoding FG loops were amplified by PCR using oligonucleotides SDG10 (SEQ ID NO:22) and SDG24 (SEQ ID NO:23) to incorporate BsaI restriction sites and N46V and E86I variants (CEN5243).

[0173] The digested BC and FG fragments were ligated together in a single step using a 3-way ligation method. Four ligation reactions were set up from 16 possible combinations, each combining two BC loop lengths and two FG loop lengths. Each ligation contained ~300 ng of total BC fragment and 300 ng of FG fragment. These four ligation pools were then amplified by PCR using oligonucleotides POP2222 (SEQ ID NO:24) and SDG28 (SEQ ID NO:25). 7.5 μg of each reaction product was then digested with Not1 and purified using a Qiagen PCR purification column. 5.2 μg of this DNA was ligated to an equimolar amount of RepA DNA fragment (~14 μg), digested with PspOMI, and the product was amplified by PCR using oligonucleotide POP2222.

[0174] Example 2: Generation of a TENCON library with an alternative bonding surface

[0175] In a specific library design, the selection of residues to be randomized determines the overall shape of the resulting interaction surface. X-ray crystallography analysis of the FN3 domain (containing a scaffold protein that binds maltose-binding protein (MBP)) selected from a library (where the BC, DE, and FG loops were randomized) revealed a predominantly curved interface suitable for the active site of MBP (Koide et al., Proc Natl Acad Sci USA 104:6632-6637, 2007). Conversely, the ankyrin repeat scaffold protein selected for MBP binding was found to have a flatter interaction surface and binds to the outer surface of the active MBP (Binz et al., Nat Biotechnol 22:575-582, 2004). These results suggest that the shape (curved vs. flat) of the scaffold binding surface may determine which target proteins or specific epitopes on those target proteins can be effectively bound by the scaffold. Published efforts toward modified protein scaffolds incorporating FN3 domains for protein binding have relied on modifying adjacent loops for target binding, resulting in curved binding surfaces. This approach may limit the number of targets and epitopes accessible to such scaffolds.

[0176] Tencon and other FN3 domains contain two sets of CDR-like loops on opposite molecular faces: the first set consists of BC, DE, and FG loops, and the second set consists of AB, CD, and EF loops. The two sets of loops are separated by a β-chain forming the center of the FN3 structure. Rotating the image of Tencon by 90 degrees reveals an alternative surface. This slightly concave surface is formed by CD and FG loops and two antiparallel β-chains, C and Fβ chains, and is referred to herein as the C-CD-F-FG surface. The C-CD-F-FG surface can be used as a template to design libraries of protein scaffold interaction surfaces by randomizing a subset of the residues forming the surface. The β-chains have a repeating structure, with the side chains of each other residue exposed on the protein surface. Therefore, libraries can be constructed by randomizing some or all of the surface-exposed residues in the β-chains. By selecting appropriate residues in the β-chains, the intrinsic stability of the Tencon scaffold should be minimally compromised while providing a unique scaffold surface for interactions with other proteins.

[0177] The TCL14 (SEQ ID NO:7) was designed into the Tencon27 bracket (SEQ ID NO:4).

[0178] A complete description of the methods used to construct this library is provided in U.S. Publication No. US2013 / 0226834.

[0179] TCL14 Document Library (SEQ ID NO:7):

[0180] LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFX1IX2YX3EX4X5X6X7GEAIVLTVPGSERSYDLTGLKPGTEYX8VX9IX 10 GVKGGX 11 X 12 SX 13 PLSAIFTT;

[0181] in

[0182] X1, X2, X3, X4, X5, X6, X 10 X 11 X 12 and X 13 It is A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, Y, or M.

[0183] In Tencon27, the two β chains forming the C-CD-F-FG surface have a total of 9 surface-exposed residues that can be randomized: C chain: S30, L32, Q34, Q36; F chain: E66, T68, S70, Y72, and V74; CD ring has 6 potential residues: S38, E39, K40, V41, G42, and E43; and FG ring has 7 potential residues: K75, G76, G77, H78, R79, S80, and N81. If all 22 residues are randomized, the selected residues are included in the TCL14 design because the theoretical library size is larger.

[0184] Thirteen sites in the Tencon were selected for randomization: L32, Q34, and Q36 in the C chain; S38, E39, K40, and V41 in the CD ring; T68, S70, and Y72 in the F chain; and H78, R79, and N81 in the FG ring. In the C and F chains, S30 and E66 were not randomized because they are located just outside the CD and FG rings and do not appear to be part of the C-CD-F-FG surface. For the CD ring, G42 and E43 were not randomized to glycine, providing flexibility that could be valuable in the ring region, and E43 is located at the surface junction. K75, G76, G77, and S80 were excluded from the FG ring. Glycine was excluded for the reasons mentioned above, and careful examination of the crystal structure revealed that S80 makes crucial contact with the core to help form a stable FG ring. K75 is far from the surface facing the C-CD-F-FG surface and is a less attractive candidate for randomization. Although the aforementioned residues were not randomized in the initial TCL14 design, they can be included in subsequent library designs to provide additional diversity for de novo selection or, for example, selection of affinity-matured libraries of TCL14 target-specific hits.

[0185] Following the generation of TCL14, three additional Tencon libraries with similar designs were generated. These two libraries, TCL19, TCL21, and TCL23, were randomized at the same positions as TCL14 (see above), but the amino acid distribution at these positions was altered (Table 3). TCL19 and TCL21 were designed to have an equal distribution of 18 native amino acids at each position (5.55% of each position), excluding only cysteine ​​and methionine. TCL23 was designed so that each randomized position approximates the amino acid distribution found in the HCDR3 ring of functional antibodies (Birtalan et al., J Mol Biol 377:1518-1528, 2008), as described in Table 2. Similar to the TCL21 library, cysteine ​​and methionine were excluded.

[0186] A third additional library was constructed to expand the potential target-binding surfaces of the other libraries. In this library, TCL24, four additional Tencon sites were randomized compared to libraries TCL14, TCL19, TCL21, and TCL23. These sites include N46 and T48 from the D chain, and S84 and I86 from the G chain. Sites 46, 48, 84, and 86 were specifically chosen because the side chains of these residues are exposed from the D and G surfaces of the β chain and are structurally adjacent to the randomized portions of the C and F chains, thus increasing the surface area available for binding to target proteins. The amino acid distribution used at each site in TCL24 is the same as described in Table 3 for TCL19 and TCL21.

[0187] TCL24 Document Library (SEQ ID NO:8)

[0188] LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFX1IX2YX3EX4X5X6X7GEAIX8LX9VPGSERSYDLTGLKPGTEYX 10 VX 11 IX 12 GVKGGX 13 X 14 SX 15 PLX 16 AX 17 FTT;

[0189] in

[0190] X1, X2, X3, X4, X5, X6, X 10 X 11 X 12 and X 13 It is A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, or W.

[0191] Table 3. Amino acid frequencies (%) at each randomized position in TCL21, TCL23, and TCL24.

[0192]

[0193]

[0194] The creation of TCL21, TCL23 and TCL24 libraries

[0195] The TCL21 library was generated using Colibra library technology (Isogenica) to control amino acid distribution. TCL19, TCL23, and TCL24 gene fragments were generated using Slonomics technology (Morphosys) to control amino acid distribution. Each library was amplified by PCR after initial synthesis and then ligated into the RepA gene for selection using the CIS display system (Odegrip et al., Proc Natl Acad Sci USA 101:2806-2810, 2004), as described above for the cyclic libraries.

[0196] Example 3: Selecting the fibronectin type III (FN3) domain that binds to human serum albumin

[0197] Document filtering

[0198] Cis-display was used to select human serum albumin-binding domains from the TCL14, TCL19, TCL21, TCL23, and TCL24 libraries. Recombinant human and macaque serum albumin and human albumin domain II (AlbuminBiosciences) were biotinylated using standard methods and used for panning. For in vitro transcription and translation (ITT), 3 μg of library DNA was incubated with 0.1 mM of complete amino acids, 1X S30 premix, and 15 μL of S30 extract (Promega) for a total volume of 50 μL at 30 °C. One hour later, 375 μL of blocking solution (0.1% casein (Thermo Fisher, Rockford, IL), 100 mg / mL Herring Sperm DNA (Promega, Madison, WI), 1 mg / mL heparin (Sigma-Aldrich, St. Louis, MO)) was added, and the reaction was incubated on ice for 15 minutes. For selection, biotinylated antigen was added at concentrations of 400 nM (round 1), 200 nM (rounds 2 and 3), and 100 nM (rounds 4 and 5). Bound library members were recovered using neutral avidin magnetic beads (Thermo Fisher, Rockford, IL) (rounds 1, 3, and 5) or streptavidin magnetic beads (Promega, Madison, WI) (rounds 2 and 4), and the beads were washed 5–14 times with 500 μL PBST, followed by 500 μL of PBST. Two washes with PBS were performed to remove unbound library members. Additional selection rounds were conducted to identify FN3 domain molecules with enhanced affinity. In short, the output from round 5 was prepared as described above, followed by additional iterative selection rounds with the following changes: incubation time with the biotinylated antigen was reduced from 1 hour to 15 minutes, bead capture time was reduced from 20 minutes to 15 minutes, bt-HSA was reduced to 25 nM (rounds 6 and 7) or 2.5 nM (rounds 8 and 9), and an additional 1-hour wash was performed in the presence of excess non-biotinylated target protein. The goal of these changes was to simultaneously select binders with potentially faster binding rates and slower dissociation rates, resulting in significantly lower Kb. D .

[0199] After selection, the selected FN3 domain was amplified by PCR using oligonucleotides Tcon6 (SEQ ID NO:33) and Tcon5shortE86I (SEQ ID NO:34), subcloned into pET15-LIC via annealing, and transformed into BL21-GOLD(DE3) cells using standard molecular biology techniques for soluble expression in *E. coli*. Single clones were picked from 96-well deep-well plates at 37°C and cultured to saturation in 1 mL LB broth containing ampicillin. The next day, 25 μL was transferred to 1 mL fresh LB-Amp medium in 96-well deep-well plates and cultured at 37°C for 2 hours. IPTG was added to a final concentration of 1 mM, and protein expression was induced at 30°C for 16 hours. Cells were harvested by centrifugation and subsequently lysed with Bugbuster HT (EMD Chemicals, Gibbstown, NJ) supplemented with 0.2 mg / mL final egg white lysozyme (Sigma-Aldrich, St. Louis, MO). The bacterial lysate was clarified by centrifugation, and the supernatant was transferred to a new 96-well deep plate and tested for binding to the target protein by ELISA.

[0200] Selection of the FN3 domain of human serum albumin

[0201] Enzyme-linked immunosorbent assay (ELISA) was performed on single clones from selected panning outputs to identify human serum albumin binders. Maxisorp plates (Nunc, Rochester, NY) were coated overnight with 5 μg HSA, cynomolgus monkey SA, or 5 μg / ml Fc (Sigma-Aldrich, St. Louis, MO), washed with Tris-buffered saline, pH 7.4, and 0.05% Tween-20 (TBST), and blocked with Starting Block T20 (Thermo Fisher, Rockford, IL). Clarified bacterial lysates (as described above) were applied to the wells of the coated HSA, cSA, and Fc plates. The plates were incubated for 1 hour, washed with TBST, and the binding of Centyrin was detected using a Molecular Devices M5 reader with anti-V5 tag antibody and POD chemiluminescent substrate (Roche, Indianapolis, IN). A hit was defined as a binding signal of human and cynomolgus monkey serum albumin higher than Fc > 10.

[0202] As shown in Table 4 below, an FN3 domain B7 exhibited significant binding to both human and cynomolgus albumin, as well as domain II of human albumin. Further characterization of residues crucial for binding human SA was achieved by preparing variants, where each residue at the putative binding site was mutated to alanine. Table 5 shows the complete amino sequences of the FN3 domain and alanine variants binding domain II. Comparison of SEQ ID NO:51 with the sequence of H9 (SEQ ID NO:70) revealed a significantly increased half-life in cynomolgus monkeys. This was unexpected.

