A novel fibronectin type III domain that binds serum albumin

Abstract

The present invention provides novel fibronectin type III domains that bind serum albumin, and specifically relates to polypeptides comprising the tenth fibronectin type III domain (<supgt;10< / supgt;Fn3) with Antarctic loop substitutions, which polypeptides bind to serum albumin. The present invention further relates to fusion molecules comprising <supgt;10< / supgt;Fn3 that binds serum albumin linked to a heterologous protein for use in diagnostic or therapeutic applications.

Description

[0001] This application claims to be a divisional application of the patent application with application number 201580014557.3 (PCT international application number PCT / US2015 / 021535) filed on March 19, 2015 and invention name “Fibronectin type III domain binding to serum albumin”. background

[0002] The insufficient half-life of therapeutic agents often forces people to administer them at high frequencies and / or higher doses, or use sustained-release formulations, in order to maintain the serum levels necessary for efficacy. However, this is often accompanied by negative side effects. For example, frequent systemic injections cause considerable discomfort to the subject and create a high risk of administration-related infections, and may require hospitalization or frequent visits to the hospital, especially when the therapeutic agent needs to be administered intravenously. Moreover, in long-term treatment, daily intravenous injections can also lead to considerable side effects such as tissue scarring and vascular lesions caused by repeated vascular punctures. It is known that all frequent systemic administration of therapeutic agents has similar problems, such as administering insulin to diabetic patients or administering interferon drugs to patients suffering from multiple sclerosis. All of these factors lead to reduced patient compliance and increased costs for the medical system.

[0003] The application provides compounds that increase the serum half-life of various therapeutic agents, compounds with increased serum half-life, and methods for increasing the serum half-life of therapeutic agents. Such compounds and methods for increasing the serum half-life of therapeutic agents can be manufactured or implemented in a cost-effective manner, have ideal biophysical properties (e.g., Tm, substantially monomeric, or well folded), and are small enough to allow tissue penetration.

[0004] Overview

[0005] The present invention is based, at least in part, on the discovery of novel Adnectins (PKE2 Adnectins) that are south pole based, serum albumin binding, and contain the tenth fibronectin type III domain ( 10 Fn3), compared with the existing Arctic-based serum albumin binding 10 Adnectins with Fn3 domains may provide enhanced properties.

[0006] In one aspect, the present invention provides a 10 A polypeptide comprising a Fn3 domain, wherein 10 The Fn3 domain contains: a) AB, BC, CD, DE, EF, and FG loops, b) has 10The polypeptide comprises a CD loop having an amino acid sequence that is altered from the corresponding CD loop sequence of an Fn3 domain, and c) wherein the polypeptide has a K of less than 500 nM D Binds to human serum albumin.

[0007] In certain embodiments, 10 The Fn3 domain further binds to one or more of rhesus serum albumin, cynomolgus serum albumin, mouse serum albumin, and rat serum albumin. For example, 10 The Fn3 domain can bind to HSA, rhesus serum albumin, and cynomolgus serum albumin, or, 10 The Fn3 domain can bind to HSA, rhesus serum albumin, cynomolgus serum albumin, mouse serum albumin, and rat serum albumin. In some embodiments, 10 The Fn3 domain has a K of less than 500 nM. D , for example, a K of less than 100 nM D , or even less than 10 nM K D In some embodiments, 10 The Fn3 domain binds to serum albumin in the pH range of 5.5 to 7.4.

[0008] In certain embodiments, 10 The Fn3 domain binds to domains I-II of HSA.

[0009] In certain embodiments, in the presence of human serum albumin, the 10 The Fn3 domain polypeptide has a serum half-life of at least 10 hours, such as at least 20 hours, or at least 30 hours.

[0010] In certain embodiments, the CD ring comprises a 1 -X 2 -VX 3 -X 4 -X 5 -SX 6 -X 7 -GX 8 -X 9 -YX 10 -X 11 -X 12 -E (SEQ ID NO: 170), wherein

[0011] (a)X 1 Selected from R or W;

[0012] (b)X 2 Selected from H, E, D, Y, or Q;

[0013] (c)X 3 Select from Q or H;

[0014] (d)X 4 is selected from I, K, M, Q, L, or V;

[0015] (e)X 5 Selected from Y, F, or N;

[0016] (f)X 6 Selected from D, V, or E;

[0017] (g)X 7 Selected from L, W, or F;

[0018] (h)X 8 Select from P or T;

[0019] (i)X 9 Select from L or M;

[0020] (j)X 10 Selected from I or V;

[0021] (k)X 11 is selected from Y or F; and

[0022] (l)X 12 Selected from T, S, Q, N, or A.

[0023] In a preferred embodiment, (a) X 1 is R; (b) X 2 is E; (c) X 3 is Q; (d) X 4 is K; (e) X 5 is Y; (f) X 6 is D; (g) X 7 is L or W; (h) X 8 is P; (i) X 9 is L; (j) X 10 is I; (k) X 11 is Y; and (l) X 12 It is Q or N.

[0024] In yet another preferred embodiment, (a) X 1 is R; (b) X 2 is E; (c) X 3 is Q; (d) X 4 is K; (e) X 5 is Y; (f) X 6 is D; (g) X 7 is L; (h)X 8 is P; (i) X 9is L; (j) X 10 is I; (k) X 11 is Y; and (l) X 12 It's Q.

[0025] In yet another preferred embodiment, (a) X 1 is R; (b) X 2 is E; (c) X 3 is Q; (d) X 4 is K; (e) X 5 is Y; (f) X 6 is D; (g) X 7 is W; (h) X 8 is P; (i) X 9 is L; (j) X 10 is I; (k) X 11 is Y; and (l) X 12 It is N.

[0026] In certain embodiments, the CD loop comprises an amino acid sequence selected from SEQ ID NOs: 101-125. In a preferred embodiment, the CD loop comprises the amino acid sequence set forth in SEQ ID NOs: 106 or 113.

[0027] In certain embodiments, the present invention provides a 10 Fn3 domain polypeptide, the 10 The Fn3 domain comprises: (i) a CD loop comprising an amino acid sequence having the consensus sequence SEQ ID NO: 170, or an amino acid sequence of any one of SEQ ID NOs: 101-125; and (ii) an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to the non-CD loop regions of SEQ ID NOs: 23-100, 184-209 and 235-260, or that differs from one of SEQ ID NOs: 23-100, 184-209 and 235-260 in at most 1, 1-2, 1-5, 1-10 or 1-20 amino acids. In certain embodiments, the polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% different from any one of SEQ ID NOs: 23-100, 184-209 and 235-260, or has at most 1, 1-2, 1-5, 1-10 or 1-20 amino acid differences from one of SEQ ID NOs: 23-100, 184-209 and 235-260. The amino acid differences may be substitutions, additions or deletions.

[0028] In certain aspects, the present invention provides a method comprising a fibronectin type III tenth ( 10Fn3) domain and a fusion polypeptide of a heterologous protein, wherein 10 The Fn3 domain contains: a) AB, BC, CD, DE, EF, and FG loops, b) has 10 The polypeptide comprises a CD loop having an amino acid sequence that is altered from the corresponding CD loop sequence of an Fn3 domain, and c) wherein the polypeptide has a K of less than 500 nM D Binds to human serum albumin.

[0029] In certain embodiments, the fusion polypeptide comprises an albumin binding Adnectin comprising an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of SEQ ID NOs: 23-100, 184-209 and 235-260, or that differs from one of SEQ ID NOs: 23-100, 184-209 and 235-260 in at most 1, 1-2, 1-5, 1-10 or 1-20 amino acids. In a preferred embodiment, the fusion polypeptide comprises an albumin binding Adnectin comprising the amino acid sequence of SEQ ID NOs: 55, 81, 190 or 241. In yet another preferred embodiment, the fusion polypeptide comprises an albumin binding Adnectin comprising the amino acid sequence of SEQ ID NO: 62, 88, 197 or 248.

[0030] In certain embodiments, the fusion polypeptide comprises an albumin binding Adnectin and a heterologous moiety, wherein the heterologous moiety is a therapeutic moiety.

[0031] In certain embodiments, the heterologous protein comprises 10 In some embodiments, the 10 The Fn3 domain binds to a target protein other than serum albumin. 10 The Fn3 domain binds to PCSK9 (i.e., a PCSK9 Adnectin) and comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 167, or that differs from SEQ ID NO: 167 in at most 1, 1-2, 1-5, 1-10, or 1-20 amino acids.

[0032] In certain embodiments, the fusion polypeptide is a PCSK9-PKE2 tandem Adnectin comprising an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 168, 169 or 261, or that differs from the amino acid sequence of one of SEQ ID NO: 168, 169 or 261 in at most 1, 1-2, 1-5, 1-10 or 1-20 amino acids.

[0033] In certain embodiments, the serum half-life of the fusion polypeptide in the presence of mouse serum albumin is at least 10 hours. In some embodiments, the serum half-life of the fusion polypeptide in the presence of cynomolgus monkey serum albumin is at least 50 hours. In certain embodiments, the serum half-life of the fusion polypeptide in the presence of mouse or cynomolgus monkey serum albumin is 10-100 hours, such as 10-90 hours, 10-80 hours, 10-70 hours, 10-60 hours, 10-50 hours, 10-40 hours, 10-30 hours, 10-20 hours, 50-100 hours, 60-100 hours, 70-100 hours, 80-100 hours, 90-100 hours, 20-90 hours, 30-80 hours, 40-70 hours, or 50-60 hours.

[0034] In certain aspects, the invention provides a PKE2 Adnectin or a PCSK9-PKE2 tandem Adnectin comprising an amino acid sequence selected from SEQ ID NO: 23-100, 168, 169, 184-209, 235-260, and 261.

[0035] In certain aspects, the invention provides a composition comprising: any albumin binding Adnectin as described herein or a fusion protein comprising such an Adnectin as described herein, and a carrier.

[0036] In certain aspects, the invention provides isolated nucleic acid molecules encoding any of the albumin binding Adnectins or fusion proteins containing the Adnectins as described herein (e.g., those set forth in SEQ ID NOs: 126-151 and 172), vectors encoding these nucleic acid molecules, and cells comprising these nucleic acid molecules. Also provided are nucleic acids comprising a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to any of these nucleotide sequences described herein, or differs therefrom by at most 1-5, 1-10, 1-50, or 1-100 nucleotides.

[0037] In certain aspects, the invention provides methods of producing an albumin binding Adnectin as described herein, or a fusion protein containing such an Adnectin, the method comprising culturing a cell comprising a nucleic acid molecule encoding the protein under conditions suitable for expression of the Adnectin or fusion protein, and purifying the protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 A schematic diagram of the competitive alpha screen assay described in Example 6 is shown.

[0039] Figure 2 is a line graph depicting competition of various Adnectins with the human FcRn receptor for binding to human serum albumin (HSA).

[0040] Figure 3 is a line graph depicting the plasma half-life of 2629_E06 and 2630_D02 PKE2 Adnectins in WT mice.

[0041] Figure 4 is a graph depicting T cell proliferation results for the percent antigenicity and the magnitude of the proliferative response for the 2629_E06 and 2630_D02 Adnectins, and the parental 2270_C01 molecule.

[0042] Figure 5 Depicted is a comparison of the modularity of tandem Adnectins. Adnectin 1318_H04 corresponds to a north pole-based serum albumin binding Adnectin. "X" refers to the configuration of a non-PKE target-specific Adnectin (i.e., myostatin; "myo", or PCSK9). The figure below depicts the EC corresponding to binding to HSA as determined by direct binding ELISA in each box. 50 Gray shaded icons for tandem:single Adnectin ratios (i.e., the darker the gray shade, the stronger the binding to HSA).

[0043] Figure 6 Bio-Layer Interferometry sensorgrams of the binding of PCSK9-PKE2 tandem Adnectin to hPCSK9 in the presence or absence of HSA are shown.

[0044] Figure 7 is a Biacore sensorgram showing the binding of 4472_C06 PCSK9-PKE2 tandem Adnectin first to HSA and then to PCSK9 after injection of the corresponding proteins.

[0045] Figure 8is a line graph depicting the in vivo PK profile of the tandem PCSK9-PKE2 Adnectin 4772_C06 in wild-type C57 Bl / 6 mice.

[0046] Fig. 9 Shown are free PCSK9 levels following administration of vehicle or 0.5 mg / kg or 2 mg / kg of PCSK9-PKE2 Adnectin 4472_C06 in hPCSK9 transgenic mice.

[0047] Fig.10 is a line graph showing the plasma PK profile and half-life of PKE2 Adnectin 2629_E06, PCSK9-PKE2 tandem 5190_E01 Adnectin, and PEGylated PCSK9 in cynomolgus monkeys.

[0048] Fig.11 is a line graph showing the plasma half-life of PKE2 Adnectin 2270_C01 in cynomolgus monkeys.

[0049] Fig.12 is a line graph showing the pharmacodynamic profile of LDL-c and PCSK9 in cynomolgus monkeys following administration of PCSK9-PKE2 tandem Adnectin 5190_E01 to cynomolgus monkeys. The profile demonstrated robust reductions in LDL-c, inhibition of free PCSK9, and increases in total PCSK9, all of which returned to baseline at the end of the study.

[0050] Fig.13 is a line graph showing the LDL-c lowering effect of PCSK9-PKE2 tandem Adnectin 5190_E01 and PEGylated PCSK9 Adnectin reference, along with 2629_E06 PKE2 control in cynomolgus monkeys.

[0051] Fig.14 Shown are target engagement of two different concentrations of the tandem PCSK9-PKE2 Adnectin in cynomolgus monkeys, compared to PEGylated PCSK9 Adnectin and PKE2 Adnectin 2629_E06.

[0052] Fig.15 Shown are total PCSK9 levels over time following administration of tandem PCSK9-PKE2 Adnectin, PEGylated PCSK9 Adnectin, or PKE2 Adnectin 2629_E06 in cynomolgus monkeys.

[0053] Fig.16Is a line graph depicting T cell proliferation results for the percentage and intensity of proliferation responses for PCSK9-PKE2 tandem Adnectins 4472_F08, 4472_E06, and 4472_C06, as well as component PKE2 Adnectin 2629_E06 and component PCSK9 Adnectin 2382_D09. The leftmost bars of the graph correspond to control proteins with low, medium, and high antigenicity.

[0054] Fig.17 The amino acid sequences of the PKE2 Adnectins described herein are shown.

[0055] Figures 18A-18C The nucleic acid sequences of the PKE2 Adnectins and PCSK9-PKE2 tandem Adnectins described herein are shown. DETAILED DESCRIPTION OF THE INVENTION

[0056] definition

[0057] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by a skilled person. Preferred methods and compositions are described below, but any methods and compositions similar to or equivalent to those described herein can be used in the practice or testing of the present invention.

[0058] As used herein, "polypeptide" refers to any sequence of two or more amino acids, regardless of length, post-translational modification, or function. "Polypeptide", "peptide", and "protein" are used interchangeably herein. Polypeptides may include natural amino acids and non-natural amino acids, such as those described in U.S. Patent No. 6,559,126, which is incorporated herein by reference. Polypeptides may also be modified using any of a variety of standard chemical methods (e.g., amino acids may be modified with protecting groups; carboxyl terminal amino acids may be made into terminal amide groups; amino terminal residues may be modified with groups, for example, to enhance lipophilicity; or, polypeptides may be chemically glycosylated or otherwise modified to increase stability or in vivo half-life). Polypeptide modifications may include attachment of another structure such as a cyclic compound or other molecule to the polypeptide, and may also include polypeptides containing one or more amino acids in an altered configuration (i.e., R or S; or, L or D).

[0059] As used herein, "polypeptide chain" refers to a polypeptide in which each domain is linked to other domains by peptide bonds, rather than non-covalent interactions or disulfide bonds.

[0060] An "isolated" polypeptide is one that has been identified and separated and / or recovered from components of its natural environment. Contaminating components of its natural environment are substances that would interfere with the diagnostic or therapeutic use of the polypeptide and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In preferred embodiments, the polypeptide will be purified to: (1) greater than 95% by weight of the polypeptide as determined by the Lowry Method, and most preferably greater than 99% by weight, (2) to a degree sufficient to obtain at least the N-terminal residues or internal amino acid sequence by use of a spinning cup sequenator, or (3) to a degree of homogeneity following SDS-PAGE performed under reducing or non-reducing conditions, using Coomassie Brilliant Blue, or preferably, silver stain. An isolated polypeptide includes the polypeptide in situ within recombinant cells, since at least one component of the polypeptide's natural environment will not be present. In general, however, an isolated polypeptide will be prepared by at least one purification step.

[0061] As used in this article 10 The "region" of the Fn3 domain refers to the human 10 The loops (AB, BC, CD, DE, EF and FG), beta strands (A, B, C, D, E, F and G), N-terminus (corresponding to amino acid residues 1-7 of SEQ ID NO: 1), or C-terminus (corresponding to amino acid residues 93-94 of SEQ ID NO: 1) of the Fn3 domain.

[0062] "Arctic loop" refers to human fibronectin type 3 ten ( 10 Fn3) domain of fibronectin BC, DE and FG loops.

[0063] "Antarctic ring" refers to human fibronectin type 3 ten ( 10 Fn3) domain of fibronectin AB, CD and EF loops.

[0064] The "bracket area" refers to the 10 Any non-loop region of the Fn3 domain. The scaffold region includes the A, B, C, D, E, F and G beta strands and the N-terminal region (corresponding to the amino acids of residues 1-8 of SEQ ID NO: 1) and the C-terminal region (corresponding to the amino acids of residues 93-94 of SEQ ID NO: 1 and optionally including the 7 amino acids that constitute the natural linker between the 10th and 11th repeats of the Fn3 domain in human fibronectin).

[0065] " Amino acid sequence identity percentage (%) " herein is defined as after carrying out sequence alignment with selected sequence in candidate sequence, and introducing room (if necessary) to reach maximum sequence identity percentage, and not considering any conservative substitution as a part for sequence identity, the percentage of the amino acid residue in candidate sequence that is identical with the amino acid residue in selected sequence. In order to determine the amino acid sequence identity percentage, various methods well known to those skilled in the art can be used to implement comparison, for example, using computer software available to the public, such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for measuring comparison, including any algorithm required for maximum comparison on the total length of the sequence compared. However, for the purposes of this article, by using the sequence comparison computer program ALIGN-2, obtain amino acid sequence identity % values ​​as described below. The ALIGN-2 sequence comparison computer program was created by Genentech, Inc., and has been filed with the U.S. Copyright Office (US Copyright Office, Washington DC, 20559) with user files, registered with U.S. Copyright Registration No. TXU510087 in the U.S. Copyright Office, and is available to the public through Genentech, Inc. (South San Francisco, Calif). The ALIGN-2 program should be compiled for use on a UNIX operating system, preferably digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not change.

[0066] For the purposes herein, the % amino acid sequence identity of a given amino acid sequence A relative to (with, or against) a given amino acid sequence B (or may be expressed as a given amino acid sequence A having or comprising a certain % amino acid sequence identity relative to (with, or against) a given amino acid sequence B) is calculated as follows: 100 multiplied by a fraction X / Y, where X is the number of amino acid residues that are scored as identical matches by the ALIGN-2 program in an alignment of A and B by the program, and where Y is the total number of amino acid residues in B. It should be understood that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A relative to B will not be equal to the % amino acid sequence identity of B relative to A.

[0067] The terms "specifically binds", "specific binding", "selectively binds", and "selectively binds" as used interchangeably herein refer to an Adnectin that exhibits affinity for serum albumin and does not significantly bind (e.g., less than about 10% binding) to another different polypeptide as measured by techniques available in the art, such as, but not limited to, Scatchard analysis and / or competitive binding assays (e.g., competition ELISA, BIACORE assays). For example, the term also applies where the binding domain of an Adnectin of the invention is specific for serum albumin.

[0068] The "half-life" of a polypeptide may generally be defined as the time taken for the serum concentration of the polypeptide to decrease by 50% in vivo, for example, due to degradation of the polypeptide by natural mechanisms and / or clearance or sequestration of the polypeptide. The half-life may be determined in any manner known per se, for example by pharmacokinetic analysis. Suitable techniques will be clear to those skilled in the art and may, for example, generally involve the following steps: administering a suitable dose of the polypeptide to a primate; collecting blood samples or other samples from the primate at regular intervals; determining the level of polypeptide concentration in the blood sample; and calculating, from (a plot of) the data thus obtained, the time elapsed until the level or concentration of the polypeptide decreases by 50% compared to the initial level at the time of administration. Methods for determining half-life can be found, for example, in Kenneth et al., Chemical Stability of Pharmaceuticals: A Handbook for Pharmacists (1986); Peters et al., Pharmacokinetic analysis: A Practical Approach (1996); and "Pharmacokinetics", M Gibaldi & D Perron, published by Marcel Dekker, 2nd revised edition (1982).

[0069] The half-life can be expressed using a term such as t l / 2 -α, t l / 2 In some embodiments, the increase in half-life refers to an increase in t1 / 2-β, with or without t l / 2 - Increase in alpha and / or AUC or both.

[0070] As used herein, the term “K D" refers to the dissociation equilibrium constant of a particular Adnectin-protein interaction or the affinity of an Adnectin for a protein (e.g., serum albumin) as measured using a surface plasmon resonance assay or a cell binding assay. As used herein, "desired K D ” refers to the K of an Adnectin that is sufficient for its intended purpose D For example, the expected K D Can refer to the K of an Adnectin required to elicit a functional effect in an in vitro assay, such as a cell-based luciferase assay. D .

[0071] As used herein, the term “k ass ” refers to the association rate constant for the binding of an Adnectin into an Adnectin / protein complex.

[0072] As used herein, the term “k diss ” refers to the dissociation rate constant for the dissociation of an Adnectin from an Adnectin / protein complex.

[0073] As used herein, the term “IC 50 ” refers to the concentration of an Adnectin that inhibits a response in an in vitro or in vivo assay to a level that is 50% of the maximal inhibitory response (ie, halfway between the maximal inhibitory response and the untreated response).

[0074] The term "therapeutically effective amount" refers to an amount of a drug effective to treat a disease or disorder in a mammal and / or to alleviate to some extent one or more of the symptoms associated with the disorder.

[0075] As used herein, "preventing" a disease or condition refers to reducing the probability of occurrence of a disease state in a statistical sample relative to an untreated control sample, or delaying the onset of or reducing the severity of one or more symptoms of the disease or condition relative to an untreated control sample. Patients may be selected for preventive treatment based on factors known to increase the risk of developing a clinical disease state compared to the general population. As used herein, the term "treating" includes (a) inhibiting a disease state, i.e., preventing its development; and / or (b) alleviating a disease state, i.e., causing the disease state to regress once established.

[0076] Overview

[0077] The novel fibronectin-based scaffold polypeptides described herein can bind to serum albumin from a variety of species and can be coupled to additional molecules, such as other proteins that bind to different targets. 10 Fn3 domains, or polypeptides for which increased half-life is beneficial.

[0078] A. General structure of fibronectin-based scaffolds

[0079] Fn3 refers to the type III domain from fibronectin. The Fn3 domain is small, monomeric, soluble, and stable. It lacks disulfide bonds and is therefore stable under reducing conditions. The overall structure of Fn3 is similar to the immunoglobulin fold. The Fn3 domain contains, in order from N-terminus to C-terminus: β or β-like strand A; loop AB; β or β-like strand B; loop BC; β or β-like strand C; loop CD; β or β-like strand D; loop DE; β or β-like strand E; loop EF; β or β-like strand F; loop FG; and β or β-like strand G. Seven antiparallel β strands are arranged into two β sheets, forming a stable core while generating two "faces" consisting of loops connecting β or β-like strands. Loops AB, CD, and EF are located on one face ("south pole") and loops BC, DE, and FG are located on the opposite face ("north pole"). Loops AB, BC, CD, DE, EF, and FG can all participate in ligand binding. There are at least 15 different Fn3 modules in human fibronectin. Although the sequence homology between modules is low, they all have high similarity in tertiary structure.

[0080] In some embodiments, the Fn3 domain is derived from the wild-type tenth module of the human fibronectin type III domain ( 10 Fn3) derived Fn3 domain:

[0081] VSDVPRDLEVVAA TPTS LLISWDAPAVTVRYYRITY GETGGNSPVQ E FTVPGSKSTATISGL KVD YTITVYAVTGRGDSPASSKPISINYRT (SEQ ID NO: 1) (AB, CD, and EF loops are underlined).

[0082] In some embodiments, 10 The non-ligand binding sequence of Fn3, i.e., " 10 Fn3 bracket" can be changed, provided that 10 Fn3 retains ligand binding function and / or structural stability. A variety of mutants have been reported 10 Fn3 scaffold. In one aspect, one or more of Asp7, Glu 9, and Asp 23 are replaced by another amino acid, such as a non-negatively charged amino acid residue (e.g., Asn, Lys, etc.). These mutations have been reported to promote the expression of mutants at neutral pH values ​​compared to the wild-type form. 10 Fn3 has a stronger stabilizing effect (see, e.g., PCT Publication No. WO 02 / 04523). 10Beneficial or neutral changes in the Fn3 scaffold. See, for example, Batori et al., Protein Eng., 15(12): 1015-1020 (December 2002); Koide et al., Biochemistry, 40(34): 10326-10333 (Aug. 28, 2001).

[0083] Variant and wild type 10 Fn3 proteins are all characterized by the same structure, i.e., seven β-chain domain sequences named A to G, and six loop regions (AB loop, BC loop, CD loop, DE loop, EF loop, and FG loop) connecting the seven β-chain domain sequences. The β-chain located closest to the N-terminus and C-terminus can adopt a β-like conformation in solution. In SEQ ID NO: 1, the AB loop corresponds to residues 14-17, the BC loop corresponds to residues 23-31, the CD loop corresponds to residues 37-47, the DE loop corresponds to residues 51-56, the EF loop corresponds to residues 63-67, and the FG loop corresponds to residues 76-87.

[0084] Thus, in some embodiments, the serum albumin binding Adnectin of the invention is identical to the human SEQ ID NO: 10 The Fn3 domains are at least 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% identical 10 Fn3 polypeptides. The variability generally occurs mostly in one or more loops. 10 Each β or β-like strand of the Fn3 polypeptide may consist essentially of an amino acid sequence that is at least 80%, 85%, 90%, 95% or 100% identical to the corresponding β or β-like strand sequence of SEQ ID NO: 1, provided that such changes do not destroy the stability of the polypeptide under physiological conditions.

[0085] Additionally, insertions and deletions can be made in the loop regions while still producing high affinity serum binding. 10 Fn3 binding domain. Thus, in some embodiments, one or more loops selected from AB, BC, CD, DE, EF, and FG are 10The corresponding ring in Fn3 can be extended or shortened in length. In any given polypeptide, one or more rings can be extended in length, one or more rings can be shortened in length, or a combination thereof. In some embodiments, the length of a given ring can be extended by 2-25, 2-20, 2-15, 2-10, 2-5, 5-25, 5-20, 5-15, 5-10, 10-25, 10-20 or 10-15 amino acids. In some embodiments, the length of a given ring can be shortened by 1-15, 1-11, 1-10, 1-5, 1-3, 1-2, 2-10 or 2-5 amino acids.

[0086] As described above, the amino acid residues corresponding to residues 14-17, 23-30, 37-47, 51-56, 63-67, and 76-87 of SEQ ID NO: 1 define the AB, BC, CD, DE, EF, and FG loops, respectively. However, it should be understood that in order to achieve a strong affinity for a desired target, 10 In the Fn3 binding domain, not every residue in the loop region needs to be modified. In some embodiments, only one loop, such as the residues in the CD loop, is modified to produce a high affinity binding target. 10 Fn3 domain.

[0087] In some embodiments, the present invention provides a 10 Fn3 domain polypeptide, wherein the 10 The Fn3 domain comprises AB, BC, CD, DE, and FG loops, and at least one loop selected from AB, CD, and EF loops has a relative sequence to that of human SEQ ID NO: 1. 10 The amino acid sequence of the corresponding loop sequence change of the Fn3 domain. In some embodiments, AB, CD, and EF loops are changed. In certain embodiments, only the AB loop is changed. In certain embodiments, only the CD loop is changed. In certain embodiments, only the EF loop is changed. In certain embodiments, both the AB loop and the CD loop are changed. In certain embodiments, both the AB loop and the EF loop are changed. In certain embodiments, both the CD loop and the EF loop are changed. In some embodiments, one or more special scaffold changes are combined with one or more loop changes. With respect to "change" it is meant one or more amino acid sequence changes relative to the template sequence (i.e., the corresponding wild-type human fibronectin domain), including amino acid additions, deletions, and substitutions.

[0088] In some embodiments, the fibronectin-based scaffold protein comprises a combination of altered north and south pole rings. 10Fn3 domain. For example, one or more of loops AB, CD, and EF, in combination with one or more of loops BC, DE, and FG, can be expressed relative to the human version of SEQ ID NO: 1. 10 The corresponding loops of the Fn3 domain are altered.