[0203] Table 4:

[0204]

[0205] Table 5. Amino acid sequences of the selected FN3 domains

[0206]

[0207]

[0208]

[0209] FN3 domain binding to human serum albumin was expressed and purified on a small scale.

[0210] The FN3 domain clone was selected and cultured to saturation at 37°C in 1 mL Luria Broth (LB) (LB-Amp medium) supplemented with 100 μg / mL ampicillin in a 96-well deep plate. The next day, 25 μL was transferred to 5 mL of fresh LB-Amp medium in a 24-well deep plate and cultured at 37°C for 2 hours. IPTG was added to a final concentration of 1 mM, and protein expression was induced at 30°C for 16 hours. Cells were harvested by centrifugation and lysed with Bugbuster HT (EMD Chemicals, Gibbstown, NJ) supplemented with 0.2 mg / mL final egg white lysozyme (Sigma-Aldrich, St. Louis, MO). The bacterial lysate was clarified by centrifugation, and the supernatant was transferred to a new 96-well deep plate. The His-tagged FN3 domain was purified using a 96-well Ni-NTA Multitrap Plate according to the manufacturer's recommendations (GE Lifesciences, Piscataway, NJ).

[0211] Size exclusion chromatography analysis

[0212] Size exclusion chromatography (SOC) was used to determine the aggregation state of the FN3 domains bound to human serum albumin. Aliquots (10 μL) of each purified FN3 domain were injected into a Superdex 75 5 / 150 column (GE Healthcare) at a flow rate of 0.3 mL / min in a mobile phase of PBS (pH 7.4). Elution was monitored by absorbance at 280 nm. FN3 domains exhibiting high levels of aggregation as determined by SEC were excluded from further analysis.

[0213] Example 4: Characterization of the FN3 domain binding to human serum albumin

[0214] Immunoprecipitation of human serum albumin-FN3 domain complex

[0215] To evaluate the function of the binder, the ability of selected FN3 domains to complex with endogenous albumin in normal serum was tested. The method for determining this is provided in U.S. Application No. 15 / 611296, which is incorporated herein by reference. Most sequences, even those with substitutions, were found to bind and pull down albumin. Some sites may be intolerant to alanine substitutions, but other substitutions can occur at these sites without eliminating albumin interactions. The results are shown in Table 6.

[0216] Table 6.

[0217]

[0218]

[0219] FN3 proteins were also tested in an ELISA assay to determine their ability to bind to immobilized human or cynomolgus monkey albumin. The binding of 667 nM B7 and B7 variants to human or cynomolgus monkey albumin is shown in Table 7. A negative control was wtTencon.

[0220] Table 7.

[0221]

[0222]

[0223] Pharmacokinetics of the FN3 domain binding albumin in cynomolgus monkeys:

[0224] In vivo studies were conducted using B7 and H9. Cynomolgus monkeys were administered B7 or H9 via intravenous (IV) bolus injection at a dose of 5 mg / kg. The method of administration and the method of determining protein concentration were determined according to the method described in U.S. Patent Application No. 15 / 611296, which is incorporated herein by reference. Figure 1and Figure 2 The pharmacokinetics are shown, including the increase in the half-life of the albumin FN3 domain, known as B7 or ALB40.

[0225] Sequence information

[0226] SEQ ID NO:1 = Original Tencon sequence

[0227] LPAPKNLVVSEVTEDSLRLSWTAPDAAFDSFLIQYQESEKVGEAINLTVPGSERSYDLTGLKPGTEYTVSIYGVKGGHRSNPLSAEFTT

[0228] SEQ ID NO:2 = TCL1 Library

[0229] LPAPKNLVVSEVTEDSLRLSWTAPDAAFDSFLIQYQESEKVGEAINLTVPGSERSYDLTGLKPGTEYTVSIYGV(X) 7-12 PLSAEFTT;

[0230] in

[0231] X1, X2, X3, X4, X5, X6, and X7 are any amino acids; and

[0232] X8, X9, X 10 X 11 and X 12 It can be any amino acid or a deletion.

[0233] SEQ ID NO:3 = TCL2 Library

[0234] LPAPKNLVVSEVTEDSLRLSWX1X2X3X4X5X6X7X8SFLIQYQESEKVGEAINLTVPGSERSYDLTGLKPGTEYTVSIYGVX9X 10 X 11 X 12 X 13 SX 14 X 15 LSAEFTT;

[0235] in

[0236] X1 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr, or Val;

[0237] X2 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr, or Val;

[0238] X3Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr or Val;

[0239] X4 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr, or Val;

[0240] X5 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr, or Val;

[0241] X6 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr, or Val;

[0242] X7 is Phe, Ile, Leu, Val, or Tyr;

[0243] X8 is Asp, Glu, or Thr;

[0244] X9 is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr, or Val;

[0245] X 10 It is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr, or Val;

[0246] X 11 It is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr, or Val;

[0247] X 12It is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr, or Val;

[0248] X 13 It is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr, or Val;

[0249] X 14 It is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr, or Val; and

[0250] X 15 It is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr, or Val;

[0251] SEQ ID NO:4 = Stable Tencon (Tencon 27)

[0252] LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFLIQYQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVKGGHRSNPLSAIFTT

[0253] SEQ ID NO:5 = TCL7 (FG and BC rings)

[0254] LPAPKNLVVSRVTEDSARLSWX1X2X3X4X5X6X7X8X9FDSFLIQYQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIYGVX 10 X 11 X 12 X 13 X 14 X 15 X 16 X 17 X 18 X 19 SNPLSAIFTT;

[0255] in

[0256] X1, X2, X3, X4, X5, X6, X 10 X 11X 12 X 13 X 14 X 15和 X 16 It is A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, or Y; and

[0257] X7, X8, X9, X 17 X 18 and X 19 It is A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, Y or missing.

[0258] SEQ ID NO:6 = TCL9 (FG ring)

[0259] LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFLIQYQESEKVGEAIVLTVPGSERSYDLTGLKPGTEYTVSIYGV X1X2X3X4X5X6X7X8X9X 10 X 11 X 12 SNPLSAIFTT;

[0260] in

[0261] X1, X2, X3, X4, X5, X6, and X7 are A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, or Y; and

[0262] X8, X9, X 10 X 11 and X 12 It is A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, Y or missing.

[0263] SEQ ID NO:7 = TCL14 Library

[0264] LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFX1IX2YX3EX4X5X6X7GEAIVLTVPGSERSYDLTGLKPGTEYX8VX9IX 10 GVKGGX 11 X 12 SX 13 PLSAIFTT;

[0265] in

[0266] X1, X2, X3, X4, X5, X6, X 10 X 11, X 12 and X 13 is A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, Y or M.

[0267] SEQ ID NO:8 = TCL24 library

[0268] LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFX1IX2YX3EX4X5X6X7GEAIX8LX9VPGSERSYDLTGLKPGTEYX 10 VX 11 IX 12 GVKGGX 13 X 14 SX 15 PLX 16 AX 17 FTT;

[0269] where

[0270] X1, X2, X3, X4, X5, X6, X 10 , X 11 , X 12 and X 13 is A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V or W.

[0271] SEQ ID NO:9 = Sloning-FOR

[0272] GTGACACGGCGGTTAGAAC

[0273] SEQ ID NO:10 = Sloning-REV

[0274] GCCTTTGGGAAGCTTCTAAG

[0275] SEQ ID NO:11 = POP2250

[0276] CGGCGGTTAGAACGCGGCTACAATTAATAC

[0277] SEQ ID NO:12 = DigLigRev

[0278] CATGATTACGCCAAGCTCAGAA

[0279] SEQ ID NO:13 = BC9

[0280] GTGACACGGCGGTTAGAACGCGGCTACAATTAATACATAACCCCATCCCCCTGTTGACAATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGGATCTACCATGCTGCCGGCGCCGAAAAACCTGGTTGTTTCTGAAGTTACCGAAGACTCTCTGCGTCTGTCTTGGNNNNNNNNNNNNNNNNNNNNNNNNNNNTTYGACTCTTTCCTGATCCAGTACCAGGAATCTGAAAAAGTTGGTGAAGCGATCAACCTGACCGTTCCGGGTTCTGAACGTTCTTACGACCTGACCGGTCTGAAACCGGGTACCGAATACACCGTTTCTATCTACGGTGTTCTTAGAAGCTTCCCAAAGGC

[0281] SEQ ID NO:14=BC8

[0282] GTGACACGGCGGTTAGAACGCGGCTACAATTAATACATAACCCCATCCCCCTGTTGACAATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGGATCTACCATGCTGCCGGCGCCGAAAAACCTGGTTGTTTCTGAAGTTACCGAAGACTCTCTGCGTCTGTCTTGGNNNNNNNNNNNNNNNNNNNNNNNNTTYGACTCTTTCCTGATCCAGTACCAGGAATCTGAAAAAGTTGGTGAAGCGATCAACCTGACCGTTCCGGGTTCTGAACGTTCTTACGACCTGACCGGTCTGAAACCGGGTACCGAATACACCGTTTCTATCTACGGTGTTCTTAGAAGCTTCCCAAAGGC

[0283] SEQ ID NO:15=BC7

[0284] GTGACACGGCGGTTAGAACGCGGCTACAATTAATACATAACCCCATCCCCCTGTTGACAATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGGATCTACCATGCTGCCGGCGCCGAAAAACCTGGTTGTTTCTGAAGTTACCGAAGACTCTCTGCGTCTGTCTTGGNNNNNNNNNNNNNNNNNNNNNTTYGACTCTTTCCTGATCCAGTACCAGGAATCTGAAAAAGTTGGTGAAGCGATCAACCTGACCGTTCCGGGTTCTGAACGTTCTTACGACCTGACCGGTCTGAAACCGGGTACCGAATACACCGTTTCTATCTACGGTGTTCTTAGAAGCTTCCCAAAGGC

[0285] SEQ ID NO:16=BC6

[0286] GTGACACGGCGGTTAGAACGCGGCTACAATTAATACATAACCCCATCCCCCTGTTGACAATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGGATCTACCATGCTGCCGGCGCCGAAAAACCTGGTTGTTTCTGAAGTTACCGAAGACTCTCTGCGTCTGTCTTGGNNNNNNNNNNNNNNNNNNTTYGACTCTTTCCTGATCCAGTACCAGGAATCTGAAAAAGTTGGTGAAGCGATCAACCTGACCGTTCCGGGTTCTGAACGTTCTTACGACCTGACCGGTCTGAAACCGGGTACCGAATACACCGTTTCTATCTACGGTGTTCTTAGAAGCTTCCCAAAGGC

[0287] SEQ ID NO:17=130mer-L17A

[0288] CGGCGGTTAGAACGCGGCTACAATTAATACATAACCCCATCCCCCTGTTGACAATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGGATCTACCATGCTG

[0289] SEQ ID NO:18POP222ext

[0290] CGG CGG TTA GAA CGC GGC TAC YOU ARE TAC

[0291] SEQ ID NO:19(LS1114).

[0292] CCA AGA CAG ACG GGC AGA GTC TTC GGT AAC GCG AGA AAC AAC CAG GTT TTTCGG CGC CGG CAG CAT GGT AGA TCC TGT TTC

[0293] SEQ ID NO:20(LS1115).

[0294] CCG AAG ACT CTG CCC GTC TGT CTT GG

[0295] SEQ ID NO:21(LS1117).

[0296] CAG TGG TCT CAC GGA TTC CTG GTA CTG GAT CAG GAA AGA GTC GAA

[0297] SEQ ID NO:22(SDG10).

[0298] CATGCGGTCTCTTCCGAAAAAGTTGGTGAAGCGATCGTCCTGACCGTTCCGGGT

[0299] SEQ ID NO:23(SDG24).