[0089] In some embodiments, the polypeptide comprises 10 Fn3 domain: 10 The Fn3 domain comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to the non-loop region and / or non-modified loop region of SEQ ID NO: 1, wherein at least one loop selected from AB, CD, and EF is altered. For example, in certain embodiments, the AB loop may have up to 4 amino acid substitutions, up to 10 amino acid insertions, up to 3 amino acid insertions, or a combination thereof; the CD loop may have up to 6 amino acid substitutions, up to 10 amino acid insertions, up to 4 amino acid deletions, or a combination thereof; and the EF loop may have up to 5 amino acid substitutions, up to 10 amino acid insertions, up to 3 amino acid deletions, or a combination thereof; and / or the FG loop may have up to 12 amino acid substitutions, up to 11 amino acid deletions, up to 25 amino acid insertions, or a combination thereof.

[0090] In some embodiments, one or more residues in the integrin binding motif "arginine-glycine-aspartic acid" (RGD) (amino acids 78-80 of SEQ ID NO: 1) can be substituted to disrupt integrin binding. In some embodiments, the FG loop of the polypeptides provided herein does not contain an RGD integrin binding site. In one embodiment, the RGD sequence is replaced with a polar amino acid-neutral amino acid-acidic amino acid sequence (in an N-terminal to C-terminal direction). In certain embodiments, the RGD sequence is replaced with SGE. In certain embodiments, the RGD sequence is replaced with RGE.

[0091] In certain embodiments, the fibronectin-based scaffold protein comprises 10 Fn3 domain, the 10 The Fn3 domain is generally defined by the following sequence:

[0092] VSDVPRDLEVVAA(X) u LLISW(X) v YRITY(X) w FTV(X) x ATISGL(X) y YTITVYA(X) z ISI NYRT (SEQ ID NO: 2)

[0093] In SEQ ID NO:2, the AB loop is represented by (X) u Indicates that the BC ring is (X) v Indicates that the CD ring is (X) w Indicates that the DE ring is (X) x Indicates that the EF ring is (X) y Indicated by X z Indicated. X represents any amino acid, and the subscript below X represents an integer of the number of amino acids. Specifically, u, v, w, x, y and z can each independently be selected from 2-20, 2-15, 2-10, 2-8, 5-20, 5-15, 5-10, 5-8, 6-20, 6-15, 6-10, 6-8, 2-7, 5-7, or 6-7 amino acids. The β chain sequence (underlined) can have any one of from 0 to 10, from 0 to 8, from 0 to 6, from 0 to 5, from 0 to 4, from 0 to 3, from 0 to 2, or from 0 to 1 substitutions, deletions or additions relative to the corresponding amino acids shown in SEQ ID NO: 2 on all 7 scaffold regions. In some embodiments, the beta strand sequence may have any of from 0 to 10, from 0 to 8, from 0 to 6, from 0 to 5, from 0 to 4, from 0 to 3, from 0 to 2, or from 0 to 1 conservative substitutions on all 7 scaffold regions relative to the corresponding amino acids shown in SEQ ID NO: 2. In certain embodiments, the hydrophobic core amino acid residues (the bold residues in SEQ ID NO: 2 above) are fixed, and any substitutions, conservative substitutions, deletions, or additions occur at residues other than the hydrophobic core amino acid residues. In some embodiments, the hydrophobic core residues of the polypeptides provided herein are relative to wild-type human 10 The Fn3 domain (SEQ ID NO: 1) was unmodified.

[0094] In some embodiments, the amino acid sequence of the N-terminal and / or C-terminal regions of the polypeptides provided herein can be determined by comparing the amino acid sequence of the polypeptides to that of wild-type human 10 The Fn3 domain (SEQ ID NO: 1) is modified by deletion, substitution or insertion of the amino acid sequence of the corresponding region. 10 Fn3 domains generally begin with amino acid number 1 of SEQ ID NO: 1. However, the present invention also encompasses domains having amino acid deletions. Additional sequences may also be added to the amino acid sequence of SEQ ID NO: 1. 10 The N-terminus or C-terminus of the Fn3 domain. For example, in some embodiments, the N-terminal extension consists of an amino acid sequence selected from the group consisting of: M, MG, and G.

[0095] In an exemplary embodiment, an alternative N-terminal region of 1-20, 1-15, 1-10, 1-8, 1-5, 1-4, 1-3, 1-2, or 1 amino acid in length can be added to the N-terminal region of SEQ ID NO: 1. Exemplary alternative N-terminal regions include (represented by single letter amino acid codes) M, MG, G, MGVSDVPRDL (SEQ ID NO: 3), and GVSDVPRDL (SEQ ID NO: 4). Other suitable alternative N-terminal regions include, for example, X n SDVPRDL (SEQ ID NO: 5), X n DVPRDL (SEQ ID NO: 6), X n VPRDL (SEQ ID NO: 7), X n PRDL (SEQ ID NO: 8) X n RDL (SEQ ID NO: 9), X n DL (SEQ ID NO: 10), or X n L, wherein n=0, 1 or 2 amino acids, wherein when n=1, X is Met or Gly, and when n=2, X is Met-Gly. When the Met-Gly sequence is added to 10 When the N-terminus of the Fn3 domain is cleaved, the M is often cleaved off, leaving a G at the N-terminus. In certain embodiments, the alternative N-terminal region comprises the amino acid sequence MASTSG (SEQ ID NO: 11).

[0096] In an exemplary embodiment, an alternative C-terminal region having a length of 1-20, 1-15, 1-10, 1-8, 1-5, 1-4, 1-3, 1-2, or 1 amino acids can be added to the C-terminal region of SEQ ID NO: 1. Specific examples of alternative C-terminal region sequences include, for example, polypeptides comprising, or consisting essentially of, EIEK (SEQ ID NO: 12), EGSGC (SEQ ID NO: 13), EIEKPCQ (SEQ ID NO: 14), EIEKPSQ (SEQ ID NO: 15), EIEKP (SEQ ID NO: 16), EIEKPS (SEQ ID NO: 17), or EIEKPC (SEQ ID NO: 18). In some embodiments, the alternative C-terminal region comprises EIDK (SEQ ID NO: 19), and in specific embodiments, the alternative C-terminal region is EIDKPCQ (SEQ ID NO: 20) or EIDKPSQ (SEQ ID NO: 21). Additional suitable alternative C-terminal regions include those shown in Table 20 and SEQ ID NOs: 210-220.

[0097] In certain embodiments, the C-terminal extension sequence comprises E and D residues and can be between 8 and 50, 10 and 30, 10 and 20, 5 and 10, and 2 and 4 amino acids in length. In some embodiments, the tail sequence comprises an ED-based linker, wherein the sequence comprises tandem repeats of ED. In exemplary embodiments, the tail sequence comprises 2-10, 2-7, 2-5, 3-10, 3-7, 3-5, 3, 4, or 5 ED repeats. In certain embodiments, the ED-based tail sequence may also comprise additional amino acid residues, such as EI, EID, ES, EC, EGS, and EGC. Such sequences are based in part on known Adnectin tail sequences, such as EIDKPSQ (SEQ ID NO: 21), in which residues D and K have been removed. In exemplary embodiments, the ED-based tail comprises E, I, or EI residues before the ED repeats.

[0098] In certain embodiments, an alternative C-terminal moiety, which may be linked to the C-terminal amino acid RT of any of the Adnectins provided herein (i.e., amino acids 93-94 of SEQ ID NO: 1), comprises amino acids P m X n , wherein P is proline, X is any amino acid, m is an integer of at least 1, and n is 0 or an integer of at least 1. In certain embodiments, the alternative C-terminal moiety comprises the amino acid PC. In certain embodiments, the alternative C-terminal portion comprises amino acids PI, PC, PID, PIE, PIDK (SEQ ID NO: 221), PIEK (SEQ ID NO: 222), PIDKP (SEQ ID NO: 223), PIEKP (SEQ ID NO: 224), PIDKPS (SEQ ID NO: 225), PIEKPS (SEQ ID NO: 226), PIDKPC (SEQ ID NO: 227), PIEKPC (SEQ ID NO: 228), PIDKPSQ (SEQ ID NO: 229), PIEKPSQ (SEQ ID NO: 230), PIDKPCQ (SEQ ID NO: 231), PIEKPCQ (SEQ ID NO: 232), PHHHHHH (SEQ ID NO: 233), and PCHHHHHH (SEQ ID NO: 234).

[0099] In certain embodiments, the fibronectin-based scaffold protein comprises a protein having both an alternative N-terminal region sequence and an alternative C-terminal region sequence. 10 Fn3 domain.

[0100] B. Serum albumin conjugates with modified antarctic loops

[0101] because10 Fn3 domains are small in size, approximately 10 kDa, and are rapidly cleared from the circulation by renal filtration and degradation (t 1 / 2 = 15-45 minutes; 3 hours in monkeys). In certain aspects, the present application provides a protein that specifically binds to serum albumin, such as human serum albumin (HSA). 10 The Fn3 domain has a south pole modification that extends 10 Fn3 domain t 1 / 2 .

[0102] In humans, HSA has a serum concentration of 600 μM. 1 / 2 19 days. Extended HSA t 1 / 2 Partly due to its recycling via neonatal Fc receptor (FcRn). After HSA is taken up into endothelial cells through the endosomal, it binds to FcRn in a pH-dependent manner; this interaction recycles HSA back into the bloodstream, thereby allowing it to avoid lysosomal degradation. FcRn is widely expressed and this recycling pathway is considered to be constitutive. In most cell types, most of the FcRn resides in the intracellular sorting endosome. HSA is easily internalized by a nonspecific liquid phase endocytosis mechanism and is rescued by FcRn from degradation in the lysosome. At the acidic pH in the endosome, HSA increases its affinity for FcRn (5 μM at pH 6.0). Once bound to FcRn, HSA avoids lysosomal degradation, is transcytosed to the cell surface, and is released on the cell surface.

[0103] Arctic-based serum albumin binding Adnectins, referred to herein as "first generation" serum albumin binding Adnectins, have been described, for example, in WO2011140086. Some of the first generation Arctic-based serum albumin binding Adnectins (SABAs) do not bind to mouse or rat serum albumin, do not have high affinity for serum albumin across species, and are not readily available in multivalent based 10 To improve upon the Fn3 platform, which is not always compatible, second generation South Pole-based serum albumin binding Adnectins (PKE2 Adnectins) with modified South Pole loops were developed, as described in the Examples.

[0104] Therefore, in one aspect, the present invention provides such 10 Fn3 domains having: (i) relative to wild-type human 10 The corresponding loop of the Fn3 domain (SEQ ID NO: 1) is modified in the amino acid sequence of at least one south pole loop selected from the group consisting of AB, CD, and EF loops, wherein 10The Fn3 domain binds to serum albumin (e.g., human serum albumin). The modified Antarctic loops contribute to binding to the same target. Various combinations of modified Antarctic loops are contemplated. For example, 10 Fn3 may comprise one modified South Pole loop, two modified South Pole loops, or even all three South Pole loops are modified. In certain embodiments, one or more modified South Pole loops may be combined with one or more modified North Pole loops (i.e., one or more of the BC, DE, and FG loops). The modified loops may have sequence modifications throughout the loop or only in a portion of the loop. In addition, one or more modified loops may have insertions or deletions such that the length of the loop varies relative to the length of the corresponding loop of the wild-type sequence (i.e., SEQ ID NO: 1). In certain embodiments, in 10 Other regions in the Fn3 domain (i.e., in addition to the South Pole loop), such as the β-strand, N-terminal, and / or C-terminal regions, may also undergo changes relative to wild-type 10 Sequence modifications of the Fn3 domains, and such additional modifications may also contribute to binding to the target. In certain embodiments, the Antarctic loop is the only modified domain. In specific embodiments, the CD loop is the only modified domain. In certain embodiments, serum binding 10 The Fn3 domain may be modified to include an N-terminal extension sequence and / or a C-terminal extension sequence, as described above.

[0105] In one embodiment, the invention provides an Adnectin that binds to serum albumin relative to wild-type human 10 Fn3 domains (e.g., those shown in SEQ ID NOs: 23-100, 184-209, and 235-260) 10 In some embodiments, the albumin binding Adnectin comprises, or lacks, a 6X histidine tail. In some embodiments, the albumin binding Adnectin corresponds to a core Adnectin lacking an N-terminal leader sequence and a C-terminal tail, as set forth in SEQ ID NOs: 75-100.

[0106] In an exemplary embodiment, the serum albumin-binding 10 The Fn3 protein has a K of less than 3 μM, 2.5 μM, 2 μM, 1.5 μM, 1 μM, 500 nM, 100 nM, 50 nM, 10 nM, 1 nM, 500 pM, 100 pM, 100 pM, 50 pM, or 10 pM. DBinds to human serum albumin. The Kd can, for example, be in the range of 0.1 nM to 50 nM, 0.1 nM to 100 nM, 0.1 nM to 1 μM, 0.5 nM to 50 nM, 0.5 nM to 100 nM, 0.5 nM to 1 μM, 1 nM to 50 nM, 1 nM to 100 nM, or 1 nM to 1 μM.

[0107] In certain embodiments, the albumin-binding Adnectins (or 10 Fn3 protein) can also bind serum albumin from one or more of cynomolgus monkey, rhesus monkey, rat, or mouse.

[0108] In certain embodiments, the serum albumin-binding 10 The Fn3 protein has a K of less than 3 μM, 2.5 μM, 2 μM, 1.5 μM, 1 μM, 500 nM, 100 nM, 50 nM, 10 nM, 1 nM, 500 pM, 100 pM or 100 pM. D Binds to rhesus serum albumin (RhSA) or cynomolgus serum albumin (CySA). D It may, for example, be in the range of 0.1 nM to 50 nM, 0.1 nM to 100 nM, 0.1 nM to 1 μM, 0.5 nM to 50 nM, 0.5 nM to 100 nM, 0.5 nM to 1 μM, 1 nM to 50 nM, 1 nM to 100 nM, or 1 nM to 1 μM.

[0109] In certain embodiments, the serum albumin-binding 10 The Fn3 protein has a K of less than 3 μM, 2.5 μM, 2 μM, 1.5 μM, 1 μM, 500 nM, 100 nM, 50 nM, 10 nM, 1 nM, 500 pM, 100 pM or 100 pM. D Binds to rhesus serum albumin (RhSA), cynomolgus serum albumin (CySA), and mouse serum albumin (MSA). D It may, for example, be in the range of 0.1 nM to 50 nM, 0.1 nM to 100 nM, 0.1 nM to 1 μM, 0.5 nM to 50 nM, 0.5 nM to 100 nM, 0.5 nM to 1 μM, 1 nM to 50 nM, 1 nM to 100 nM, or 1 nM to 1 μM.