[0300] GGTGGTGAAGATCGCAGACAGCGGGTTAG

[0301] SEQ ID NO:24(POP2222).

[0302] CGGCGGTTAGAACGCGGCTAC

[0303] SEQ ID NO:25=SDG28

[0304] AAGATCAGTTGCGGCCGCTAGACTAGAACCGCTGCCACCGCCGGTGGTGAAGATCGCAGAC

[0305] SEQ ID NO:26=FG12

[0306] GTGACACGGCGGTTAGAACGCGGCTACAATTAATACATAACCCCATCCCCCTGTTGACAATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGGATCTACCATGCTGCCGGCGCCGAAAAACCTGGTTGTTTCTCGCGTTACCGAAGACTCTGCGCGTCTGTCTTGGACCGCGCCGGACGCGGCGTTCGACTCTTTCCTGATCCAGTACCAGGAATCTGAAAAAGTTGGTGAAGCGATCGTGCTGACCGTTCCGGGTTCTGAACGTTCTTACGACCTGACCGGTCTGAAACCGGGTACCGAATACACCGTTTCTATCTACGGTGTTNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNTCTAACCCGCTGTCTGCGATCTTCACCACCGGCGGTCACCATCACCATCACCATGGCAGCGGTTCTAGTCTAGCGGCCGCAACTGATCTTGGC

[0307] SEQ ID NO:27=FG11

[0308] GTGACACGGCGGTTAGAACGCGGCTACAATTAATACATAACCCCATCCCCCTGTTGACAATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGGATCTACCATGCTGCCGGCGCCGAAAAACCTGGTTGTTTCTCGCGTTACCGAAGACTCTGCGCGTCTGTCTTGGACCGCGCCGGACGCGGCGTTCGACTCTTTCCTGATCCAGTACCAGGAATCTGAAAAAGTTGGTGAAGCGATCGTGCTGACCGTTCCGGGTTCTGAACGTTCTTACGACCTGACCGGTCTGAAACCGGGTACCGAATACACCGTTTCTATCTACGGTGTTNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNTCTAACCCGCTGTCTGCGATCTTCACCACCGGCGGTCACCATCACCATCACCATGGCAGCGGTTCTAGTCTAGCGGCCGCAACTGATCTTGGC

[0309] SEQ ID NO:28=FG10

[0310] GTGACACGGCGGTTAGAACGCGGCTACAATTAATACATAACCCCATCCCCCTGTTGACAATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGGATCTACCATGCTGCCGGCGCCGAAAAACCTGGTTGTTTCTCGCGTTACCGAAGACTCTGCGCGTCTGTCTTGGACCGCGCCGGACGCGGCGTTCGACTCTTTCCTGATCCAGTACCAGGAATCTGAAAAAGTTGGTGAAGCGATCGTGCTGACCGTTCCGGGTTCTGAACGTTCTTACGACCTGACCGGTCTGAAACCGGGTACCGAATACACCGTTTCTATCTACGGTGTTNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNTCTAACCCGCTGTCTGCGATCTTCACCACCGGCGGTCACCATCACCATCACCATGGCAGCGGTTCTAGTCTAGCGGCCGCAACTGATCTTGGC

[0311] SEQ ID NO:29=FG9

[0312] GTGACACGGCGGTTAGAACGCGGCTACAATTAATACATAACCCCATCCCCCTGTTGACAATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGGATCTACCATGCTGCCGGCGCCGAAAAACCTGGTTGTTTCTCGCGTTACCGAAGACTCTGCGCGTCTGTCTTGGACCGCGCCGGACGCGGCGTTCGACTCTTTCCTGATCCAGTACCAGGAATCTGAAAAAGTTGGTGAAGCGATCGTGCTGACCGTTCCGGGTTCTGAACGTTCTTACGACCTGACCGGTCTGAAACCGGGTACCGAATACACCGTTTCTATCTACGGTGTTNNNNNNNNNNNNNNNNNNNNNNNNNNNTCTAACCCGCTGTCTGCGATCTTCACCACCGGCGGTCACCATCACCATCACCATGGCAGCGGTTCTAGTCTAGCGGCCGCAACTGATCTTGGC

[0313] SEQ ID NO:30=FG8

[0314] GTGACACGGCGGTTAGAACGCGGCTACAATTAATACATAACCCCATCCCCCTGTTGACAATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGGATCTACCATGCTGCCGGCGCCGAAAAACCTGGTTGTTTCTCGCGTTACCGAAGACTCTGCGCGTCTGTCTTGGACCGCGCCGGACGCGGCGTTCGACTCTTTCCTGATCCAGTACCAGGAATCTGAAAAAGTTGGTGAAGCGATCGTGCTGACCGTTCCGGGTTCTGAACGTTCTTACGACCTGACCGGTCTGAAACCGGGTACCGAATACACCGTTTCTATCTACGGTGTTNNNNNNNNNNNNNNNNNNNNNNNNTCTAACCCGCTGTCTGCGATCTTCACCACCGGCGGTCACCATCACCATCACCATGGCAGCGGTTCTAGTCTAGCGGCCGCAACTGATCTTGGCSEQ ID NO:31=FG7

[0315] GTGACACGGCGGTTAGAACGCGGCTACAATTAATACATAACCCCATCCCCCTGTTGACAATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGGATCTACCATGCTGCCGGCGCCGAAAAACCTGGTTGTTTCTCGCGTTACCGAAGACTCTGCGCGTCTGTCTTGGACCGCGCCGGACGCGGCGTTCGACTCTTTCCTGATCCAGTACCAGGAATCTGAAAAAGTTGGTGAAGCGATCGTGCTGACCGTTCCGGGTTCTGAACGTTCTTACGACCTGACCGGTCTGAAACCGGGTACCGAATACACCGTTTCTATCTACGGTGTTNNNNNNNNNNNNNNNNNNNNNTCTAACCCGCTGTCTGCGATCTTCACCACCGGCGGTCACCATCACCATCACCATGGCAGCGGTTCTAGTCTAGCGGCCGCAACTGATCTTGGC

[0316] SEQ ID NO:32 Tencon's FG ring

[0317] KGGHRSN

[0318] SEQ ID NO:33=Tcon 6

[0319] AAGAAGGAGAACCGGTATGCTGCCGGCGCCGAAAAAC

[0320] SEQ ID NO:34=Tcon5E86I short

[0321] GAG CCG CCG CCA CCG GTT TAA TGG TGA TGG TGA TGG TGA CCA CCG GTG GTGAAG ATC GCA GAC AG

[0322] SEQ ID NO:35 Original tencon C-chain

[0323] sfliqyqe

[0324] SEQ ID NO:36ALB-E05 C-chain

[0325] sfQiEyWe

[0326] SEQ ID NO:37ALB-E07 C-chain

[0327] sfKiLyEe

[0328] SEQ ID NO:38ALB-H9 C-chain

[0329] sfHiEyWe

[0330] SEQ ID NO:39 Original Tencon CD-Ring

[0331] sekvge

[0332] SEQ ID NO:40ALB-E05 CD-ring

[0333] DDVGge

[0334] SEQ ID NO:41ALB-E07 CD-ring

[0335] YLVFge

[0336] SEQ ID NO:42ALB-H9 CD-ring

[0337] QSIVge

[0338] SEQ ID NO:43 from the original F-chain

[0339] eytvsiygvk

[0340] SEQ ID NO:44ALB-E05 F-chain

[0341] eyDvYiLgvk

[0342] SEQ ID NO:45ALB-E07 F-chain

[0343] eyWvAiWgvk

[0344] SEQ ID NO:46ALB-H9 F-chain

[0345] eyRvWiYgvk

[0346] SEQ ID NO:47 from the original FG-loop

[0347] gghrsnp

[0348] SEQ ID NO:48ALB-E05 FG-loop

[0349] ggWEsGP

[0350] SEQ ID NO:49ALB-E07 FG-loop

[0351] ggQVsGT

[0352] SEQ ID NO:50ALB-H9 FG-loop

[0353] ggNDsWP

[0354] B7-SEQ ID NO:51

[0355] MLPAKNLVASRVTEDSARLSWTAPDAAFDSFNIAYWEPGIGGEAIWLRVPGSERSYDLTGLKPGTEYKVWIHGVKGGASSPPLIARFTT

[0356] A10V SEQ ID NO:52

[0357] MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFNIAYWEPGIGGEAIWLRVPGSERSYDLTGLKPGTEYKVWIHGVKGGASSPPLIARFTT

[0358] A10V,N33A SEQ ID NO:53

[0359] MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFAIAYWEPGIGGEAIWLRVPGSERSYDLTGLKPGTEYKVWIHGVKGGASSPPLIARFTT

[0360] A10V,A35S SEQ ID NO:54

[0361] MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFNISYWEPGIGGEAIWLRVPGSERSYDLTGLKPGTEYKVWIHGVKGGASSPPLIARFTT

[0362] A10V,W37A SEQ ID NO:55

[0363] MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFNIAYAEPGIGGEAIWLRVPGSERSYDLTGLKPGTEYKVWIHGVKGGASSPPLIARFTT

[0364] A10V,P39A SEQ ID NO:56

[0365] MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFNIAYWEAGIGGEAIWLRVPGSERSYDLTGLKPGTEYKVWIHGVKGGASSPPLIARFTT

[0366] A10V,G40A SEQ ID NO:57

[0367] MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFNIAYWEPAIGGEAIWLRVPGSERSYDLTGLKPGTEYKVWIHGVKGGASSPPLIARFTT

[0368] A10V,I41A SEQ ID NO:58

[0369] MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFNIAYWEPGAGGEAIWLRVPGSERSYDLTGLKPGTEYKVWIHGVKGGASSPPLIARFTT

[0370] A10V,G42A SEQ ID NO:59

[0371] MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFNIAYWEPGIAGEAIWLRVPGSERSYDLTGLKPGTEYKVWIHGVKGGASSPPLIARFTT

[0372] A10V,W47A SEQ ID NO:60

[0373] MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFNIAYWEPGIGGEAIALRVPGSERSYDLTGLKPGTEYKVWIHGVKGGASSPPLIARFTT

[0374] A10V,R49A SEQ ID NO:61

[0375] MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFNIAYWEPGIGGEAIWLAVPGSERSYDLTGLKPGTEYKVWIHGVKGGASSPPLIARFTT

[0376] A10V,K69A SEQ ID NO:62

[0377] MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFNIAYWEPGIGGEAIWLRVPGSERSYDLTGLKPGTEYAVWIHGVKGGASSPPLIARFTT

[0378] A10V,W71A SEQ ID NO:63

[0379] MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFNIAYWEPGIGGEAIWLRVPGSERSYDLTGLKPGTEYKVAIHGVKGGASSPPLIARFTT

[0380] A10V,H73A SEQ ID NO:64

[0381] MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFNIAYWEPGIGGEAIWLRVPGSERSYDLTGLKPGTEYKVWIAGVKGGASSPPLIARFTT

[0382] A10V,A79S SEQ ID NO:65

[0383] MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFNIAYWEPGIGGEAIWLRVPGSERSYDLTGLKPGTEYKVWIHGVKGGSSSPPLIARFTT

[0384] A10V,S80A SEQ ID NO:66

[0385] MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFNIAYWEPGIGGEAIWLRVPGSERSYDLTGLKPGTEYKVWIHGVKGGAASPPLIARFTT

[0386] A10V,P82A SEQ ID NO:67

[0387] MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFNIAYWEPGIGGEAIWLRVPGSERSYDLTGLKPGTEYKVWIHGVKGGASSAPLIARFTT

[0388] A10V,I85A SEQ ID NO:68

[0389] MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFNIAYWEPGIGGEAIWLRVPGSERSYDLTGLKPGTEYKVWIHGVKGGASSPPLAARFTT

[0390] A10V,R87A SEQ ID NO:69

[0391] MLPAPKNLVVSRVTEDSARLSWTAPDAAFDSFNIAYWEPGIGGEAIWLRVPGSERSYDLTGLKPGTEYKVWIHGVKGGASSPPLIAAFTT

[0392] SEQ ID NO:70 ALB-H9

[0393] LPAPKNLVVSRVTEDSARLSWTAPDAAFDSFHIEYWEQSIVGEAIVLTVPGSERSYDLTGLKPGTEYRVWIYGVKGGNDSWPLSAIFTT

[0394] SEQ ID NO:71(GS)2

[0395] GSGS

[0396] SEQ ID NO:72(GGGS)2

[0397] GGGSGGGS

[0398] SEQ ID NO:73(GGGGS)2

[0399] GGGGSGGGGSGGGGSGGGGSGGGGS

[0400] SEQ ID NO:74(AP)2

[0401] APAP

[0402] SEQ ID NO:75(AP)5

[0403] APAPAPAPAP

[0404] SEQ ID NO:76(AP) 10

[0405] APAPAPAPAPAPAPAPAPAP

[0406] SEQ ID NO:77(AP) 20

[0407] APAPAPAPAPAPAPAPAPAPAPAPAPAPAPAPAP

[0408] SEQ ID NO:78A(EAAAK)5AAA

[0409] AEAAAKEAAAKEAAAKEAAAKEAAAKAAA

[0410] SEQ ID NO:79 Human serum albumin

[0411] KWVTFISLLFLFSSAYSRGVFRRDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL

[0412] SEQ ID NO:80 Cynomolgus monkey serum albumin

[0413] KWVTFISLLFLFSSAYSRGVFRRDTHKSEVAHRFKDLGEEHFKGLVLVAFSQYLQQCPFEEHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPPLVRPEVDVMCTAFHDNEATFLKKYLYEVARRHPYFYAPELLFFAARYKAAFAECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGDRAFKAWAVARLSQKFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYMCENQDSISSKLKECCDKPLLEKSHCLAEVENDEMPADLPSLAADYVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVMLLLRLAKAYEATLEKCCAAADPHECYAKVFDEFQPLVEEPQNLVKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGAKCCKLPEAKRMPCAEDYLSVVLNRLCVLHEKTPVSEKVTKCCTESLVNRRPCFSALELDEAYVPKAFNAETFTFHADMCTLSEKEKQVKKQTALVELVKHKPKATKEQLKGVMDNFAAFVEKCCKADDKEACFAEEGPKFVAASQAALA

[0414] SEQ ID NO:81 Tenascin C (TN3)

[0415] DAPSQIEVKDVTDTTALITWFKPLAEIDGIELTYGIKDVPGDRTTIDLTEDENQYSIGNLKPDTEYEVSLISRRGDMSSNPAKETFTT

[0416] SEQ ID NO:82 Fibcon

[0417] LDAPTDLQVTNVTDTSITVSWTPPSATITGYRITYTPSNGPGEPKELTVPPSSTSVTITGLTPGVEYVVSLYALKDNQESPPLVGTQTT

[0418] SEQ ID NO:83 FN10

[0419] VSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFTVPGSKSTATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRT. sequence list <110> ARO Biotherapeutics Company <120> Fibronectin type III domain bound to serum albumin and its application <130> 145965.02402 <160> 83 <170> PatentIn version 3.5 <210> 1 <211> 89 <212> PRT <213> Homo sapiens <400> 1 Leu Pro Ala Pro Lys Asn Leu Val Val Ser Glu Val Thr Glu Asp Ser 1 5 10 15 Leu Arg Leu Ser Trp Thr Ala Pro Asp Ala Ala Phe Asp Ser Phe Leu 20 25 30 Ile Gln Tyr Gln Glu Ser Glu Lys Val Gly Glu Ala Ile Asn Leu Thr 35 40 45 Val Pro Gly Ser Glu Arg Ser Tyr Asp Leu Thr Gly Leu Lys Pro Gly 50 55 60 Thr Glu Tyr Thr Val Ser Ile Tyr Gly Val Lys Gly Gly His Arg Ser 65 70 75 80 Asn Pro Leu Ser Ala Glu Phe Thr Thr 85 <210> 2 <211> 94 <212> PRT <213> Artificial Sequence <220> <223> synthetic sequence <220> <221> MISC_FEATURE <222> (75) (81) <223> Each X is any amino acid. <220> <221> MISC_FEATURE <222> (82)..(86) <223> Each X represents any amino acid or deletion. <400> 2 Leu Pro Ala Pro Lys Asn Leu Val Val Ser Glu Val Thr Glu Asp Ser 1 5 10 15 Leu Arg Leu Ser Trp Thr Ala Pro Asp Ala Ala Phe Asp Ser Phe Leu 20 25 30 Ile Gln Tyr Gln Glu Ser Glu Lys Val Gly Glu Ala Ile Asn Leu Thr 35 40 45 Val Pro Gly Ser Glu Arg Ser Tyr Asp Leu Thr Gly Leu Lys Pro Gly 50 55 60 Thr Glu Tyr Thr Val Ser Ile Tyr Gly Val Xaa Xaa Xaa Xaa Xaa Xaa 65 70 75 80 Xaa Xaa Xaa Xaa Xaa Xaa Pro Leu Ser Ala Glu Phe Thr Thr 85 90 <210> 3 <211> 89 <212> PRT <213> Artificial Sequence <220> <223> synthetic sequence <220> <221> MISC_FEATURE <222> (22)..(27) <223> Each X is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. <220> <221> MISC_FEATURE <222> (28)..(28) <223> X is Phe, Ile, Leu, Val, or Tyr <220> <221> MISC_FEATURE <222> (29)..(29) <223> X is Asp, Glu, or Thr. <220> <221> MISC_FEATURE <222> (75) (79) <223> Each X is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. <220> <221> MISC_FEATURE <222> (81) (82) <223> Each X is Ala, Arg, Asn, Asp, Glu, Gln, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. <400> 3 Leu Pro Ala Pro Lys Asn Leu Val Val Ser Glu Val Thr Glu Asp Ser 1 5 10 15 Leu Arg Leu Ser Trp Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ser Phe Leu 20 25 30 Ile Gln Tyr Gln Glu Ser Glu Lys Val Gly Glu Ala Ile Asn Leu Thr 35 40 45 Val Pro Gly Ser Glu Arg Ser Tyr Asp Leu Thr Gly Leu Lys Pro Gly 50 55 60 Thr Glu Tyr Thr Val Ser Ile Tyr Gly Val Xaa Xaa Xaa Xaa Xaa Ser 65 70 75 80 Xaa Xaa Leu Ser Ala Glu Phe Thr Thr 85 <210> 4 <211> 89 <212> PRT <213> Artificial Sequence <220> <223> Synthetic sequence <400> 4 Leu Pro Ala Pro Lys Asn Leu Val Val Ser Arg Val Thr Glu Asp Ser 1 5 10 15 Ala Arg Leu Ser Trp Thr Ala Pro Asp Ala Ala Phe Asp Ser Phe Leu 20 25 30 Ile Gln Tyr Gln Glu Ser Glu Lys Val Gly Glu Ala Ile Val Leu Thr 35 40 45 Val Pro Gly Ser Glu Arg Ser Tyr Asp Leu Thr Gly Leu Lys Pro Gly 50 55 60 Thr Glu Tyr Thr Val Ser Ile Tyr Gly Val Lys Gly Gly His Arg Ser 65 70 75 80 Asn Pro Leu Ser Ala Ile Phe Thr Thr 85 <210> 5 <211> 97 <212> PRT <213> Artificial Sequence <220> <223> synthetic sequence <220> <221> MISC_FEATURE <222> (22)..(27) <223> Each X is A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, or Y. <220> <221> MISC_FEATURE <222> (28) (30) <223> Each X is A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, Y, or missing. <220> <221> MISC_FEATURE <222> (78) (84) <223> Each X is A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, or Y. <220> <221> MISC_FEATURE <222> (85) (87) <223> Each X is A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, Y, or missing. <400> 5 Leu Pro Ala Pro Lys Asn Leu Val Val Ser Arg Val Thr Glu Asp Ser 1 5 10 15 Ala Arg Leu Ser Trp Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Phe Asp 20 25 30 Ser Phe Leu Ile Gln Tyr Gln Glu Ser Glu Lys Val Gly Glu Ala Ile 35 40 45 Val Leu Thr Val Pro Gly Ser Glu Arg Ser Tyr Asp Leu Thr Gly Leu 50 55 60 Lys Pro Gly Thr Glu Tyr Thr Val Ser Ile Tyr Gly Val Xaa Xaa Xaa 65 70 75 80 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ser Asn Pro Leu Ser Ala Ile Phe Thr 85 90 95 Thr <210> 6 <211> 96 <212> PRT <213> Artificial Sequence <220> <223> synthetic sequence <220> <221> MISC_FEATURE <222> (75) (81) <223> Each X is A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, or Y. <220> <221> MISC_FEATURE <222> (82)..(86) <223> Each X is A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, Y, or missing. <400> 6 Leu Pro Ala Pro Lys Asn Leu Val Val Ser Arg Val Thr Glu Asp Ser 1 5 10 15 Ala Arg Leu Ser Trp Thr Ala Pro Asp Ala Ala Phe Asp Ser Phe Leu 20 25 30 Ile Gln Tyr Gln Glu Ser Glu Lys Val Gly Glu Ala Ile Val Leu Thr 35 40 45 Val Pro Gly Ser Glu Arg Ser Tyr Asp Leu Thr Gly Leu Lys Pro Gly 50 55 60 Thr Glu Tyr Thr Val Ser Ile Tyr Gly Val Xaa Xaa Xaa Xaa Xaa Xaa 65 70 75 80 Xaa Xaa Xaa Xaa Xaa Xaa Ser Asn Pro Leu Ser Ala Ile Phe Thr Thr 85 90 95 <210> 7 <211> 89 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Sequence <220> <221> MISC_FEATURE <222> (32)..(32) <223> X is A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, Y or M <220> <221> MISC_FEATURE <222> (34)..(34) <223> X is A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, Y or M <220> <221> MISC_FEATURE <222> (36)..(36) <223> X is A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, Y, or M. <220> <221> MISC_FEATURE <222> (38) (41) <223> Each X is A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, Y, or M. <220> <221> MISC_FEATURE <222> (68)..(68) <223> X is A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, Y, or M. <220> <221> MISC_FEATURE <222> (70)..(70) <223> X is A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, Y, or M. <220> <221> MISC_FEATURE <222> (72)..(72) <223> X is A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, Y, or M. <220> <221> MISC_FEATURE <222> (78) (79) <223> Each X is A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, Y, or M. <220> <221> MISC_FEATURE <222> (81)..(81) <223> X is A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, W, Y, or M. <400> 7 Leu Pro Ala Pro Lys Asn Leu Val Val Ser Arg Val Thr Glu Asp Ser 1 5 10 15 Ala Arg Leu Ser Trp Thr Ala Pro Asp Ala Ala Phe Asp Ser Phe Xaa 20 25 30 Ile Xaa Tyr Xaa Glu Xaa Xaa Xaa Xaa Gly Glu Ala Ile Val Leu Thr 35 40 45 Val Pro Gly Ser Glu Arg Ser Tyr Asp Leu Thr Gly Leu Lys Pro Gly 50 55 60 Thr Glu Tyr Xaa Val Xaa Ile Xaa Gly Val Lys Gly Gly Xaa Xaa Ser 65 70 75 80 Xaa Pro Leu Ser Ala Ile Phe Thr Thr 85 <210> 8 <211> 89 <212> PRT <213> Artificial Sequence <220> <223> synthetic sequence <220> <221> MISC_FEATURE <222> (32)..(32) <223> X is A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, or W. <220> <221> MISC_FEATURE <222> (34)..(34) <223> X is A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, or W. <220> <221> MISC_FEATURE <222> (36)..(36) <223> X is A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, or W. <220> <221> MISC_FEATURE <222> (38) (41) <223> Each X is A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, or W. <220> <221> MISC_FEATURE <222> (46)..(46) <223> X is A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, or W. <220> <221> MISC_FEATURE <222> (48)..(48) <223> X is A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, or W. <220> <221> MISC_FEATURE <222> (68)..(68) <223> X is A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, or W. <220> <221> MISC_FEATURE <222> (70)..(70) <223> X is A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, or W. <220> <221> MISC_FEATURE <222> (72)..(72) <223> X is A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V, or W. <220> <221> MISC_FEATURE <222> (78) (79) <223> Each X is A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V or W <220> <221> MISC_FEATURE <222> (81)..(81) <223> X is A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V or W <220> <221> MISC_FEATURE <222> (84)..(84) <223> X is A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V or W <220> <221> MISC_FEATURE <222> (86)..(86) <223> X is A, D, E, F, G, H, I, K, L, N, P, Q, R, S, T, V or W <-- 400 --> 8 Leu Pro Ala Pro Lys Asn Leu Val Val Ser Arg Val Thr Glu Asp Ser 1 5 10 15 Ala Arg Leu Ser Trp Thr Ala Pro Asp Ala Ala Phe Asp Ser Phe Xaa 20 25 30 Ile Xaa Tyr Xaa Glu Xaa Xaa Xaa Xaa Gly Glu Ala Ile Xaa Leu Xaa 35 40 45 Val Pro Gly Ser Glu Arg Ser Tyr Asp Leu Thr Gly Leu Lys Pro Gly 50 55 60 Thr Glu Tyr Xaa Val Xaa Ile Xaa Gly Val Lys Gly Gly Xaa Xaa Ser 65 70 75 80 Xaa Pro Leu Xaa Ala Xaa Phe Thr Thr 85 <210> 9 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> synthetic sequence <400> 9 gtgacacggc ggttagaac 19 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> synthetic sequence <400> 10 gcctttggga agcttctaag 20 <210> 11 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> synthetic sequence <400> 11 cggcggttag aacgcggcta caattaatac 30 <210> 12 <211> twenty two <212> DNA <213> Artificial Sequence <220> <223> synthetic sequence <400> 12 catgattacg ccaagctcag aa 22 <210> 13 <211> 385 <212> DNA <213> Artificial Sequence <220> <223> synthetic sequence <220> <221> misc_feature <222> (198)..(224) <223> N is any base <400> 13 gtgacacggc ggttagaacg cggctacaat taatacataa ccccatcccc ctgttgacaa 60 ttaatcatcg gctcgtataa tgtgtggaat tgtgagcgga taacaatttc acacaggaaa 120 caggatctac catgctgccg gcgccgaaaa acctggttgt ttctgaagtt accgaagact 180 ctctgcgtct gtcttggnnnn nnnnnnnnnn nnnnnnnnnn nnnnttygac tctttcctga 240 tccagtacca ggaatctgaa aaagttggtg aagcgatcaa cctgaccgtt ccgggttctg 300 aacgttctta cgacctgacc ggtctgaaac cgggtaccga atacaccgtt tctatctacg 360 gtgttcttag aagcttccca aaggc 385 <210> 14 <211> 382 <212> DNA <213> Artificial Sequence <220> <223> synthetic sequence <220> <221> misc_feature <222> (198)..(221) <223> N is any base <400> 14 gtgacacggc ggttagaacg cggctacaat taatacataa ccccatcccc ctgttgacaa 60 ttaatcatcg gctcgtataa tgtgtggaat tgtgagcgga taacaatttc acacaggaaa 120 caggatctac catgctgccg gcgccgaaaa acctggttgt ttctgaagtt accgaagact 180 ctctgcgtct gtcttggnnn nnnnnnnnnn nnnnnnnnnn nttygactct ttcctgatcc 240 agtaccagga atctgaaaaa gttggtgaag cgatcaacct gaccgttccg ggttctgaac 300 gttcttacga cctgaccggt ctgaaaccgg gtaccgaata caccgtttct atctacggtg 360 ttcttagaag cttcccaaag gc 382 <210> 15 <211> 379 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Sequence <220> <221> misc_feature <222> (198)..(218) <223> N is any base <(400)> 15 gtgacacggc ggttagaacg cggctacaat taatacataa ccccatcccc ctgttgacaa 60 ttaatcatcg gctcgtataa tgtgtggaat tgtgagcgga taacaatttc acacaggaaa 120 caggatctac catgctgccg gcgccgaaaa acctggttgt ttctgaagtt accgaagact 180 ctctgcgtct gtcttggnnn nnnnnnnnnn nnnnnnnntt ygactctttc ctgatccagt 240 accaggaatc tgaaaaagtt ggtgaagcga tcaacctgac cgttccgggt tctgaacgtt 300 cttacgacct gaccggtctg aaaccgggta ccgaatacac cgtttctatc tacggtgttc 360 ttagaagctt cccaaaggc 379 <210> 16 <211> 376 <212> DNA <213> Artificial Sequence <220> <223> Synthetic sequence <220> <221> misc_feature <222> (198)..(215) <223> N is any base <400> 16 gtgacacggc ggttagaacg cggctacaat taatacataa ccccatcccc ctgttgacaa 60 ttaatcatcg gctcgtataa tgtgtggaat tgtgagcgga taacaatttc acacaggaaa 120 caggatctac catgctgccg gcgccgaaaa acctggttgt ttctgaagtt accgaagact 180 ctctgcgtct gtcttggnnn nnnnnnnnnn nnnnnttyga ctctttcctg atccagtacc 240 aggaatctga aaaagttggt gaagcgatca acctgaccgt tccgggttct gaacgttctt 300 acgacctgac cggtctgaaa ccgggtaccg aatacaccgt ttctatctac ggtgttctta 360 gaagcttccc aaaggc 376 <210> 17 <211> 131 <212> DNA <213> Artificial Sequence <220> <223> synthetic sequence <400> 17 cggcggttag aacgcggcta caattaatac ataaccccat ccccctgttg acaattaatc 60 atcggctcgt ataatgtgtg gaattgtgag cggataacaa tttcacacag gaaacaggat 120 ctaccatgct g 131 <210> 18 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> synthetic sequence <400> 18 cggcggttag aacgcggcta caattaatac 30 <210> 19 <211> 81 <212> DNA <213> Artificial Sequence <220> <223> synthetic sequence <400> 19 ccaagacaga cgggcagagt cttcggtaac gcgagaaaca accaggtttt tcggcgccgg 60 cagcatggta gatcctgttt c 81 <210> 20 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> synthetic sequence <400> 20 ccgaagactc tgcccgtctg tcttgg 26 <210> twenty one <211> 45 <212> DNA <213> Artificial Sequence <220> <223> synthetic sequence <400> twenty one cagtggtctc acggattcct ggtactggat caggaaagag tcgaa 45 <210> twenty two <211> 54 <212> DNA <213> Artificial Sequence <220> <223> synthetic sequence <400> twenty two catgcggtct cttccgaaaa agttggtgaa gcgatcgtcc tgaccgttcc gggt 54 <210> twenty three <211> 29 <212> DNA <213> Artificial Sequence <220> <223> synthetic sequence <400> twenty three ggtggtgaag atcgcagaca gcgggttag 29 <210> twenty four <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> synthetic sequence <400> twenty four cggcggttag aacgcggcta c 21 <210> 25 <211> 61 <212> DNA <213> Artificial Sequence <220> <223> synthetic sequence <400> 25 aagatcagtt gcggccgcta gactagaacc gctgccaccg ccggtggtga agatcgcaga 60 c 61 <210> 26 <211> 485 <212> DNA <213> Artificial Sequence <220> <223> synthetic sequence <220> <221> misc_feature <222> (357) (392) <223> N is any base <400> 26 gtgacacggc ggttagaacg cggctacaat taatacataa ccccatcccc ctgttgacaa 60 ttaatcatcg gctcgtataa tgtgtggaat tgtgagcgga taacaatttc acacaggaaa 120 caggatctac catgctgccg gcgccgaaaa acctggttgt ttctcgcgtt accgaagact 180 ctgcgcgtct gtcttggacc gcgccggacg cggcgttcga ctctttcctg atccagtacc 240 aggaatctga aaaagttggt gaagcgatcg tgctgaccgt tccgggttct gaacgttctt 300 acgacctgac cggtctgaaa ccgggtaccg aatacaccgt ttctatctac ggtgttnnnn 360 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nntctaaccc gctgtctgcg atcttcacca 420 ccggcggtca ccatcaccat caccatggca gcggttctag tctagcggcc gcaactgatc 480 ttggc 485 <210> 27 <211> 482 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Sequence <220> <221> misc_feature <222> (357)..(389) <223> N is any base <400> 27 gtgacacggc ggttagaacg cggctacaat taatacataa ccccatcccc ctgttgacaa 60 ttaatcatcg gctcgtataa tgtgtggaat tgtgagcgga taacaatttc acacaggaaa 120 caggatctac catgctgccg gcgccgaaaa acctggttgt ttctcgcgtt accgaagact 180 ctgcgcgtct gtcttggacc gcgccggacg cggcgttcga ctctttcctg atccagtacc 240 aggaatctga aaaagttggt gaagcgatcg tgctgaccgt tccgggttct gaacgttctt 300 acgacctgac cggtctgaaa ccgggtaccg aatacaccgt ttctatctac ggtgttnnnn 360 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnt ctaacccgct gtctgcgatc ttcaccaccg 420 gcggtcacca tcaccatcac catggcagcg gttctagtct agcggccgca actgatcttg 480 gc 482 <210> 28 <211> 479 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Sequence <220> <221> misc_feature <222> (357)..(386) <223> N is any base <400> 28 gtgacacggc ggttagaacg cggctacaat taatacataa ccccatcccc ctgttgacaa 60 ttaatcatcg gctcgtataa tgtgtggaat tgtgagcgga taacaatttc acacaggaaa 120 caggatctac catgctgccg gcgccgaaaa acctggttgt ttctcgcgtt accgaagact 180 ctgcgcgtct gtcttggacc gcgccggacg cggcgttcga ctctttcctg atccagtacc 240 aggaatctga aaaagttggt gaagcgatcg tgctgaccgt tccgggttct gaacgttctt 300 acgacctgac cggtctgaaa ccgggtaccg aatacaccgt ttctatctac ggtgttnnnn 360 nnnnnnnnnn nnnnnnnnnn nnnnnntcta acccgctgtc tgcgatcttc accaccggcg 420 gtcaccatca ccatcaccat ggcagcggtt ctagtctagc ggccgcaact gatcttggc 479[[ID=eleven]] <210> 29 <211> 476 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Sequence <220> <221> misc_feature <222> (357)..(383) <223> N is any base <400> 29 gtgacacggc ggttagaacg cggctacaat taatacataa ccccatcccc ctgttgacaa 60 ttaatcatcg gctcgtataa tgtgtggaat tgtgagcgga taacaatttc acacaggaaa 120 caggatctac catgctgccg gcgccgaaaa acctggttgt ttctcgcgtt accgaagact 180 ctgcgcgtct gtcttggacc gcgccggacg cggcgttcga ctctttcctg atccagtacc 240 aggaatctga aaaagttggt gaagcgatcg tgctgaccgt tccgggttct gaacgttctt 300 acgacctgac cggtctgaaa ccgggtaccg aatacaccgt ttctatctac ggtgttnnnn 360 nnnnnnnnnn nnnnnnnnnn nnntctaacc cgctgtctgc gatcttcacc accggcggtc 420 accatcacca tcaccatggc agcggttcta gtctagcggc cgcaactgat cttggc 476 <210> 30 <211> 473 <212> DNA <213> Artificial Sequence <220> <223> Synthetic sequence <220> <221> misc_feature <222> (357)..(380) <223> N is any base <400> 30 gtgacacggc ggttagaacg cggctacaat taatacataa ccccatcccc ctgttgacaa 60 ttaatcatcg gctcgtataa tgtgtggaat tgtgagcgga taacaatttc acacaggaaa 120 caggatctac catgctgccg gcgccgaaaa acctggttgt ttctcgcgtt accgaagact 180 ctgcgcgtct gtcttggacc gcgccggacg cggcgttcga ctctttcctg atccagtacc 240 aggaatctga aaaagttggt gaagcgatcg tgctgaccgt tccgggttct gaacgttctt 300 acgacctgac cggtctgaaa ccgggtaccg aatacaccgt ttctatctac ggtgttnnnn 360 nnnnnnnnnn nnnnnnnnnn tctaacccgc tgtctgcgat cttcaccacc ggcggtcacc 420 atcaccatca ccatggcagc ggttctagtc tagcggccgc aactgatctt ggc 473 <210> 31 <211> 470 <212> DNA <213> Artificial Sequence <220> <223> Synthetic sequence <220> <221> misc_feature <222> (357)..(377) <223> N is any base <400> 31 gtgacacggc ggttagaacg cggctacaat taatacataa ccccatcccc ctgttgacaa 60 ttaatcatcg gctcgtataa tgtgtggaat tgtgagcgga taacaatttc acacaggaaa 120 caggatctac catgctgccg gcgccgaaaa acctggttgt ttctcgcgtt accgaagact 180 ctgcgcgtct gtcttggacc gcgccggacg cggcgttcga ctctttcctg atccagtacc 240 aggaatctga aaaagttggt gaagcgatcg tgctgaccgt tccgggttct gaacgttctt 300 acgacctgac cggtctgaaa ccgggtaccg aatacaccgt ttctatctac ggtgttnnnn 360 nnnnnnnnnn nnnnnnntct aacccgctgt ctgcgatctt caccaccggc ggtcaccatc 420 accatcacca tggcagcggt tctagtctag cggccgcaac tgatcttggc 470 <210> 32 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> synthetic sequence <400> 32 Lys Gly Gly His Arg Ser Asn 1 5 <210> 33 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> synthetic sequence <400> 33 aagaaggaga accggtatgc tgccggcgcc gaaaaac 37 <210> 34 <211> 65 <212> DNA <213> Artificial Sequence <220> <223> synthetic sequence <400> 34 gagccgccgc caccggttta atggtgatgg tgatggtgac caccggtggt gaagatcgca 60 gacag 65 <210> 35 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> synthetic sequence <400> 35 Ser Phe Leu Ile Gln Tyr Gln Glu 1 5 <210> 36 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> synthetic sequence <400> 36 Ser Phe Gln Ile Glu Tyr Trp Glu 1 5 <210> 37 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> synthetic sequence <400> 37 Ser Phe Lys Ile Leu Tyr Glu Glu 1 5 <210> 38 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> synthetic sequence <400> 38 Ser Phe His Ile Glu Tyr Trp Glu 1 5 <210> 39 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> synthetic sequence <400> 39 Ser Glu Lys Val Gly Glu 1 5 <210> 40 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> synthetic sequence <400> 40 Asp Asp Val Gly Gly Glu 1 5 <210> 41 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> synthetic sequence <400> 41 Tyr Leu Val Phe Gly Glu 1 5 <210> 42 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> synthetic sequence <400> 42 Gln Ser Ile Val Gly Glu 1 5 <210> 43 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> synthetic