[0110] In certain embodiments, the serum albumin-binding 10 The Fn3 protein has a K of less than 3 μM, 2.5 μM, 2 μM, 1.5 μM, 1 μM, 500 nM, 100 nM, 50 nM, 10 nM, 1 nM, 500 pM, 100 pM or 100 pM. DBinds to rhesus serum albumin (RhSA), cynomolgus serum albumin (CySA), mouse serum albumin (MSA), and rat serum albumin (RSA). D It may, for example, be in the range of 0.1 nM to 50 nM, 0.1 nM to 100 nM, 0.1 nM to 1 μM, 0.5 nM to 50 nM, 0.5 nM to 100 nM, 0.5 nM to 1 μM, 1 nM to 50 nM, 1 nM to 100 nM, or 1 nM to 1 μM.

[0111] In certain embodiments, the albumin binding Adnectins described herein bind to serum albumin in the pH range of 5.5 to 7.4.

[0112] In certain embodiments, the albumin binding Adnectins described herein bind to domains I-II of human serum albumin.

[0113] In certain embodiments, the serum half-life of an albumin binding Adnectin of the invention, or an albumin binding Adnectin linked to a heterologous moiety (e.g., a second Adnectin), 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, 40 hours, 50 hours, 60 hours, 70 hours, 80 hours, 90 hours, 100 hours, 110 hours, 120 hours, 130 hours, 135 hours, 140 hours, 150 hours, 160 hours, or 200 hours. In certain embodiments, the serum half-life of an albumin binding Adnectin, or the serum half-life of an albumin binding Adnectin linked to a heterologous moiety (e.g., a second Adnectin), is 2-200 hours, 5-200 hours, 10-200 hours, 25-200 hours, 50-200 hours, 100-200 hours, 150-200 hours, 2-150 hours, 2-100 hours, 2-50 hours, 2-25 hours, 2-10 hours, 2-5 hours, 5-150 hours, 10-100 hours, or 25-50 hours.

[0114] In certain embodiments, the albumin-binding Adnectin comprises a wild-type 10 The Fn3 domain (SEQ ID NO: 1) has a sequence that is at least 40%, 50%, 60%, 70%, 75%, 80% or 85% identical. In one embodiment, at least one of the AB, CD, or EF loops is expressed relative to the wild-type 10The Fn3 domain is modified. In certain embodiments, at least two of the AB, CD, or EF loops undergo a modification relative to wild-type 10 The Fn3 domain is modified. In certain embodiments, all three of the AB, CD, or EF loops are modified relative to wild-type 10 In certain embodiments, the Fn3 domains are modified. 10 The Fn3 domain comprises a sequence that is at least 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of SEQ ID NOs: 23-100, 184-209 and 235-260.

[0115] In certain embodiments, the serum albumin-binding 10 The Fn3 domain (or Adnectin) may comprise a sequence as set forth in SEQ ID NO: 2, wherein the CD loop is represented by (X) w , and replaced by the CD loop from any of the 26 core PKE2 Adnectin sequences (i.e., SEQ ID NOs: 75-100). Such albumin-binding Adnectin scaffold regions may have 0 to 20, 0 to 15, 0 to 10, 0 to 8, 0 to 6, 0 to 5, 0 to 4, 0 to 3, 0 to 2, or 0 to 1 substitutions, conservative substitutions, deletions, or additions relative to the scaffold amino acid residues of SEQ ID NO: 1. As long as the albumin-binding Adnectin is able to bind with the desired K D Binding to serum albumin, such as HSA, can result in such scaffold modifications.

[0116] In some embodiments, the CD loop regions of the albumin binding Adnectins of the invention may be described based on consensus sequences.

[0117] Thus, in some embodiments, the CD loop consists of the consensus sequence GX 1 -X 2 -VX 3 -X 4 -X 5 -SX 6 -X 7 -GX 8 -X 9 -YX 10 -X 11 -X 12 -E (SEQ ID NO: 170), wherein

[0118] (a)X 1 Selected from R or W;

[0119] (b)X 2 Selected from H, E, D, Y, or Q;

[0120] (c)X 3 Select from Q or H;

[0121] (d)X 4 is selected from I, K, M, Q, L, or V;

[0122] (e)X 5 Selected from Y, F, or N;

[0123] (f)X 6 Selected from D, V, or E;

[0124] (g)X 7 Selected from L, W, or F;

[0125] (h)X 8 Select from P or T;

[0126] (i)X 9 Select from L or M;

[0127] (j)X 10 Selected from I or V;

[0128] (k)X 11 is selected from Y or F; and

[0129] (l)X 12 Selected from T, S, Q, N, or A.

[0130] In certain preferred embodiments,

[0131] (a)X 1 It is R;

[0132] (b)X 2 It is E;

[0133] (c)X 3 It is Q;

[0134] (d)X 4 It is K;

[0135] (e)X 5 It is Y;

[0136] (f)X 6 It is D;

[0137] (g)X 7 is L or W;

[0138] (h)X 8 It is P;

[0139] (i)X9 It is L;

[0140] (j)X 10 is I;

[0141] (k)X 11 is Y; and

[0142] (l)X 12 It is Q or N.

[0143] In a preferred embodiment, X 7 is L and X 12 It's Q.

[0144] In another preferred embodiment, X 7 is W and X 12 It is N.

[0145] In some embodiments, the albumin binding Adnectins of the invention comprise a CD loop having a sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the CD loop sequences set forth in SEQ ID NOs: 101-125, or having at most 1, 1-2, or 1-3 amino acid differences (i.e., substitutions, e.g., deletions, additions, or conservative substitutions). The scaffold region of such an albumin binding Adnectin may comprise 0 to 20, 0 to 15, 0 to 10, 0 to 8, 0 to 6, 0 to 5, 0 to 4, 0 to 3, 0 to 2, or 0 to 1 substitutions, conservative substitutions, deletions, or additions relative to the scaffold amino acid residues of SEQ ID NO: 1. As long as the Adnectin is capable of expressing the desired K D Binding to serum albumin can result in such scaffold modifications.

[0146] In a preferred embodiment, the CD loop of the albumin binding Adnectin of the invention comprises an amino acid sequence selected from the group consisting of:

[0147] GRHVQIYSDLGPLYIYTE(SEQ ID NO:101)、

[0148] GRHVHIYSDWGPMYIYTE(SEQ ID NO:102)、

[0149] GREVQKYSVLGPLYIYTE(SEQ ID NO:103)、

[0150] GREVQMYSDLGPLYVYSE(SEQ ID NO:104)、

[0151] GREVQKFSDWGPLYIYTE(SEQ ID NO:105)、

[0152] GREVQKYSDLGPLYIYQE(SEQ ID NO:106)、

[0153] GREVHQYSDWGPMYIYNE(SEQ ID NO:107)、

[0154] GREVHKNSDWGTLYIYTE(SEQ ID NO:108)、

[0155] GREVQKYSDLGPLYIYAE(SEQ ID NO:109)、

[0156] GREVHLYSDWGPMYIYTE(SEQ ID NO:110)、

[0157] GRHVQMYSDLGPLYIFSE(SEQ ID NO:111)、

[0158] GREVHMYSDFGPMYIYTE(SEQ ID NO:112)、

[0159] GREVQKYSDWGPLYIYNE(SEQ ID NO:113)、

[0160] GREVQMYSDLGPLYIYNE(SEQ ID NO:114)、

[0161] GREVQMYSDLGPLYIYTE(SEQ ID NO:115)、

[0162] GRHVQIYSDLGPLYIYNE(SEQ ID NO:116)、

[0163] GREVQIYSDWGPLYIYNE(SEQ ID NO:117)、

[0164] GREVQKYSDWGPLYIYQE(SEQ ID NO:118)、

[0165] GRHVHLYSEFGPMYIYNE(SEQ ID NO:119)、

[0166] GRDVHMYSDWGPMYIYQE(SEQ ID NO:120)、

[0167] GRHVQIYSDWGPLYIYNE(SEQ ID NO:121)、

[0168] GRYVQLYSDWGPMYIYTE(SEQ ID NO:122)、

[0169] GRQVQVFSDLGPLYIYNE(SEQ ID NO:123)、

[0170] GRQVQIYSDWGPLYIYNE(SEQ ID NO:124), and

[0171] GRQVQMYSDWGPLYIYAE (SEQ ID NO: 125).

[0172] In some embodiments, the albumin binding Adnectin comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to any one of SEQ ID NOs: 23-100, 184-209 and 235-260, or has at most 1, 1-2, 1-3, 1-5, 1-10, or 1-20 amino acid differences, e.g., amino acid deletions, additions, or substitutions (e.g., conservative substitutions). In certain embodiments, the albumin binding molecule comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to the non-CD loop regions of SEQ ID NOs: 23-100, 184-209 and 235-260.

[0173] In a preferred embodiment, the albumin binding Adnectin comprises the amino acid sequence set forth in any one of SEQ ID NOs: 29, 55, 81, 190, and 241. In another preferred embodiment, the albumin binding Adnectin comprises the amino acid sequence set forth in any one of SEQ ID NOs: 36, 62, 88, 197, and 248.

[0174] In some embodiments, the invention provides albumin-binding Adnectin molecules with mutations that introduce cysteine ​​residues at specific positions. Exemplary cysteine ​​mutations are A12C, A26C, S55C, T56C, and T58C (see Table 7 in the Examples). In a preferred embodiment, the cysteine ​​mutations do not substantially alter the binding of the albumin-binding Adnectin to serum albumin.

[0175] In certain embodiments, 10A proline residue is introduced at the C-terminus of the Fn3 domain, e.g., as shown in SEQ ID NOs: 184-209 and 235-260. In certain embodiments, a proline residue is introduced at the C-terminus of the tandem albumin binding Adnectin, e.g., as shown in SEQ ID NOs: 168 and 261. The addition of a proline residue does not prevent the addition of additional amino acid sequences at the C-terminus of the albumin binding Adnectin or the tandem albumin binding Adnectins.

[0176] C. Cross-competing Adnectins and / or Adnectins that bind to the same Adnectin binding site

[0177] Provided herein are proteins, such as Adnectins, antibodies or antigen-binding fragments thereof, small molecules, peptides, and the like, that compete (e.g., cross-compete) for binding to serum albumin (e.g., HSA) with a particular PKE2 Adnectin described herein. Such competing proteins, such as Adnectins, can be identified based on their ability to competitively inhibit the binding of an Adnectin described herein to serum albumin (e.g., HSA) in a standard serum albumin binding assay. For example, a standard ELISA assay can be used in which recombinant serum albumin proteins are immobilized on a plate, one of the proteins is fluorescently labeled, and the ability of the unlabeled protein to compete away the binding of the labeled protein is assessed.

[0178] The following exemplary competition assay is provided in the context of competition between an Adnectin and one of the PKE2 proteins described herein for binding to serum albumin. The same assay is also useful in testing competition with non-Adnectin proteins. In one embodiment, a competitive ELISA can be performed to determine whether two serum albumin Adnectins bind to overlapping Adnectin binding sites (epitopes) on serum albumin (e.g., HSA). In one format, Adnectin #1 is coated on a plate, which is then blocked and washed. Serum albumin alone or pre-incubated with a saturating concentration of Adnectin #2 is added to this plate. After an appropriate incubation period, the plate is washed and probed with a polyclonal anti-serum albumin antibody, followed by detection with a streptavidin-HRP conjugate and a standard tetramethylbenzidine development procedure. If the OD signal is the same with or without pre-incubation with Adnectin #2, the binding of the two Adnectins is independent of each other and their Adnectin binding sites do not overlap. However, if the OD signals of the wells receiving the serum albumin / Adnectin #2 mixture are lower than those wells receiving serum albumin alone, it is confirmed that the binding of Adnectin #2 blocks the binding of Adnectin #1 to serum albumin.

[0179] Alternatively, similar experiments are performed by surface plasmon resonance (SPR, e.g., BIAcore). Adnectin #1 is immobilized on the surface of an SPR chip, followed by injection of serum albumin alone or serum albumin pre-incubated with a saturated concentration of Adnectin #2. If the binding signal of the serum albumin / Adnectin #2 mixture is the same or higher than that of serum albumin alone, the binding of the two Adnectins is independent of each other, and their Adnectin binding sites do not overlap. However, if the binding signal for the serum albumin / Adnectin #2 mixture is lower than the binding signal of serum albumin alone, the binding of Adnectin #2 is confirmed to block the binding of Adnectin #1 to serum albumin. A feature of these experiments is the use of a saturated concentration of Adnectin #2. If serum albumin is not saturated with Adnectin #2, the above conclusion does not hold. Similar experiments can be used to determine whether any two proteins that bind serum albumin bind to overlapping Adnectin binding sites.

[0180] The two assays exemplified above can also be performed in the reverse order, where Adnectin #2 is immobilized and serum albumin-Adnectin #1 is added to the plate. Alternatively, Adnectin #1 and / or #2 can be replaced with monoclonal antibodies and / or soluble receptor-Fc fusion proteins.

[0181] In certain embodiments, competition can be determined using an HTRF sandwich assay.

[0182] In certain embodiments, a competing Adnectin is an Adnectin that binds to the same Adnectin binding site on serum albumin as a specific PKE2 Adnectin described herein. Standard mapping techniques, such as protease mapping, mutational analysis, X-ray crystallography, and 2-dimensional nuclear magnetic resonance can be used to determine whether an Adnectin binds to the same Adnectin binding site as a reference Adnectin (see, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, GE Morris ed. (1996)).

[0183] Competitive albumin binding candidate proteins, e.g., Adnectins, may inhibit the binding of a PKE2 Adnectin of the invention to serum albumin (e.g., HSA) by at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99%. The % competition may be determined using the methods described above.

[0184] D. Multivalent / Tandem Adnectins

[0185] Provided herein are methods comprising two or more molecules that specifically bind to a target 10 For example, a multivalent protein may comprise 2, 3 or more covalently linked Fn3 domains. 10 In an exemplary embodiment, the multivalent protein is a protein comprising two 10 In certain embodiments, the multivalent protein comprises a first specific or dimeric protein that binds to serum albumin (e.g., human serum albumin). 10 The Fn3 domain and a second target molecule (e.g., PCSK9) 10 Fn3 domains. When the first and second target molecules are serum albumin, the first and second 10 The Fn3 domains may bind to the same or different epitopes. 10 The modified regions in the Fn3 domains that are associated with target binding may be the same or different. In an exemplary embodiment, each of the multivalent fibronectin-based protein scaffolds 10 The Fn3 domain has a K of less than 500 nM, 100 nM, 50 nM, 1 nM, 500 pM, 100 pM or less. D In some embodiments, each of the multivalent fibronectin-based protein scaffolds 10 The Fn3 domains are expressed at a K between 1 pM and 1 μM, between 100 pM and 500 nM, between 1 nM and 500 nM, or between 1 nM and 100 nM. D In an exemplary embodiment, each of the multivalent fibronectin-based protein scaffolds 10 Fn3 domain is not 10 Fn3 domains (especially wild-type human 10 Fn3 domain) binds specifically to the target bound by it.