sequence <400> 43 Glu Tyr Thr Val Ser Ile Tyr Gly Val Lys 1 5 10 <210> 44 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> synthetic sequence <400> 44 Glu Tyr Asp Val Tyr Ile Leu Gly Val Lys 1 5 10 <210> 45 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> synthetic sequence <400> 45 Glu Tyr Trp Val Ala Ile Trp Gly Val Lys 1 5 10 <210> 46 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> synthetic sequence <400> 46 Glu Tyr Arg Val Trp Ile Tyr Gly Val Lys 1 5 10 <210> 47 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> synthetic sequence <400> 47 Gly Gly His Arg Ser Asn Pro 1 5 <210> 48 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> synthetic sequence <400> 48 Gly Gly Trp Glu Ser Gly Pro 1 5 <210> 49 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> synthetic sequence <400> 49 Gly Gly Gln Val Ser Gly Thr 1 5 <210> 50 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> synthetic sequence <400> 50 Gly Gly Asn Asp Ser Trp Pro 1 5 <210> 51 <211> 90 <212> PRT <213> Artificial Sequence <220> <223> synthetic sequence <400> 51 Met Leu Pro Ala Pro Lys Asn Leu Val Ala Ser Arg Val Thr Glu Asp 1 5 10 15 Ser Ala Arg Leu Ser Trp Thr Ala Pro Asp Ala Ala Phe Asp Ser Phe 20 25 30 Asn Ile Ala Tyr Trp Glu Pro Gly Ile Gly Gly Glu Ala Ile Trp Leu 35 40 45 Arg Val Pro Gly Ser Glu Arg Ser Tyr Asp Leu Thr Gly Leu Lys Pro 50 55 60 Gly Thr Glu Tyr Lys Val Trp Ile His Gly Val Lys Gly Gly Ala Ser 65 70 75 80 Ser Pro Pro Leu Ile Ala Arg Phe Thr Thr 85 90 <210> 52 <211> 90 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 52 Met Leu Pro Ala Pro Lys Asn Leu Val Val Ser Arg Val Thr Glu Asp 1 5 10 15 Ser Ala Arg Leu Ser Trp Thr Ala Pro Asp Ala Ala Phe Asp Ser Phe 20 25 30 Asn Ile Ala Tyr Trp Glu Pro Gly Ile Gly Gly Glu Ala Ile Trp Leu 35 40 45 Arg Val Pro Gly Ser Glu Arg Ser Tyr Asp Leu Thr Gly Leu Lys Pro 50 55 60 Gly Thr Glu Tyr Lys Val Trp Ile His Gly Val Lys Gly Gly Ala Ser 65 70 75 80 Ser Pro Pro Leu Ile Ala Arg Phe Thr Thr 85 90 <210> 53 <211> 90 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 53 Met Leu Pro Ala Pro Lys Asn Leu Val Val Ser Arg Val Thr Glu Asp 1 5 10 15 Ser Ala Arg Leu Ser Trp Thr Ala Pro Asp Ala Ala Phe Asp Ser Phe 20 25 30 Ala Ile Ala Tyr Trp Glu Pro Gly Ile Gly Gly Glu Ala Ile Trp Leu 35 40 45 Arg Val Pro Gly Ser Glu Arg Ser Tyr Asp Leu Thr Gly Leu Lys Pro 50 55 60 Gly Thr Glu Tyr Lys Val Trp Ile His Gly Val Lys Gly Gly Ala Ser 65 70 75 80 Ser Pro Pro Leu Ile Ala Arg Phe Thr Thr 85 90 <210> 54 <211> 90 <212> PRT <213> Artificial Sequence <220> <223> synthetic sequence <400> 54 Met Leu Pro Ala Pro Lys Asn Leu Val Val Ser Arg Val Thr Glu Asp 1 5 10 15 Ser Ala Arg Leu Ser Trp Thr Ala Pro Asp Ala Ala Phe Asp Ser Phe 20 25 30 Asn Ile Ser Tyr Trp Glu Pro Gly Ile Gly Gly Glu Ala Ile Trp Leu 35 40 45 Arg Val Pro Gly Ser Glu Arg Ser Tyr Asp Leu Thr Gly Leu Lys Pro 50 55 60 Gly Thr Glu Tyr Lys Val Trp Ile His Gly Val Lys Gly Gly Ala Ser 65 70 75 80 Ser Pro Pro Leu Ile Ala Arg Phe Thr Thr 85 90 <210> 55 <211> 90 <212> PRT <213> Artificial Sequence <220> <223> synthetic sequence <400> 55 Met Leu Pro Ala Pro Lys Asn Leu Val Val Ser Arg Val Thr Glu Asp 1 5 10 15 Ser Ala Arg Leu Ser Trp Thr Ala Pro Asp Ala Ala Phe Asp Ser Phe 20 25 30 Asn Ile Ala Tyr Ala Glu Pro Gly Ile Gly Gly Glu Ala Ile Trp Leu 35 40 45 Arg Val Pro Gly Ser Glu Arg Ser Tyr Asp Leu Thr Gly Leu Lys Pro 50 55 60 Gly Thr Glu Tyr Lys Val Trp Ile His Gly Val Lys Gly Gly Ala Ser 65 70 75 80 Ser Pro Pro Leu Ile Ala Arg Phe Thr Thr 85 90 <210> 56 <211> 90 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 56 Met Leu Pro Ala Pro Lys Asn Leu Val Val Ser Arg Val Thr Glu Asp 1 5 10 15 Ser Ala Arg Leu Ser Trp Thr Ala Pro Asp Ala Ala Phe Asp Ser Phe 20 25 30 Asn Ile Ala Tyr Trp Glu Ala Gly Ile Gly Gly Glu Ala Ile Trp Leu 35 40 45 Arg Val Pro Gly Ser Glu Arg Ser Tyr Asp Leu Thr Gly Leu Lys Pro 50 55 60 Gly Thr Glu Tyr Lys Val Trp Ile His Gly Val Lys Gly Gly Ala Ser 65 70 75 80 Ser Pro Pro Leu Ile Ala Arg Phe Thr Thr 85 90 <210> 57 <211> 90 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 57 Met Leu Pro Ala Pro Lys Asn Leu Val Val Ser Arg Val Thr Glu Asp 1 5 10 15 Ser Ala Arg Leu Ser Trp Thr Ala Pro Asp Ala Ala Phe Asp Ser Phe 20 25 30 Asn Ile Ala Tyr Trp Glu Pro Ala Ile Gly Gly Glu Ala Ile Trp Leu 35 40 45 Arg Val Pro Gly Ser Glu Arg Ser Tyr Asp Leu Thr Gly Leu Lys Pro 50 55 60 Gly Thr Glu Tyr Lys Val Trp Ile His Gly Val Lys Gly Gly Ala Ser 65 70 75 80 Ser Pro Pro Leu Ile Ala Arg Phe Thr Thr 85 90 <210> 58 <211> 90 <212> PRT <213> Artificial Sequence <220> <223> synthetic sequence <400> 58 Met Leu Pro Ala Pro Lys Asn Leu Val Val Ser Arg Val Thr Glu Asp 1 5 10 15 Ser Ala Arg Leu Ser Trp Thr Ala Pro Asp Ala Ala Phe Asp Ser Phe 20 25 30 Asn Ile Ala Tyr Trp Glu Pro Gly Ala Gly Gly Glu Ala Ile Trp Leu 35 40 45 Arg Val Pro Gly Ser Glu Arg Ser Tyr Asp Leu Thr Gly Leu Lys Pro 50 55 60 Gly Thr Glu Tyr Lys Val Trp Ile His Gly Val Lys Gly Gly Ala Ser 65 70 75 80 Ser Pro Pro Leu Ile Ala Arg Phe Thr Thr 85 90 <210> 59 <211> 90 <212> PRT <213> Artificial Sequence <220> <223> synthetic sequence <400> 59 Met Leu Pro Ala Pro Lys Asn Leu Val Val Ser Arg Val Thr Glu Asp 1 5 10 15 Ser Ala Arg Leu Ser Trp Thr Ala Pro Asp Ala Ala Phe Asp Ser Phe 20 25 30 Asn Ile Ala Tyr Trp Glu Pro Gly Ile Ala Gly Glu Ala Ile Trp Leu 35 40 45 Arg Val Pro Gly Ser Glu Arg Ser Tyr Asp Leu Thr Gly Leu Lys Pro 50 55 60 Gly Thr Glu Tyr Lys Val Trp Ile His Gly Val Lys Gly Gly Ala Ser 65 70 75 80 Ser Pro Pro Leu Ile Ala Arg Phe Thr Thr 85 90 <210> 60 <211> 90 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 60 Met Leu Pro Ala Pro Lys Asn Leu Val Val Ser Arg Val Thr Glu Asp 1 5 10 15 Ser Ala Arg Leu Ser Trp Thr Ala Pro Asp Ala Ala Phe Asp Ser Phe 20 25 30 Asn Ile Ala Tyr Trp Glu Pro Gly Ile Gly Gly Glu Ala Ile Ala Leu 35 40 45 Arg Val Pro Gly Ser Glu Arg Ser Tyr Asp Leu Thr Gly Leu Lys Pro 50 55 60 Gly Thr Glu Tyr Lys Val Trp Ile His Gly Val Lys Gly Gly Ala Ser 65 70 75 80 Ser Pro Pro Leu Ile Ala Arg Phe Thr Thr 85 90 <210> 61 <211> 90 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 61 Met Leu Pro Ala Pro Lys Asn Leu Val Val Ser Arg Val Thr Glu Asp 1 5 10 15 Ser Ala Arg Leu Ser Trp Thr Ala Pro Asp Ala Ala Phe Asp Ser Phe 20 25 30 Asn Ile Ala Tyr Trp Glu Pro Gly Ile Gly Gly Glu Ala Ile Trp Leu 35 40 45 Ala Val Pro Gly Ser Glu Arg Ser Tyr Asp Leu Thr Gly Leu Lys Pro 50 55 60 Gly Thr Glu Tyr Lys Val Trp Ile His Gly Val Lys Gly Gly Ala Ser 65 70 75 80 Ser Pro Pro Leu Ile Ala Arg Phe Thr Thr 85 90 <210> 62 <211> 90 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 62 Met Leu Pro Ala Pro Lys Asn Leu Val Val Ser Arg Val Thr Glu Asp 1 5 10 15 Ser Ala Arg Leu Ser Trp Thr Ala Pro Asp Ala Ala Phe Asp Ser Phe 20 25 30 Asn Ile Ala Tyr Trp Glu Pro Gly Ile Gly Gly Glu Ala Ile Trp Leu 35 40 45 Arg Val Pro Gly Ser Glu Arg Ser Tyr Asp Leu Thr Gly Leu Lys Pro 50 55 60 Gly Thr Glu Tyr Ala Val Trp Ile His Gly Val Lys Gly Gly Ala Ser 65 70 75 80 Ser Pro Pro Leu Ile Ala Arg Phe Thr Thr 85 90 <210> 63 <211> 90 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 63 Met Leu Pro Ala Pro Lys Asn Leu Val Val Ser Arg Val Thr Glu Asp 1 5 10 15 Ser Ala Arg Leu Ser Trp Thr Ala Pro Asp Ala Ala Phe Asp Ser Phe 20 25 30 Asn Ile Ala Tyr Trp Glu Pro Gly Ile Gly Gly Glu Ala Ile Trp Leu 35 40 45 Arg Val Pro Gly Ser Glu Arg Ser Tyr Asp Leu Thr Gly Leu Lys Pro 50 55 60 Gly Thr Glu Tyr Lys Val Ala Ile His Gly Val Lys Gly Gly Ala Ser 65 70 75 80 Ser Pro Pro Leu Ile Ala Arg Phe Thr Thr 85 90 <210> 64 <211> 90 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 64 Met Leu Pro Ala Pro Lys Asn Leu Val Val Ser Arg Val Thr Glu Asp 1 5 10 15 Ser Ala Arg Leu Ser Trp Thr Ala Pro Asp Ala Ala Phe Asp Ser Phe 20 25 30 Asn Ile Ala Tyr Trp Glu Pro Gly Ile Gly Gly Glu Ala Ile Trp Leu 35 40 45 Arg Val Pro Gly Ser Glu Arg Ser Tyr Asp Leu Thr Gly Leu Lys Pro 50 55 60 Gly Thr Glu Tyr Lys Val Trp Ile Ala Gly Val Lys Gly Gly Ala Ser 65 70 75 80 Ser Pro Pro Leu Ile Ala Arg Phe Thr Thr 85 90 <210> 65 <211> 90 <212> PRT <213> Artificial Sequence <220> <223> Synthetic sequence <400> 65 Met Leu Pro Ala Pro Lys Asn Leu Val Val Ser Arg Val Thr Glu Asp 1 5 10 15 Ser Ala Arg Leu Ser Trp Thr Ala Pro Asp Ala Ala Phe Asp Ser Phe 20 25 30 Asn Ile Ala Tyr Trp Glu Pro Gly Ile Gly Gly Glu Ala Ile Trp Leu 35 40 45 Arg Val Pro Gly Ser Glu Arg Ser Tyr Asp Leu Thr Gly Leu Lys Pro 50 55 60 Gly Thr Glu Tyr Lys Val Trp Ile His Gly Val Lys Gly Gly Ser Ser 65 70 75 80 Ser Pro Pro Leu Ile Ala Arg Phe Thr Thr 85 90 <210> 66 <211> 90 <212> PRT <213> Artificial Sequence <220> <223> Synthetic sequence <400> 66 Met Leu Pro Ala Pro Lys Asn Leu Val Val Ser Arg Val Thr Glu Asp 1 5 10 15 Ser Ala Arg Leu Ser Trp Thr Ala Pro Asp Ala Ala Phe Asp Ser Phe 20 25 30 Asn Ile Ala Tyr Trp Glu Pro Gly Ile Gly Gly Glu Ala Ile Trp Leu 35 40 45 Arg Val Pro Gly Ser Glu Arg Ser Tyr Asp Leu Thr Gly Leu Lys Pro 50 55 60 Gly Thr Glu Tyr Lys Val Trp Ile His Gly Val Lys Gly Gly Ala Ala 65 70 75 80 Ser Pro Pro Leu Ile Ala Arg Phe Thr Thr 85 90 <210> 67 <211> 90 <212> PRT <213> Artificial Sequence <220> <223> Synthetic sequence <400> 67 Met Leu Pro Ala Pro Lys Asn Leu Val Val Ser Arg Val Thr Glu Asp 1 5 10 15 Ser Ala Arg Leu Ser Trp Thr Ala Pro Asp Ala Ala Phe Asp Ser Phe 20 25 30 Asn Ile Ala Tyr Trp Glu Pro Gly Ile Gly Gly Glu Ala Ile Trp Leu 35 40 45 Arg Val Pro Gly Ser Glu Arg Ser Tyr Asp Leu Thr Gly Leu Lys Pro 50 55 60 Gly Thr Glu Tyr Lys Val Trp Ile His Gly Val Lys Gly Gly Ala Ser 65 70 75 80 Ser Ala Pro Leu Ile Ala Arg Phe Thr Thr 85 90 <210> 68 <211> 90 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 68 Met Leu Pro Ala Pro Lys Asn Leu Val Val Ser Arg Val Thr Glu Asp 1 5 10 15 Ser Ala Arg Leu Ser Trp Thr Ala Pro Asp Ala Ala Phe Asp Ser Phe 20 25 30 Asn Ile Ala Tyr Trp Glu Pro Gly Ile Gly Gly Glu Ala Ile Trp Leu 35 40 45 Arg Val Pro Gly Ser Glu Arg Ser Tyr Asp Leu Thr Gly Leu Lys Pro 50 55 60 Gly Thr Glu Tyr Lys Val Trp Ile His Gly Val Lys Gly Gly Ala Ser 65 70 75 80 Ser Pro Pro Leu Ala Ala Arg Phe Thr Thr 85 90 <210> 69 <211> 90 <212> PRT <213> Artificial Sequence <220> <223> synthetic sequence <400> 69 Met Leu Pro Ala Pro Lys Asn Leu Val Val Ser Arg Val Thr Glu Asp 1 5 10 15 Ser Ala Arg Leu Ser Trp Thr Ala Pro Asp Ala Ala Phe Asp Ser Phe 20 25 30 Asn Ile Ala Tyr Trp Glu Pro Gly Ile Gly Gly Glu Ala Ile Trp Leu 35 40 45 Arg Val Pro Gly Ser Glu Arg Ser Tyr Asp Leu Thr Gly Leu Lys Pro 50 55 60 Gly Thr Glu Tyr Lys Val Trp Ile His Gly Val Lys Gly Gly Ala Ser 65 70 75 80 Ser Pro Pro Leu Ile Ala Ala Phe Thr Thr 85 90 <210> 70 <211> 89 <212> PRT <213> Artificial Sequence <220> <223> synthetic sequence <400> 70 Leu Pro Ala Pro Lys Asn Leu Val Val Ser Arg Val Thr Glu Asp Ser 1 5 10 15 Ala Arg Leu Ser Trp Thr Ala Pro Asp Ala Ala Phe Asp Ser Phe His 20 25 30 Ile Glu Tyr Trp Glu Gln Ser Ile Val Gly Glu Ala Ile Val Leu Thr 35 40 45 Val Pro Gly Ser Glu Arg Ser Tyr Asp Leu Thr Gly Leu Lys Pro Gly 50 55 60 Thr Glu Tyr Arg Val Trp Ile Tyr Gly Val Lys Gly Gly Asn Asp Ser 65 70 75 80 Trp Pro Leu Ser Ala Ile Phe Thr Thr 85 <210> 71 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 71 Gly Ser Gly Ser 1 <210> 72 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 72 Gly Gly Gly Ser Gly Gly Gly Ser 1 5 <210> 73 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> synthetic sequence <400> 73 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser Gly Gly Gly Gly Ser 20 25 <210> 74 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> synthetic sequence <400> 74 Ala Pro Ala Pro 1 <210> 75 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> synthetic sequence <400> 75 Ala Pro Ala Pro Ala Pro Ala Pro Ala Pro 1 5 10 <210> 76 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> synthetic sequence <400> 76 Ala For Ala For Ala For Ala For Ala For Ala For Ala For Ala For 1 5 10 15 Ala Pro Ala Pro 20 <210> 77 <211> 40 <212> PRT <213> Artificial Sequence <220> <223> synthesis sequence <400> 77 Ala For Ala For Ala For Ala For Ala For Ala For Ala For Ala For 1 5 10 15 Ala For Ala For Ala For Ala For Ala For Ala For Ala For Ala For 20 25 30 Ala Pro Ala Pro Ala Pro Ala Pro 35 40 <210> 78 <211> 29 <212> PRT <213> Artificial Sequence <220> <223> synthesis sequence <400> 78 Ala Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys 1 5 10 15 Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Ala Ala Ala 20 25 <210> 79 <211> 608 <212> PRT <213> Homo sapiens <400> 79 Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala Tyr 1 5 10 15 Ser Arg Gly Val Phe Arg Arg Asp Ala His Lys Ser Glu Val Ala His 20 25 30 Arg Phe Lys Asp Leu Gly Glu Glu Asn Phe Lys Ala Leu Val Leu Ile 35 40 45 Ala Phe Ala Gln Tyr Leu Gln Gln Cys Pro Phe Glu Asp His Val Lys 50 55 60 Leu Val Asn Glu Val Thr Glu Phe Ala Lys Thr Cys Val Ala Asp Glu 65 70 75 80 Ser Ala Glu Asn Cys Asp Lys Ser Leu His Thr Leu Phe Gly Asp Lys 85 90 9� Leu Cys Thr Val Ala Thr Leu Arg Glu Thr Tyr Gly Glu Met Ala Asp 100 105 110 Cys Cys Ala Lys Gln Glu Pro Glu Arg Asn Glu Cys Phe Leu Gln His 115 120 125 Lys Asp Asp Asn Pro Asn Leu Pro Arg Leu Val Arg Pro Glu Val Asp 130 135 140 Val Met Cys Thr Ala Phe His Asp Asn Glu Glu Thr Phe Leu Lys Lys 145 150 155 160 Tyr Leu Tyr Glu Ile Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro Glu 165 170 175 Thr Glu Cys Cys 180 185 190 Gln Ala Asp Lys Ala Cys Ala Leu Leu Pro Lys Leu Asp Glu Leu 195 200 205 Arg Asp Glu Gly Lys Ser Ser Ala Lys Ala Gln Arg Leu Lys Cys Ala 210 215 220 Ser Leu Gln Lys Phe Gly Glu Arg Ala Phe Lys Ala Trp Ala Val Ala 225 230 235 240 Arg Leu Ser Gln Arg Phe Pro Lys Ala Glu Phe Ala Glu Val Ser Lys 245 250 255 Leu Val Thr Asp Leu Thr Lys Val His Thr Glu Cys Cys His Gly Asp 260 265 270 Leu Leu Glu Cys Ala Asp Arg Ala Asp Leu Ala Lys Tyr Ile Cys 275 280 285 Glu Asn Gln Asp Ser Ile Ser Ser Lys Leu Lys Glu Cys Glu Lys 290,295,300 Pro Leu Leu Glu Lys Ser His Cys Ile Ala Glu Val Glu Asn Asp Glu 305 310 315 320 Met Pro Ala Asp Leu Pro Ser Leu Ala Ala Asp Phe Val Glu Ser Lys 325 330 335 Asp Val Cys Lys Asn Tyr Ala Glu Ala Lys Asp Val Phe Leu Gly Met 340 345 350 Phe Leu Tyr Glu Tyr Ala Arg Arg His Pro Asp Tyr Ser Val Val Leu 355 360 365 From Arg to Lys Thr Tyr Glu Thr Thr from Lys Cys Cys Glu 370 375 380 Ala Ala Ala Asp Pro His Glu Cys Tyr Ala Lys Val Phe Asp Glu Phe 385 390 395 400 Lys Pro Leu Val Glu Glu Pro Gln Asn Leu And Lys Gln Asn Cys Glu 405 410 415 Leu Phe Glu Gln Leu Gly Glu Tyr Lys Phe Gln Asn Ala Leu Leu Val 420 425 430 Arg Tyr Thr Lys Lys Val Pro Gln Val Ser Thr Pro Thr Leu Val Glu 435 440 445 Val Ser Arg Asn Leu Gly Lys Val Gly Ser Lys Cys Cys Lys His Pro 450 455 460 Glu Ala Lys Arg Met Pro Cys Ala Glu Asp Tyr Leu Ser Val Val Leu 465 470 475 480 Asn Gln Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Asp Arg Val 485 490 495 Thr Lys Cys Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe Ser 500 505 510 Ala Leu Glu Val Asp Glu Thr Tyr Val Pro Lys Glu Phe Asn Ala Glu 515 520 525 Thr Phe Thr Phe His Ala Asp Ile Cys Thr Leu Ser Glu Lys Glu Arg 530 535 540 Gln Ile Lys Lys Gln Thr Ala Leu Val Glu Leu Val Lys His Lys Pro 545 550 555 560 Lys Ala Thr Lys Glu Gln Leu Lys Ala Val Met Asp Asp Phe Ala Ala 565 570 575 Phe Val Glu Lys Cys Cys Lys Ala Asp Asp Lys Glu Thr Cys Phe Ala 580 585 590 Glu Glu Gly Lys Lys Leu Val Ala Ala Ser Gln Ala Ala Leu Gly Leu 595 600 605 <210> 80 <211> 607 <212> PRT <213> Cynomolgus monkey (Macaca fascicularis) <400> 80 Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala Tyr 1 5 10 15 Ser Arg Gly Val Phe Arg Arg Asp Thr His Lys Ser Glu Val Ala His 20 25 30 Arg Phe Lys Asp Leu Gly Glu Glu His Phe Lys Gly Leu Val Leu Val 35 40 45 Ala Phe Ser Gln Tyr Leu Gln Gln Cys Pro Phe Glu Glu His Val Lys 50 55 60 Leu Val Asn Glu Val Thr Glu Phe Ala Lys Thr Cys Val Ala Asp Glu 65 70 75 80 Ser Ala Glu Asn Cys Asp Lys Ser Leu His Thr Leu Phe Gly Asp Lys 85 90 95 Leu Cys Thr Val Ala Thr Leu Arg Glu Thr Tyr Gly Glu Met Ala Asp 100 105 110 Cys Cys Ala Lys Gln Glu Pro Glu Arg Asn Glu Cys Phe Leu Gln His 115 120 125 Lys Asp Asp Asn Pro Asn Leu Pro Pro Leu Val Arg Pro Glu Val Asp 130 135 140 Val Met Cys Thr Ala Phe His Asp Asn Glu Ala Thr Phe Leu Lys Lys 145 150 155 160 Tyr Leu Tyr Glu Val Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro Glu 165 170 175 Leu Leu Phe Phe Ala Arg Tyr Lys Ala Phe Ala Glu Cys Cys 180 185 190 Gln Ala Asp Lys Ala Cys Ala Leu Leu Pro Lys Leu Asp Glu Leu 195 200 205 Arg Asp Glu Gly Lys Ser Ser Ala Lys Ala Gln Arg Leu Lys Cys Ala 210 215 220 Ser Leu Gln Lys Phe Gly Asp Arg Ala Phe Lys Ala Trp Ala Val Ala 225 230 235 240 Arg Leu Ser Gln Lys Phe Pro Lys Ala Glu Phe Ala Glu Val Ser Lys 245 250 255 Leu Val Thr Asp Leu Thr Lys Val His Thr Glu Cys Cys His Gly Asp 260 265 270 Leu Leu Glu Cys Ala Asp Arg Ala Asp Leu Ala Lys Tyr Met Cys 275 280 285 Glu Asn Gln Asp Ser Ile Ser Ser Lys Leu Lys Glu Cys Asp Lys 290,295,300 Pro Leu Leu Glu Lys Ser His Cys Leu Ala Glu Val Glu Asn Asp Glu 305 310 315 320 Met Pro Ala Asp Leu Pro Ser Leu Ala Ala Asp Tyr Val Glu Ser Lys 325 330 335 Asp Val Cys Lys Asn Tyr Ala Glu Ala Lys Asp Val Phe Leu Gly Met 340 345 350 Phe Leu Tyr Glu Tyr Ala Arg Arg His Pro Asp Tyr Ser Val Met Leu 355 360 365 Arg and Lys and Tyr Glu and Thr and Lys Cys Cys 370 375 380 Ala Ala Ala Asp Pro His Glu Cys Tyr Ala Lys Val Phe Asp Glu Phe 385 390 395 400 Gln Pro Leu Will Glu Glu Pro Gln Asn Leu Will Lys Gln Asn Cys Glu 405 410 415 Leu Phe Glu Gln Leu Gly Glu Tyr Lys Phe Gln Asn Ala Leu Leu Val 420 425 430 Arg Tyr Thr Lys Lys Val Pro Gln Val Ser Thr Pro Thr Leu Val Glu 435 440 445 Val Ser Arg Asn Leu Gly Lys Val Gly Ala Lys Cys Cys Lys Leu Pro 450 455 460 Glu Ala Lys Arg Met Pro Cys Ala Glu Asp Tyr Leu Ser Val Val Leu 465 470 475 480 Asn Arg Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Glu Lys Val 485,490,495 Thr Lys Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe Ser 500 505 510 Ala Leu Glu Leu Asp Glu Ala Tyr Val Pro Lys Ala Phe Asn Ala Glu 515,520,525 Thr Phe Thr Phe His Ala Asp Met Cys Thr Leu Ser Glu Lys Glu Lys 530 535 540 Gln Val Lys Lys Gln Thr Ala Leu Val Glu Leu Val Lys Lys Pro 545 550 555 560 Lys Ala Thr Lys Glu Gln Leu Lys Gly Val Met Asp Asn Phe Ala Ala 565,570,575 Phe Val Glu Lys Cys Lys Ala Asp Asp Lys Glu Ala Cys Phe Ala 580,585,590 Glu Glu Gly Pro Lys Phe Val Ala Ser Gln Ala Ala Leu Ala 595,600,605 <210> 81 <211> 88 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 81 Asp Ala Pro Ser Gln Ile Glu Val Lys Asp Val Thr Asp Thr Thr Ala 1 5 10 15 Leu Ile Thr Trp Phe Lys Pro Leu Ala Glu Ile Asp Gly Ile Glu Leu 20 25 30 Thr Tyr Gly Ile Lys Asp Val Pro Gly Asp Arg Thr Thr Ile Asp Leu 35 40 45 Thr Glu Asp Glu Asn Gln Tyr Ser Ile Gly Asn Leu Lys Pro Asp Thr 50 55 60 Glu Tyr Glu Val Ser Leu Ile Ser Arg Arg Gly Asp Met Ser Ser Asn 65 70 75 80 Pro Ala Lys Glu Thr Phe Thr Thr 85 <210> 82 <211> 89<212> PRT <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 82 Leu Asp Ala Pro Thr Asp Leu Gln Val Thr Asn Val Thr Asp Thr Ser 1 5 10 15 Ile Thr Val Ser Trp Thr Pro Pro Ser Ala Thr Ile Thr Gly Tyr Arg ​20 25 30 Ile Thr Tyr Thr Pro Ser Asn Gly Pro Gly Glu Pro Lys Glu Leu Thr 35 40 45 Val Pro Pro Ser Ser Thr Ser Val Thr Ile Thr Gly Leu Thr Pro Gly 50 55 60 Val Glu Tyr Val Val Ser Leu Tyr Ala Leu Lys Asp Asn Gln Glu Ser 65 70 75 80 Pro Pro Leu Val Gly Thr Gln Thr Thr 85 <210> 83 <211> 94 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Sequence <400> 83 Val Ser Asp Val Pro Arg Asp Leu Glu Val Val Ala Ala Thr Pro Thr 1 5 10 15 Ser Leu Leu Ile Ser Trp Asp Ala Pro Ala Val Thr Val Arg Tyr Tyr 20 25 30 Arg Ile Thr Tyr Gly Glu Thr Gly Gly Asn Ser Pro Val Gln Glu Phe 35 40 45 Thr Val Pro Gly Ser Lys Ser Thr Ala Thr Ile Ser Gly Leu Lys Pro 50 55 60 Gly Val Asp Tyr Thr Ile Thr Val Tyr Ala Val Thr Gly Arg Gly Asp 65 70 75 80 Ser Pro Ala Ser Ser Lys Pro Ile Ser Ile Asn Tyr Arg Thr 85 90