[0186] Multivalent fibronectin-based scaffold proteins 10The Fn3 domains can be linked via a polypeptide linker. Exemplary polypeptide linkers include polypeptides having 1-20, 1-15, 1-10, 1-8, 1-5, 1-4, 1-3, or 1-2 amino acids. Suitable polypeptides for linking 10 The linker of the Fn3 domain allows each domain to fold independently of each other, forming a three-dimensional structure that allows high affinity binding to the target molecule. Specific examples of suitable linkers include glycine-serine based linkers, glycine-proline based linkers, proline-alanine based linkers, and linkers with the amino acid sequence PSTPPTPSPSTPPTPSPS (SEQ ID NO: 152). In some embodiments, the linker is a glycine-serine based linker. In some embodiments, the linker is a glycine-serine based linker. These linkers contain glycine and serine residues and can be between 8 and 50, 10 and 30, and 10 and 20 amino acids in length. Examples include linkers with the amino acid sequence (GS) 7 (SEQ ID NO: 153), G (GS) 6 (SEQ ID NO: 154), and G (GS) 7 G (SEQ ID NO: 155). Other linkers contain glutamic acid and include, for example, (GSE) 5 (SEQ ID NO: 156) and GGSEGGSE (SEQ ID NO: 157). Other exemplary glycine-serine linkers include (GS) 4 (SEQ ID NO: 158), (GGGGS) 7 (SEQ ID NO: 159), (GGGGS) 5 (SEQ ID NO: 160), and (GGGGS) 3 G (SEQ ID NO: 161). In some embodiments, the linker is a glycine-proline based linker. These linkers contain glycine and proline residues and can be between 3 and 30, 10 and 30, and 3 and 20 amino acids in length. Examples include having the amino acid sequence (GP) 3 G (SEQ ID NO: 162), (GP) 5 G (SEQ ID NO: 163), and GPG. In certain embodiments, the linker can be a proline-alanine based linker having a length between 3 and 30, 10 and 30, and 3 and 20 amino acids. Examples of proline-alanine based linkers include, for example, (PA) 3 (SEQ ID NO: 164), (PA) 6 (SEQ ID NO: 165) and (PA) 9(SEQ ID NO: 166). It is contemplated that the optimal linker length and amino acid composition can be determined by routine experimentation using methods well known in the art. In an exemplary embodiment, the linker does not contain any Asp-Lys (DK) pairs.

[0187] In certain embodiments, the linker has the amino acid sequence PSPEPPTPEP (SEQ ID NO: 173), PSPEPPTPEPPSPEPPTPEP (SEQ ID NO: 174), PSPEPPTPEPPSPEPPTPEPPSPEPPTPEP (SEQ ID NO: 175), or PSPEPPTPEPPSPEPPTPEPPSPEPPTPEPPSPEPPTPEP (SEQ ID NO: 176). Generally, the linker may comprise the amino acid sequence (PSPEPPTPEP) n (SEQ ID NO: 262), wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1-5 or 1-10. In certain embodiments, the linker has the amino acid sequence EEEEDE (SEQ ID NO: 177), EEEDEEEEEEDE (SEQ ID NO: 178), EEEEDEEEEDEEEEDEEEEDE (SEQ ID NO: 179), EEEEDEEEEDEEEEDEEEEDEEEEDEEEEDE (SEQ ID NO: 180). Typically, the linker may comprise the sequence (EEEEDE) n E (SEQ ID NO: 263), wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1-5 or 1-10. In certain embodiments, the linker has the amino acid sequence RGGEEKKKEKEKEEQEERETKTP (SEQ ID NO: 181). Such a linker can be used to connect an albumin-binding Adnectin to another polypeptide (e.g., another Adnectin). An exemplary use of the PSPEPPTPEP (SEQ ID NO: 173) linker is shown below.

[0188] N-terminal Adnectin linked to a C-terminal peptide:

[0189] ...NYRTPGPSPEPPTPEP-polypeptide (SEQ ID NO: 182)

[0190] N-terminal peptide linked to C-terminal Adnectin:

[0191] Polypeptide-PSPEPPTPEPGVSDV... (SEQ ID NO: 183)

[0192] In some embodiments, the multivalent Adnectin is a protein comprising a first protein that binds to serum albumin (e.g., a PKE2 Adnectin). 10 Fn3 domain, and a second domain that binds to a specific target 10 Tandem Adnectins with Fn3 domains. Tandem Adnectins can have the configuration of albumin-binding Adnectin-X and X-albumin-binding Adnectin, where X is the target specific 10 Fn3 domain. The skilled artisan is familiar with methods for testing the functional activity of such tandem Adnectin molecules and assessing their biophysical properties.

[0193] In one aspect, the present invention provides a method comprising a first fibronectin type III tenth ( 10 Fn3) domain and the second 10 A fusion polypeptide of Fn3 domain, wherein the first 10 The Fn3 domain contains: a) AB, BC, CD, DE, EF, and FG loops, b) has 10 The polypeptide comprises a CD loop having an amino acid sequence that is altered from the corresponding CD loop sequence of an Fn3 domain, and c) wherein the polypeptide has a K of less than 500 nM D Binds to human serum albumin. The "first" domain and the second "domain" may be in an N- to C-terminal or C- to N-terminal orientation.

[0194] In some embodiments, for example, in embodiments of multivalent Adnectins, the first 10 The Fn3 domain comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 23-100, 184-209, and 235-260, or that differs therefrom in at most 1, 1-2, 1-5, 1-10, or 1-20 amino acids, e.g., amino acid deletions, additions, or substitutions (e.g., conservative amino acid substitutions).

[0195] In some embodiments, the first 10 The Fn3 domain comprises the amino acid sequence of any one of SEQ ID NOs: 23-100, 184-209, and 235-260.

[0196] In a preferred embodiment, the first 10 The Fn3 domain comprises the amino acid sequence of SEQ ID NO: 29, 55, 81, 190 or 241. In another preferred embodiment, the first 10 The Fn3 domain comprises the amino acid sequence of SEQ ID NO: 36, 62, 88, 197 or 248.

[0197] In some embodiments, the multivalent Adnectin comprises a second 10 Fn3 domains that specifically bind to target proteins other than serum albumin 10 Fn3 domain.

[0198] In a preferred embodiment, the second 10 The Fn3 domain specifically binds to PCSK9.

[0199] Therefore, in one embodiment, the second 10 The Fn3 domain comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 168 or 261, or has at most 1, 1-2, 1-5, 1-10 or 1-20 amino acid differences therefrom (e.g., amino acid deletions, additions or substitutions (e.g., conservative amino acid substitutions)). Additional suitable amino acids that bind to PCSK9 10 Fn3 domains are disclosed, for example, in WO2011 / 130354, the contents of which are incorporated herein by reference.

[0200] In one embodiment, the second 10 The Fn3 domain has the amino acid sequence shown in SEQ ID NO: 168 or 261.

[0201] In certain embodiments, the invention provides PCSK9-serum albumin binding tandem Adnectins comprising the amino acid sequence set forth in SEQ ID NO: 168 or 261, and PCSK9-serum albumin tandem Adnectins having an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to, or that has at most 1, 1-2, 1-5, 1-10 or 1-20 amino acid differences (e.g., amino acid deletions, additions or substitutions (e.g., conservative amino acid substitutions)) from SEQ ID NO: 168 or 261, wherein the tandem Adnectin retains binding to PCSK9 and serum albumin.

[0202] In one embodiment, the invention provides a nucleic acid encoding a tandem Adnectin that binds PCSK9-serum albumin having a nucleic acid sequence set forth in SEQ ID NO: 172; and a nucleic acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 172, wherein the encoded tandem Adnectin that binds PCSK9-serum albumin retains binding to PCSK9 and serum albumin. In some embodiments, the nucleotide substitutions do not change the resulting translated amino acid sequence (i.e., silent mutations).

[0203] In one aspect, the serum albumin binding based tandem Adnectins described herein (e.g., PCSK9-PKE2 tandem Adnectins) have a K of less than 3 μM, 2.5 μM, 2 μM, 1.5 μM, 1 μM, 500 nM, 100 nM, 50 nM, 10 nM, 1 nM, 500 pM, 100 pM, 100 pM, 50 pM, or 10 pM. D Binds to human serum albumin. K D It may, for example, be in the range of 0.1 nM to 50 nM, 0.1 nM to 100 nM, 0.1 nM to 1 μM, 0.5 nM to 50 nM, 0.5 nM to 100 nM, 0.5 nM to 1 μM, 1 nM to 50 nM, 1 nM to 100 nM, or 1 nM to 1 μM.

[0204] In certain embodiments, the serum albumin binding based tandem Adnectins described herein (e.g., PCSK9-PKE2 tandem Adnectins) may also bind serum albumin from one or more of cynomolgus monkeys, rhesus monkeys, rats, or mice.

[0205] In certain embodiments, the serum albumin binding based tandem Adnectins described herein (e.g., PCSK9-PKE2 tandem Adnectins) have a K of less than 3 μM, 2.5 μM, 2 μM, 1.5 μM, 1 μM, 500 nM, 100 nM, 50 nM, 10 nM, 1 nM, 500 pM, 100 pM, or 100 pM. D Binds to rhesus serum albumin (RhSA) or cynomolgus serum albumin (CySA). D It may, for example, be in the range of 0.1 nM to 50 nM, 0.1 nM to 100 nM, 0.1 nM to 1 μM, 0.5 nM to 50 nM, 0.5 nM to 100 nM, 0.5 nM to 1 μM, 1 nM to 50 nM, 1 nM to 100 nM, or 1 nM to 1 μM.

[0206] In certain embodiments, the serum albumin binding based tandem Adnectins described herein (e.g., PCSK9-PKE2 tandem Adnectins) have a K of less than 3 μM, 2.5 μM, 2 μM, 1.5 μM, 1 μM, 500 nM, 100 nM, 50 nM, 10 nM, 1 nM, 500 pM, 100 pM, or 100 pM. D Binds to rhesus serum albumin (RhSA), cynomolgus serum albumin (CySA), and mouse serum albumin (MSA). D It may, for example, be in the range of 0.1 nM to 50 nM, 0.1 nM to 100 nM, 0.1 nM to 1 μM, 0.5 nM to 50 nM, 0.5 nM to 100 nM, 0.5 nM to 1 μM, 1 nM to 50 nM, 1 nM to 100 nM, or 1 nM to 1 μM.

[0207] In certain embodiments, the serum albumin binding based tandem Adnectins described herein (e.g., PCSK9-PKE2 tandem Adnectins) have a K of less than 3 μM, 2.5 μM, 2 μM, 1.5 μM, 1 μM, 500 nM, 100 nM, 50 nM, 10 nM, 1 nM, 500 pM, 100 pM, or 100 pM. D Binds to rhesus serum albumin (RhSA), cynomolgus serum albumin (CySA), mouse serum albumin (MSA), and rat serum albumin (RSA). D It may, for example, be in the range of 0.1 nM to 50 nM, 0.1 nM to 100 nM, 0.1 nM to 1 μM, 0.5 nM to 50 nM, 0.5 nM to 100 nM, 0.5 nM to 1 μM, 1 nM to 50 nM, 1 nM to 100 nM, or 1 nM to 1 μM.

[0208] In certain embodiments, the serum albumin binding based tandem Adnectins described herein (e.g., PCSK9-PKE2 tandem Adnectins) bind to serum albumin in the pH range of 5.5 to 7.4.

[0209] In certain embodiments, the serum albumin binding based tandem Adnectins described herein (e.g., PCSK9-PKE2 tandem Adnectins) bind to domains I-II of human serum albumin.

[0210] In certain embodiments, the serum albumin binding based tandem Adnectins described herein (e.g., PCSK9-PKE2 tandem Adnectins) have a serum half-life of at least 1 hour, 2 hours, 5 hours, 10 hours, 20 hours, 30 hours, 40 hours, 50 hours, 60 hours, 70 hours, 80 hours, 90 hours, 100 hours, 150 hours, 200 hours, or at least about 300 hours in the presence of human serum albumin, cynomolgus serum albumin, rhesus serum albumin, mouse serum albumin, and / or rat serum albumin. In certain embodiments, the serum albumin binding based tandem Adnectins described herein (e.g., PCSK9-PKE2 tandem Adnectins) have, e.g., 1-300 hours, such as 1-250 hours, 1-200 hours, 1-150 hours, 1-100 hours, 1-90 hours, 1-80 hours, 1-70 hours, 1-60 hours, 1-50 hours, 1-40 hours, 1-50 hours, 1-60 hours, 1-70 hours, 1-80 hours, 1-90 hours, 1-10 ... -30 hours, 1-20 hours, 1-10 hours, 1-5 hours, 5-300 hours, 10-300 hours, 20-300 hours, 30-300 hours, 40-300 hours, 50-300 hours, 60-300 hours, 70-300 hours, 80-300 hours, 90-300 hours, 100-300 hours, 150-300 hours, 200-300 hours, 250-300 hours, 5-250 hours, 10-200 hours, 50-150 hours, or 80-120 hours of serum half-life.

[0211] In certain embodiments, the serum half-life of the partner Adnectin (e.g., in the case of PCSK9-PKE2 tandem Adnectins, a PCSK9 Adnectin) in the serum albumin-based tandem Adnectin is increased relative to the serum half-life of the partner Adnectin when not conjugated to a serum albumin binding Adnectin. In certain embodiments, the serum half-life of the serum albumin-based tandem Adnectin 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 relative to the serum half-life of the partner Adnectin when not fused to a serum albumin binding Adnectin. In certain embodiments, the serum half-life of the serum albumin-based tandem Adnectin is 20-3000% longer, such as 40-3000%, 60-3000%, 80-3000%, 100-3000%, 120-3000%, 150-3000%, 180-3000%, 200-3000%, 400-3000%, 600-3000%, 800-3000%, 1000-3000%, 1200-3000%, 1500-3000%, 1800-3000%, 1 900-3000%, 2000-3000%, 2500-3000%, 20-2500%, 20-2000%, 20-1900%, 20-1800%, 20-1500%, 20-1200%, 20-1000%, 20-800%, 20-600%, 20-400%, 20-200%, 20-180%, 20-150%, 20-120%, 20-100%, 20-80%, 20-60%, 20-40%, 50-2500%, 100-2000%, 150-1500%, 200-1000%, 400-800%, or 500-700%. In certain embodiments, the serum half-life of the serum albumin binding based tandem Adnectin is at least 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 10-fold, 12-fold, 13-fold, 15-fold, 17-fold, 20-fold, 22-fold, 25-fold, 27-fold, 30-fold, 35-fold, 40-fold, or 50-fold greater than the serum half-life of the partner Adnectin when not fused to the serum albumin binding Adnectin.In certain embodiments, the serum half-life of the serum albumin binding based tandem Adnectin is 1.5-50 fold, such as 1.5-40 fold, 1.5-35 fold, 1.5-30 fold, 1.5-27 fold, 1.5-25 fold, 1.5-22 fold, 1.5-20 fold, 1.5-17 fold, 1.5-15 fold, 1.5-13 fold, 1.5-12 fold, 1.5-10 fold, 1.5-9 fold, 1.5-8 fold, 1.5-7 fold, 1.5-8 fold, 1.5-9 fold, 1.5-10 fold, 1.5-11 fold, 1.5-12 fold, 1.5-13 fold, 1.5-14 fold, 1.5-15 fold, 1.5-16 fold, 1.5-17 fold, 1.5-18 fold, 1.5-19 fold, 1.5-20 fold, 1.5-21 fold, 1.5-22 fold, 1.5-2 1.5-4.5 times, 1.5-4 times, 1.5-3.5 times, 1.5-3 times, 1.5-2.5 times, 1.5-2 times, 2-50 times, 2.5-50 times, 3-50 times, 3.5-50 times, 4-50 times, 4.5-50 times, 5-50 times, 6-50 times, 7-50 times, 8-50 times, 10-50 times, 12-50 times, 13-50 times, 15-50 times, 17-50 times, 20-50 times, 22-50 times, 25-50 times, 27-50 times, 30-50 times, 40-50 times, 2-40 times, 5-35 times, 10-20 times, or 10-15 times. In certain embodiments, the serum half-life of the serum albumin binding based tandem Adnectin 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, 40 hours, 50 hours, 60 hours, 70 hours, 80 hours, 90 hours, 100 hours, 110 hours, 120 hours, 130 hours, 135 hours, 140 hours, 150 hours, 160 hours, or 200 hours.In certain embodiments, the serum half-life of the serum albumin binding based tandem Adnectin is 2-200 hours, 2.5-200 hours, 3-200 hours, 4-200 hours, 5-200 hours, 6-200 hours, 7-200 hours, 8-200 hours, 9-200 hours, 10-200 hours, 15-200 hours, 20-200 hours, 25-200 hours, 30-200 hours, 35-200 hours, 40-200 hours, 50-200 hours, 60-200 hours, 70-200 hours, 80-200 hours, 90-200 hours, 100-200 hours, 125-200 hours, 150-2 00 hours, 175-200 hours, 190-200 hours, 2-190 hours, 2-175 hours, 2-150 hours, 2-125 hours, 2-100 hours, 2-90 hours, 2-80 hours, 2-70 hours, 2-60 hours, 2-50 hours, 2-40 hours, 2-35 hours, 2-30 hours, 2-25 hours, 2-20 hours, 2-15 hours, 2-10 hours, 2-9 hours, 2-8 hours, 2-7 hours, 2-6 hours, 2-5 hours, 2-4 hours, 2-3 hours, 5-175 hours, 10-150 hours, 15-125 hours, 20-100 hours, 25-75 hours, or 30-60 hours.

[0212] E. Conjugates of serum albumin-binding Adnectin

[0213] Certain aspects of the invention provide conjugates comprising a serum albumin binding Adnectin and at least one additional moiety (e.g., a therapeutic moiety). The additional moiety can be used for diagnostic, imaging, or therapeutic purposes.

[0214] In some embodiments, the serum albumin binding Adnectin is fused to a second moiety, which is a small organic molecule, a nucleic acid, a peptide, or a protein. In some embodiments, the serum albumin binding Adnectin is fused to a therapeutic moiety that targets a receptor, a receptor ligand, a viral coat protein, an immune system protein, a hormone, an enzyme, an antigen, or a cell signaling protein. The fusion can be formed by attaching the second moiety to either end of the serum albumin binding Adnectin, i.e., the following arrangement: serum albumin binding Adnectin—therapeutic molecule, or therapeutic molecule—serum albumin binding Adnectin.

[0215] In certain embodiments, the serum half-life of the moiety fused to the serum albumin binding Adnectin is increased relative to the serum half-life of the moiety when not conjugated to the serum albumin binding Adnectin. In certain embodiments, the serum half-life of the serum albumin binding Adnectin 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 moiety when not fused to the serum albumin binding Adnectin. In certain embodiments, the serum half-life of the serum albumin binding based tandem Adnectin fusion is 20-3000%, such as 40-3000%, 60-3000%, 80-3000%, 100-3000%, 120-3000%, 150-3000%, 180-3000%, 200-3000%, 400-3000%, 600-3000%, 800-3000%, 1000-3000%, 1200-3000%, 1500-3000%, 1800-3000%, 1900-3000%, or 1500-3000%. 0-3000%, 2000-3000%, 2500-3000%, 20-2500%, 20-2000%, 20-1900%, 20-1800%, 20-1500%, 20-1200%, 20-1000%, 20-800%, 20-600%, 20-400%, 20-200%, 20-180%, 20-150%, 20-120%, 20-100%, 20-80%, 20-60%, 20-40%, 50-2500%, 100-2000%, 150-1500%, 200-1000%, 400-800%, or 500-700%. In certain embodiments, the serum half-life of the PKE2 Adnectin fusion is at least 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 10-fold, 12-fold, 13-fold, 15-fold, 17-fold, 20-fold, 22-fold, 25-fold, 27-fold, 30-fold, 35-fold, 40-fold, or 50-fold greater than the serum half-life of that moiety when not fused to a serum albumin binding Adnectin.In certain embodiments, the serum half-life of the PKE2 Adnectin fusion is 1.5-50 times, such as 1.5-40 times, 1.5-35 times, 1.5-30 times, 1.5-27 times, 1.5-25 times, 1.5-22 times, 1.5-20 times, 1.5-17 times, 1.5-15 times, 1.5-13 times, 1.5-12 times, 1.5-10 times, 1.5-9 times, 1.5-8 times, 1.5-7 times, 1.5-6 times, 1.5-5 times, 1.5-4.5 times, 1.5-5 times, 1.5-6 times, 1.5-7 times, 1.5-8 times, 1.5-9 times, 1.5-10 times, 1.5-11 times, 1.5-12 ... fold, 1.5-4-fold, 1.5-3.5-fold, 1.5-3-fold, 1.5-2.5-fold, 1.5-2-fold, 2-50-fold, 2.5-50-fold, 3-50-fold, 3.5-50-fold, 4-50-fold, 4.5-50-fold, 5-50-fold, 6-50-fold, 7-50-fold, 8-50-fold, 10-50-fold, 12-50-fold, 13-50-fold, 15-50-fold, 17-50-fold, 20-50-fold, 22-50-fold, 25-50-fold, 27-50-fold, 30-50-fold, 40-50-fold, 2-40-fold, 5-35-fold, 10-20-fold, or 10-15-fold. In some embodiments, the serum half-life of the serum albumin binding Adnectin fusion 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, 40 hours, 50 hours, 60 hours, 70 hours, 80 hours, 90 hours, 100 hours, 110 hours, 120 hours, 130 hours, 135 hours, 140 hours, 150 hours, 160 hours, or 200 hours.In certain embodiments, the serum half-life of the serum albumin binding Adnectin fusion is 2-200 hours, 2.5-200 hours, 3-200 hours, 4-200 hours, 5-200 hours, 6-200 hours, 7-200 hours, 8-200 hours, 9-200 hours, 10-200 hours, 15-200 hours, 20-200 hours, 25-200 hours, 30-200 hours, 35-200 hours, 40-200 hours, 50-200 hours, 60-200 hours, 70-200 hours, 80-200 hours, 90-200 hours, 100-200 hours, 125-200 hours, 150-2 00 hours, 175-200 hours, 190-200 hours, 2-190 hours, 2-175 hours, 2-150 hours, 2-125 hours, 2-100 hours, 2-90 hours, 2-80 hours, 2-70 hours, 2-60 hours, 2-50 hours, 2-40 hours, 2-35 hours, 2-30 hours, 2-25 hours, 2-20 hours, 2-15 hours, 2-10 hours, 2-9 hours, 2-8 hours, 2-7 hours, 2-6 hours, 2-5 hours, 2-4 hours, 2-3 hours, 5-175 hours, 10-150 hours, 15-125 hours, 20-100 hours, 25-75 hours, or 30-60 hours.

[0216] In certain embodiments, the serum albumin binding Adnectin fusion protein has a K of less than 3 μM, 2.5 μM, 2 μM, 1.5 μM, 1 μM, 500 nM, 100 nM, 50 nM, 10 nM, 1 nM, 500 pM, 100 pM, 100 pM, 50 pM, or 10 pM. D Binds to HSA. D It may, for example, be in the range of 0.1 nM to 50 nM, 0.1 nM to 100 nM, 0.1 nM to 1 μM, 0.5 nM to 50 nM, 0.5 nM to 100 nM, 0.5 nM to 1 μM, 1 nM to 50 nM, 1 nM to 100 nM, or 1 nM to 1 μM.

[0217] In some embodiments, the therapeutic moiety can be linked directly or indirectly to the serum albumin binding Adnectin via a polymeric linker, as described herein. A polymeric linker can be used to optimally alter the distance between the various components of the fusion to produce a protein fusion having one or more of the following characteristics: 1) reduced or increased steric hindrance of binding to one or more protein domains when bound to a protein of interest; 2) increased protein stability or solubility, 3) reduced protein aggregation, and 4) increased overall protein affinity or avidity.

[0218] In some embodiments, the fusions described herein are linked to serum albumin binding Adnectins via a polypeptide linker having a protease site that can be cleaved by proteases in the blood or target tissues. Such embodiments can be used to release therapeutic proteins for better delivery or therapeutic properties or more efficient production.

[0219] Available in 10 The C-terminus of the Fn3 domain is introduced with an additional linker or spacer so that it is located 10 between the Fn3 domain and the polypeptide linker.

[0220] In some embodiments, the therapeutic moiety is linked to the serum albumin binding Adnectin via a biocompatible polymer (e.g., a polymeric sugar). The polymeric sugar may include an enzymatic cleavage site that can be cleaved by enzymes in the blood or target tissue. Such embodiments can be used to release the therapeutic protein to achieve better delivery or therapeutic properties or more efficient production.

[0221] The serum albumin binding Adnectin fusion molecules described herein can be used to increase the half-life of the therapeutic moiety by forming a fusion between the therapeutic moiety and the serum albumin binding Adnectin. Such fusion molecules can be used to treat conditions that respond to the biological activity of the therapeutic moiety contained in the fusion. The present invention contemplates the use of serum albumin binding Fn3 fusion molecules in diseases caused by disorders of any of the following proteins or molecules.

[0222] In exemplary embodiments, the therapeutic moiety linked (C-terminus or N-terminus) to the serum albumin binding Adnectin is VEGF, VEGF-R1, VEGF-R2, VEGF-R3, Her-1, Her-2, Her-3, EGF-I, EGF-2, EGF-3, A3, cMet, ICOS, CD40L, LFA-I, c-Met, ICOS, LFA-I, IL-6, B7.1, W1.2, OX40, IL-1b, TACI, IgE, BAFF or BLys, TPO-R, CD19, CD20, CD22, CD33, CD28, IL-I-R1, TNF-α, TRAIL-R1, complement receptor 1, FGFa, osteopontin, vitronectin, ephrins A1-A5, ephrins B1-B3, alpha-2-macroglobulin, CCL1, CCL 2. CCL3, CCL4, CCL5, CCL6, CCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CCL13, CCL14, CCL15, CXCL16, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, PDGF, TGFb, GMCSF, SCF, p40 (IL12 / IL23), IL1b, IL1a, IL1ra, IL2, IL3, IL4, IL5, IL6, IL8, IL10, IL12, IL15, IL23, Fas, FasL, Flt3 ligand, 41BB, ACE, ACE-2, KGF, FGF-7, SCF, neural guidance factor 1,2, IFNa,b,g, caspase-2,3,7,8,10, ADAM S1,S5,8,9,15,TS1,TS5; adiponectin, ALCAM, ALK-I, APRIL, annexin V, angiopoietin, amphiregulin, angiopoietin-1,2,4, B7-1 / CD80, B7-2 / CD86, B7-H1, B7-H2, B7-H3, Bcl-2, BACE-I, BAK, BCAM, BDNF, bNGF, bECGF, BMP2,3,4,5,6,7,8; CRP, cadherin 6,8,11; cathepsins A,B,C,D,E,L,S,V,X; CD1 1a / LFA-1, LFA-3, GP2b3a, GH receptor, RSVF protein, IL-23 (p40, p19), IL-12, CD80, CD86, CD28, CTLA-4, α4-β1, α4-β7, TNF / lymphotoxin, IgE, CD3, CD20, IL-6, IL-6R, BLYS / BAFF, IL-2R, HER2, EGFR, CD33, CD52, digoxin, Rho(D), Varicella, Hepatitis, CMV, Tetanus, Vaccinia, Anti-snake venom serum, Botulinum toxin, Trail-R1, Trail-R2, cMet, TNF-R family, such as LA NGF-R, CD27, CD30, CD40, CD95, Lymphotoxin a / b receptor, WsI-I, TL1A / TNFSF15, BAFF, BAFF-R / TNFRSF13C, TRAIL R2 / TNFRSF10B, TRAIL R2 / TNFRSF10B, Fas / TNFRSF6 CD27 / TNFRSF7, DR3 / TNFRSF25, HVEM / TNFRSF14, TROY / TNFRSF19, CD40 ligand / TNFSF5, BCMA / TNFRSF17, CD30 / TNFRSF8, LIGHT / TNFSF14, 4-1BB / TNFRSF9, CD40 / TNFRSF5, GITR / [γ]NFRSF 18, osteoprotegerin / TNFRSF1 IB, RANK / TNFRSF1 IA, TRAIL R3 / TNFRSF10C, TRAIL / TNFSFIO, TRANCE / RANK L / TNFSF11, 4-1BB ligand / TNFSF9, TWEAK / TNFSF12, CD40 ligand / TNFSFS, Fas ligand / TNFSF6, RELT / TNFRSF19L, APRIL / TNFSF13, DcR3 / TNFRSF6B, TNF RI / TNFRSFIA, TRAIL R1 / TNFRSFIOA, TRAIL R4 / TNFRSF10D, CD30 ligand / TNFSF8, GITR ligand / TNFSF18, TNFSF18, TACI / TNFRSF13B, NGF R / TNFRSF16, OX40 ligand / TNFSF4, TRAIL R2 / TNFRSF10B, TRAIL R3 / TNFRSF10C, TWEAK R / TNFRSF12, BAFF / BLyS / TNFSF13, DR6 / TNFRSF21, TNF-α / TNFSF1 A, Pro-TNF-α / TNFSF1A, Lymphotoxin βR / TNFRSF3, Lymphotoxin βR (LTbR) / Fc chimera, TNFRI / TNFRSFIA, TNF-β / TNFSF1B, PGRP-S, TNF RI / TNFRSFIA, TNF RII / TNFRSFIB, EDA-A2, TNF-α / TNFSFIA, EDAR, XEDAR, TNF RI / TNFRSFIA.