Claims

1. A protein that binds to albumin, comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 51, 52, 54, 56, 57, 58, 59, 61, 62, 66, 67 and 69.

2. The protein of claim 1, wherein the protein is linked to other molecules.

3. The protein of claim 2, wherein the other molecule is a drug, protein, polymer, or toxin.

4. The protein of claim 2, wherein the other molecule is an antibody.

5. The protein of claim 2, wherein the other molecule is an FN3 domain that binds to molecules other than human albumin.

6. The protein of claim 5, wherein the FN3 domain binds to CD71.

7. The protein of any one of claims 2-6, wherein the protein is linked to the other molecules via a connector.

8. The protein of claim 7, wherein the linker is a peptide linker, the peptide linker comprising a subset selected from (GS)2 (SEQ ID NO:71), (GGGS)2 (SEQ ID NO:72), (GGGGS)5 (SEQ ID NO:73), (AP)2 (SEQ ID NO:74), (AP)5 (SEQ ID NO:75), and (AP). 10 (SEQ ID NO:76), (AP) 20 The sequences (SEQ ID NO:77) and A(EAAAK)5AAA(SEQ ID NO:78).

9. An isolated polynucleotide encoding the protein of any one of claims 1-8.

10. A vector comprising the polynucleotide of claim 9.

11. An isolated host cell comprising the vector of claim 10.

12. A pharmaceutical composition comprising the protein of any one of claims 1-8 and a pharmaceutically acceptable carrier.

Images