[0223] In exemplary embodiments, the therapeutic moiety attached (C-terminus or N-terminus) to the serum albumin binding Adnectin is any of the following proteins or a protein that binds thereto: 4EBP1, 14-3-3ζ, 53BP1, 2B4 / SLAMF4, CCL21 / 6Ckine, 4-1BB / TNFRSF9, 8D6A, 4-1BB Ligand / TNFSF9, 8-oxo-dG, 4-amino-1,8-naphthalimide, A2B5, aminopeptidase LRAP / ERAP2, A33, aminopeptidase N / ANPEP, Aag, aminopeptidase P2 / XPNPEP2, ABCG2, aminopeptidase P1 / XPNPEP1, ACE, aminopeptidase PILS / ARTS1 , ACE-2, anamnion, actin, amphiregulin, β-actin, AMPKα1 / 2, activin A, AMPKα1, activin AB, AMPKα2, activin B, AMPKβ1, activin C, AMPKβ2, activin RIA / ALK-2, androgen R / NR3C4, activin RIB / ALK-4, angiogenin, activin RIIA, angiogenin-1, activin RIIB, angiogenin-2, ADAMS, angiogenin-3, ADAM9, angiogenin-4, ADAM1O, angiogenin-like 1, ADAM12, angiogenin-like 2, ADAM15, angiogenin-like 3, TACE / ADAM17, angiogenin eopoietin-like 4, ADAM19, angiopoietin-like 7 / CDT6, ADAM33, angiostatin, ADAMTS4, annexin A1 / annexin I, ADAMTS5, annexin A7, ADAMTS1, annexin A10, ADAMTSL-1 / Punctin, annexin V, adiponectin / Acrp30, ANP, AEBSF, AP site, aggrecan, APAF-I, aggrecan, APC, AgRP, APE, AGTR-2, APJ, AIF, APLP-I, Akt, APLP-2, Akt1, apolipoprotein AI, Akt2, apolipoprotein B, Akt3, APP, serum albumin, APRIL / T NFSF13, ALCAM, ARC, ALK-I, Artemin, ALK-7, arylsulfatase AJARSA, alkaline phosphatase, ASAH2 / N-acylsphingosine asparaginase-2, α2u-globulin, ASC, α-1-acid glycoprotein, ASGR1, α-fetoprotein, ASK1, ALS, ATM, ameloblast ATRIP, AMICA / JAML, mitotic kinase A, AMIGO, mitotic kinase B, AMIG02, Axin-1, AMIG03, AxI, aminoacylase / ACY1, azurocidin / CAP37 / HBP, aminopeptidase A / ENPEP, B4GALT1, BIM, B7-1 / CD80,6-Biotin-17-NAD, B7-2 / CD86, BLAME / SLAMF8, B7-H1 / PD-L1, CXCL13 / BLC / BCA-1, B7-H2, BLIMP1, B7-H3, BIk, B7 -H4, BMI-I, BACE-I, BMP-1 / PCP, BACE-2, BMP-2, Bad, BMP-3, BAFF / TNFSF13B, BMP-3b / GDF-10, BAFFR / TNFRSF 13C, BMP-4, Bag-1, BMP-5, BAK, BMP-6, BAMBI / NMA, BMP-7, BARD 1. BMP-8, Bax, BMP-9, BCAM, BMP-10, Bcl-10, BMP-15 / GDF-9B, Bcl-2, BMPR-IA / ALK-3, Bcl-2-associated protein A1, BMPR-IB / ALK-6, Bcl-w, BMPR-II, Bcl-x, BNIP3L, Bcl-xL, BOC, BCMA / TNFRSF17, BOK, BDNF, BPDE, benzamide, mouse short-tail mutant phenotype, common β chain, B-Raf, βIG-H3, CXCL14 / BRAK, B cell factor, BRCA1, β-defensin 2, BRCA2, BID, BTLA, biglycan, Bub-1, Bik-like killer protein, c-jun, CD90 / Thyl, c-Rel, CD94, CCL6 / C10, CD97, CIq R1 / CD93, CD151, CIqTNF1, CD160, ClqTNF4, CD163, ClqTNF5, CD164, complement component CIr, CD200, complement component CIs, CD200R1, complement component C2, CD229 / SLAMF3, complement component C3a, CD23 / FcεR11, complement component C3d, CD2F-10 / SLAMF9, complement component C5a, CD5L, cadherin-4 / R-cadherin, CD69, cadherin-6, CDC2, cadherin-8, CDC25A, cadherin-11, CDC25B, cadherin-12, CDCP1, cadherin-13, CDO, cadherin-17, CDX4, E-cadherin, CEACAM-1 / CD66a, N-cadherin, CEACAM-6, P-cadherin, Cerberus1, VE-cadherin, CFTR, calcium binding protein D, cGMP, calcineurin A, Chem R23, calcineurin B, chemokine, calreticulin-2, chemokine sampler pack, CaM kinase II, chitinase 3-like 1, cAMP, chitotriosidase / CHIT1, cannabinoid R1, Chk1,Cannabinoids R2 / CB2 / CNR2, Chk2, CAR / NR1I3, CHL-1 / L1CAM-2, Carbonic Anhydrase I, Choline Acetyltransferase / CbAT, Carbonic Anhydrase II, Chondroitin, Carbonic Anhydrase III, Chordopoietin, Carbonic Anhydrase IV, Chordopoietin-like 1, Carbonic Anhydrase VA, Chordopoietin-like 2, Carbonic Anhydrase VB, CINC-I, Carbonic Anhydrase VI, CINC-2, Carbonic Anhydrase VII, CINC-3, Carbonic Anhydrase VIII, Claspin, Carbonic Anhydrase IX, Tight Junction Protein-6, Carbonic Anhydrase X, CLC, Carbonic Anhydrase XII, CLEC-I, Carbonic Anhydrase XIII, CLEC-2, Carbonic Anhydrase XIV, CLECSF 13 / CLEC4F, carboxymethyllysine, CLECSF8, carboxypeptidase A1 / CPA1, CLF-I, carboxypeptidase A2, CL-P1 / COLEC12, carboxypeptidase A4, clusterin, carboxypeptidase B1, clusterin-like 1, carboxypeptidase E / CPE, CMG-2, carboxypeptidase X1, CMV UL146, cardiotrophin-1, CMV UL147, carnosine dipeptidase 1, CNP, Caronte, CNTF, CART, CNTF Rα, caspase, coagulation factor II / thrombin, caspase-1, coagulation factor I11 / tissue factor, caspase-2, coagulation factor VII, caspase-3, coagulation factor X, caspase-4, coagulation factor XI, caspase-6, coagulation factor XIV / protein C, caspase-7, COCO, caspase-8, adhesion protein, caspase-9, collagen I, caspase-10, collagen II, caspase-12, collagen IV, caspase-13, common gamma chain n / IL-2Rγ, peptide inhibitor of caspase, COMP / thrombospondin-5, catalase, complement component CIrLP, beta-catenin white, complement component CIqA, cathepsin 1, complement component CIqC, cathepsin 3, complement factor D, cathepsin 6, complement factor I, cathepsin A, complement MASP3, cathepsin B, connexin 43, cathepsin C / DPPI, contactin-1, cathepsin D, contactin-2 / TAG1, cathepsin E, contactin-4, cathepsin F, contactin-5, cathepsin H, atrial natriuretic peptide converting enzyme (Corin), cathepsin L, Cornulin, cathepsin O, CORS26 / ClqTNF,3, cathepsin S, rat cortical stem cells, cathepsin V, cortisol, cathepsin XITJ?, COUP-TF I / NR2F1, CBP, COUP-TF II / NR2F2, CCI, COX-I, CCK-AR, COX-2, CCL28,CRACC / SLAMF7, CCR1, C-reactive protein, CCR2, creatine kinase, muscle / CKMM, CCR3, creatinine, CCR4, CREB, CCR5, CREG, CCR6, CRELD1, CCR7, CRELD2, CCR8, CRHBP, CCR9, CRHR-I, CCR1O, CRIM1, CD155 / PVR, Cripto, CD2, CRISP-2, CD3, CRISP-3, CD4, Crossveinless-2, CD4+ / 45RA-, CRTAM, CD4+ / 45RO, CRTH-2, C D4+ / CD62L- / CD44, CRY1, CD4+ / CD62L+ / CD44, Cryptic, CD5, CSB / ERCC6, CD6, CCL27 / CTACK, CD8, CTGF / CCN2, CD8+ / 45RA-, CTLA-4, CD8+ / 45R O-, Cubilin, CD9, CX3CR1, CD14, CXADR, CD27 / TNFRSF7, CXCL16, CD27 ligand / TNFSF7, CXCR3, CD28, CXCR4, CD30 / TNFRSF8, CXCR5, CD30 ligand / TNFSF8 、CXCR6、CD31 / PECAM-1、cyclophilin A、CD34、Cyr61 / CCN1、CD36 / SR-B3、cystatin A、CD38、cystatin B、CD40 / TNFRSF5、cystatin C、CD40 ligand / TNFSF5、cystatin D、CD43、cystatin E / M、CD44、cystatin F、CD45、cystatin H、CD46、cystatin H2、CD47、cystatin S、CD48 / SLAMF2、cystatin SA、CD55 / DAF、cystatin SN、CD58 / LFA-3、cytochrome c、CD59、deheme cytochrome c、CD68、whole cell color c, CD72, cytokeratin 8, CD74, cytokeratin 14, CD83, cytokeratin 19, CD84 / SLAMF5, Cytonin, D6, DISP1, DAN, Dkk-1, DANCE, Dkk-2, DARPP-32, Dkk-3, DAX1 / NR0B1, Dkk-4, DCC, DLEC, DCIR / CLEC4A, DLL1, DCAR, DLL4, DcR3 / TNFRSF6B, d-luciferin, DC-SIGN, DNA ligase IV, DC-SIGNR / CD299, DNA polymerase β, DcTRAIL R1 / TNFRSF23, DNAM-I, DcTRAIL R2 / TNFRSF22, DNA-PKcs, DDR1, DNER, DDR2, dopa decarboxylase / DDC,DEC-205, DPCR-I, Decapentaplegic, DPP6, Decorin, DPP A4, DCA-1 / CLEC7A, DPPA5 / ESG1, DCA-2 / CLEC6A, DPPII / QPP / DPP7, DEP-1 / CD148, DPPIV / CD26, Desert Hedgehog Factor, DR3 / TNFRSF25, Desmin, DR6 / TNFRSF21, Desmoglein-1, DSCAM, Desmoglein-2, DSCAM-L1, Desmoglein-3, DSPG3, Dishevelled-1, Dtk, Dishevelled-3 , dynamin, EAR2 / NR2F6, EphA5, ECE-I, EphA6, ECE-2, EphA7, ECF-L / CHI3L3, EphA8, ECM-I, EphB1, Ecotin, EphB2, EDA, EphB3, EDA-A2, EphB4, EDAR, EphB6, EDG-I, ephrin, EDG-5, ephrin-A1, EDG-8, ephrin-A2, eEF-2, ephrin-A3, EGF, ephrin-A4, EGF R, ephrin-A5, EGR1, ephrin-B, EG-VEGF / PK1, ephrin-B1, eIF2α, ephrin-B2, eIF4E, ephrin-B3, EIk-I, Epigen, EMAP-II, epidermal morphogen / synaptophysin 2, EMMPRIN / CD147, epiregulin, CXCL5 / ENA, EPR-1 / Xa receptor, endothelial cell-specific molecule-1 (Endocan), ErbB2, endoglin / CD105, ErbB3, tetrasaccharide (En doglycan), ErbB4, Endonuclease III, ERCC1, Endonuclease IV, ERCC3, Endonuclease V, ERK1 / ERK2, Endonuclease VIII, ERK1, Endorepellin / Perlecan, ERK2, Cortistatin, ERK3, Endothelin-1, ERK5 / BMK1, Engrailed-2, ERRα / NR3B1, EN-RAGE, ERRβ / NR3B2, Enteropeptidase / Enterokinase, ERRγ / NR3B3, CCL1 1 / eotaxin, erythropoietin, CCL24 / eotaxin-2, erythropoietin R, CCL26 / eotaxin-3, ESAM, EpCAM / TROP-1, ERα / NR3A1, EPCR, ERβ / NR3A2, Eph, exonuclease III, EphA1, exostosis-like 2 / EXTL2, EphA2,Exostosis-like 3 / EXTL3, EphA3, FABP1, FGF-BP, FABP2, FGF R1-4, FABP3, FGF R1, FABP4, FGF R2, FABP5, FGF R3, FABP7, FGF R4, FABP9, FGF R5, complement factor B, Fgr, FADD, FHR5, FAM3A, fibronectin, FAM3B, fibrin-2, FAM3C, fibrin-3, FAM3D, FITC, fibroblast activation protein α / FAP, FKBP38, Fas / TNFRSF6, 5-lipoxygenase activating protein (Flap), Fas ligand / TNFSF6, FLIP, FATP1, FLRG, FATP4, FLRT1, FA TP5, FLRT2, FcγR1 / CD64, FLRT3, FcγRIIB / CD32b, Flt-3, FcγRIIC / CD32c, Flt-3 ligand, FcγRIIA / CD32a, follistatin, FcγRIII / CD16, follistatin-like 1, FcRH1 / IRTA5, FosB / G0S3, FcRH2 / IRTA4, FoxD3, FcRH4 / IRTA1, FoxJ1, FcRH 5 / IRTA2, FoxP3, Fc receptor-like 3 / CD16-2, Fpg, FEN-I, FPR1, fetuin A, FPRL1, fetuin B, FPRL2, acidic FGF, CX3CL1 / Fractin, basic FGF, Frizzled-1, FGF-3, Frizzled-2, FGF-4, Frizzled-3, FGF-5, Frizzled-4, FGF-6, Frizzled-5, FGF-8, Frizzled-6, FGF-9, Frizzled FGF-7, FGF-IO, Frizzled-8, FGF-11, Frizzled-9, FGF-12, Frk, FGF-13, sFRP-1, FGF-16, sFRP-2, FGF-17, sFRP-3, FGF-19, sFRP-4, FGF-20, furin, FGF-21, FXR / NR1H4, FGF-22, Fyn, FGF-23, G9a / EHMT2, GFRα-3 / GDNF Rα-3, GABA-A-Rα1, GFRα-4 / GDNF Rα-4, GABA-A-Rα2, GITR / TNFRSF18, GABA-A-Rα4, GITR ligand / TNFSF18, GABA-A-Rα5, GLI-I, GABA-A-Rα6, GLI-2, GABA-A-Rβ1, GLP / EHMT1, GABA-A-Rβ2, GLP-I R, GABA-A-Rβ3, glucagon, GABA-A-Rγ2, glucosamine (N-acetyl)-6-sulfatase / GNS,GABA-B-R2, GIuR1, GAD1 / GAD67, GluR2 / 3, GAD2 / GAD65, GluR2, GADD45α, GluR3, GADD45β, Glut1, GADD45γ, Glut2, Galectin-1, Glut3, Galectin-2, Glut4, Galectin-3, Glut5, Galectin-3BP, Gluredoxin 1, Galectin-4, Glycine R, Galectin-7, Glycophorin A, Galectin-8, Glypican 2, Galectin-9, Glypican 3, GalNAc4S-6ST, Glypican 5, GAP-43, Glypican 6, GAPDH, GM-CSF, Gas1, GM-CSF Rα, Gas6, GMF-β, GASP-1 / WFIKKNRP, gpl30, GASP-2 / WFIKKN, glycogen phosphorylase BB / GPBB, GATA-I, GPR15, GATA-2, GPR39, GATA-3, GPVI, GATA-4, GR / NR3C1, GATA-5, Gr-1 / Ly-6G, GATA-6, granulysin, GBL, granzyme A, GCNF / NR6A1, granzyme B, CXCL6 / GCP-2, granzyme D, G-CSF, granzyme G, G-CSF R, granzyme H, GDF-I, GRASP, GDF-3GRB2, GDF-5, Gremlin, GDF-6, GRO, GDF-7, CXCL1 / GROα, GDF-8, CXCL2 / GROβ, GDF-9, CXCL3 / GROγ, GDF-11, growth hormone, GDF-15, growth hormone R, GDNF, GRP75 / HSPA9B, GFAP, GSK-3α / β, GFI-I, GSK-3α, GFRα-1 / GDNF Rα-1, GSK-3β, GFRα-2 / GDNF Rα-2, EZFIT, H2AX, histidine, H60, HM74A, HAI-I, HMGA2, HAI-2, HMGB1, HAI-2A, TCF-2 / HNF-1β, HAI-2B, HNF-3β / FoxA2, ​​HAND1, HNF-4α / NR2A1, HAPLN1, HNF-4γ / NR2A2, airway trypsin-like protease / HAT, HO-1 / HMOX1 / HSP32, HB-EGF, HO-2 / HMOX2, CCL 14a / HCC-1, HPRG, CCL14b / HCC-3, Hrk, CCL16 / HCC-4, HRP-I, αHCG, HS6ST2, Hck, HSD-I, HCR / CRAM-A / B, HSD-2, HDGF,HSP 10 / EPF, hemoglobin, HSP27, hepatokinin, HSP60, HES-1, HSP70, HES-4, HSP90, HGF, HTRA / proteinase Do, HGF activator, HTRA1 / PRSS11, HGF R, HTRA2 / 0ml, HIF-Iα, HVEM / TNFRSF14, HIF-2α, hyaluronic acid, HIN-1 / secretase 3A1, 4-hydroxynonenal, Hip, CCL1 / I-309 / TCA-3, IL-IO, cIAP (pan), IL-IO Rα, cIAP-1 / HIAP-2, IL-10Rβ, cIAP-2 / HIAP-1, IL-11, IBSP / sialoprotein II, EL-11Rα, ICAM-1 / CD54, IL-1 2. ICAM-2 / CD102, IL-12 / IL-23p40, ICAM-3 / CD50, IL-12Rβ1, ICAM-5, IL-12Rβ2, ICAT, IL-13, IC OS, IL-13Rα1, iduronate 2-sulfatase / EOS, IL-13Rα2, EFN, IL-15, IFN-α, IL-15Rα, IFN-α1, IL-16, IFN-α2 , IL-17, IFN-α4b, IL-17R, IFN-αA, IL-17RC, IFN-αB2, IL-17RD, IFN-αC, IL-17B, IFN-αD, IL-17B R, IFN-αF, IL-17C, IFN-αG, IL-17D, IFN-αH2, IL-17E, IFN-αI, IL-17F, IFN-αJ1, IL-18 / IL-1F4, IFN-αK, IL-18BPa, IFN-αWA , IL-18BPc, IFN-α / βR1, IL-18BPd, IFN-α / βR2, IL-18Rα / IL-1R5, IFN-β, IL-18Rβ / IL-1R7, IFN-γ, IL-19, IFN-γR1, IL-20, IF N-γR2, IL-20Rα, IFN-ω, IL-20Rβ, IgE, IL-21, IGFBP-I, IL-21R, IGFBP-2, IL-22, IGFBP-3, IL-22R, IGFBP-4, IL-22BP, IGFBP -5, IL-23, IGFBP-6, IL-23R, IGFBP-L1, IL-24, IGFBP-rp1 / IGFBP-7, IL-26 / AK155, IGFBP-rPIO, IL-27, IGF-I, EL-28A, IGF-I R, IL-28B, IGF-II, IL-29 / EFN-λ1, IGF-II R, IL-31, IgG, EL-31RA, IgM,IL-32α, IGSF2, IL-33, IGSF4A / SynCAM, ILT2 / CD85J, IGSF4B, ILT3 / CD85k, IGSF8, ILT4 / CD85d, IgY, ILT5 / CD85a, IkB-β, ILT6 / CD85e, IKKα, Indian hedgehog Hedgehog), IKKε, INSRR, EKKγ, insulin, IL-1α / IL-IF1, insulin R / CD220, IL-1β / IL-1F2, proinsulin, IL-1ra / IL-1F3, insulinolysin / EDE, IL-1F5 / FIL1δ, integrin α2 / CD49b, IL-1F6 / FIL1ε, integrin α3 / CD49c, IL-1F7 / FIL1ζ, integrin α3β1 / VLA-3, IL-1F8 / FIL1ζ, integrin α4 / CD49d, IL-1F9 / IL-1H1, integrin α5 / CD49e, IL-1F10 / IL-1HY2, integrin α5β1, IL-I RI, integrin α6 / CD49f, IL-I RII, integrin α7, IL-I R3 / IL-1R AcP, integrin α9, IL-I R4 / ST2, integrin αE / CD103, IL-I R6 / IL-1R rp2, integrin αL / CD1 Ia, IL-I R8, integrin αLβ2, IL-I R9, integrin αM / CD1 Ib, IL-2, integrin αMβ2, IL-2Rα, integrin αV / CD51, IL-2Rβ, integrin αVβ5, IL-3, integrin αVβ3, IL-3Rα, integrin αVβ6, IL-3Rβ, integrin αXJCD1 Ic, IL-4, integrin β1 / CD29, IL-4R, integrin β2 / CD18, IL-5, integrin β3 / CD61, IL-5Rα, integrin β5, IL-6, integrin β6, IL-6R, integrin β7, IL-7, CXCL10 / EP-10 / CRG-2, IL-7Rα / CD127, IRAKI, CXCR1 / IL-8RA, IRAK4, CXCR2 / IL-8RB, ERS-I, CXCL8 / IL-8, Islet-1, IL-9, CXCL1 1 / I-TAC, IL-9R, Jagged 1, JAM-4 / IGSF5, Jagged 2, JNK, JAM-A, JNK1 / JNK2, JAM-B / VE-JAM, JNK1, JAM-C, JNK2, kininogen, kallikrein 3 / PSA, kallikrein, kallikrein 4, KER / CD158, kallikrein 5, KER2D1,Kallikrein 6 / Neurosin, KIR2DL3, Kallikrein 7, KIR2DL4 / CD158d, Kallikrein 8 / Neuropsin, KIR2DS4, Kallikrein 9, KIR3DL1, Plasma Kallikrein / KLKB1, KER3DL2, Kallikrein 10, Irregular Chiasmatic Protein 2, Kallikrein 11, KLF4, Kallikrein 12, KLF5, Kallikrein 13, KLF6, Kallikrein 14, Klotho, Kallikrein 15, Klotho β, KC, KOR, Keleh-like epichlorohydrin-associated protein (Keap) 1, Kremen-1, KeI1, Kremen-2, KGF / FGF-7, LAG-3, LINGO-2, LAIR1, Lipin 2, LAIR2, lipocalin-1, laminin α4, lipocalin-2 / NGAL, laminin γ1,5-lipoxygenase, laminin I, LXRα / NR1H3, laminin S, LXRβ / NR1H2, laminin-1, inhibitor of apoptosis (Livin), laminin-5, LEX, LAMP, LMIR1 / CD300A, Langerin, LMIR2 / CD300c, LAR, LMIR3 / CD300LF, latex, LMIR5 / CD300LB, hyaluronic acid receptor (Layilin), LMIR6 / CD300LE, LBP, LMO2, LDL R, LOX-1 / SR-E1, LECT2, LRH-1 / NR5A2, LEDGF, LRIG1, Lefty, LRIG3, Lefty-1, LRP-I, Lefty-A, LRP-6, legumin, LSECtin / CLEC4G, leptin, perlecan, leptin R, CXCL15 / Lungkine, leukotriene B4, XCL1 / lymphocyte chemoattractant factor, leukotriene B4R1, lymphotoxin, LEF, lymphotoxin β / TNFSF3, LIF Rα, lymphotoxin βR / TNFRSF3, LIGHT / TNFSF14, Lyn, Limitin, Lyp, LIMPII / SR-B2, lysyl oxidase homolog 2, LIN-28, LYVE-I, LINGO-I, α2-macroglobulin, CXCL9 / MIG, MAD2L1, osteoinductive factor (Mimecan), MAdCAM-1, Mindin, MafB, mineralocorticoid R / NR3C2, MafF, CCL3L1 / MIP-1α isoform LD78β, MafG, CCL3 / MIP-1α,MafK, CCL4L1 / LAG-1, MAG / sialic acid-binding immunoglobulin-like lectin (Siglec)-4-a, CCL4 / MIP-1β, MANF, CCL15 / MEP-1δ, MAP2, CCL9 / 10 / MIP-1γ, MAPK, MIP-2, Marapsin / Pancreasin, CCL19 / MIP-3β, MARCKS, CCL20 / MIP-3α, MARCO, MIP-I, Mash1, MIP-II, cartilage matrix protein-2, MIP-III, cartilage matrix protein-3, MIS / AMH, cartilage matrix protein-4, MIS RII, proteinase / ST14, MIXL1, MBL, MKK3 / MKK6, MBL-2, MKK3, melanocortin 3R / MC3R, MKK4, MCAM / CD146, MKK6, MCK-2, MKK7, McI-I, MKP-3, MCP-6, MLH-I, CCL2 / MCP-1, MLK4α, MCP-11, MMP, CCL8 / MCP-2, MMP-1, CCL7 / MCP-3 / MARC, MMP-2, CCL13 / MCP-4, MMP-3, CCL12 / MCP-5, MMP-7, M-CSF, MMP-8, M-CSF R, MMP-9, MCV type II, MMP-IO, MD-I, MMP-I 1, MD-2, MMP-12, CCL22 / MDC, MMP-13, MDL-1 / CLEC5A, MMP-14, MDM2, MMP-15, MEA-I, MMP-16 / MT3-MMP, MEK1 / MEK2, MMP-24 / MT5-MMP, MEK1, MMP-25 / MT6-MMP, MEK2, MMP-26, integrin β1 binding protein (Melusin), MMR, MEPE, MOG, menoprotein α, CCL23 / MPIF-1, menoprotein β, M-Ras / R-Ras3, Mer, Mrel 1, mesothelin, MRP1, MSK1 / MSK2, methionine aminopeptidase 1, MSK1, methionine aminopeptidase, MSK2, methionine aminopeptidase 2, MSP, MFG-E8, MSP R / Ron, MFRP, Mug, MgcRacGAP, MULT-I, MGL2, Musashi-1, MGMT, Musashi-2, MIA, MuSK, MICA, MutY DNA glycosylase, MICB, MyD88, MICL / CLEC12A, myeloperoxidase, β2 microglobulin, myocardin, midkine, actin, MIF,Myoglobin, NAIP NGFI-Bγ / NR4A3, Nanog, NgR2 / NgRH1, CXCL7 / NAP-2, NgR3 / NgRH2, Nbsl, Nestin-1 / Entramin, NCAM-1 / CD56, Nestin-2, NCAM-L1, Nitric Oxide, Nectin-1, Nitrotyrosine, Nectin-2 / CD1 12, NKG2A, Nectin-3, NKG2C, Nectin-4, NKG2D, Regeneration Protein, NKp30, Neprilysin / CDIO, NKp44, Neprilysin-2 / MMEL1 / MMEL2, NKp46 / NCR1, Nestin, NKp80 / KLRF1, NETO2, NKX2.5, Netrin-1, NMDAR, NR1 subunit, Netrin-2, NMDA R, NR2A subunit, Plectin-4, NMDAR, NR2B subunit, Plectin-Gla, NMDAR, NR2C subunit, Plectin-G2a, N-Me-6,7-diOH-TIQ, Neuregulin-1 / NRG1, Nodal, Neuregulin-3 / NRG3, Noggin, Neuritin, Nogo receptor, NeuroD1, Noggin-A, Neurofascin, NOMO, Neurogenic protein-1, Nope, Neurogenic protein-2, Norrin, Neurogenic protein-3, eNOS, Neurolytic protein, iNOS, Optomelanocortin II, nNOS, Neuro Pilin-1, Notch-1, Neuropilin-2, Notch-2, Neuropoietin, Notch-3, Neurotrimin, Notch-4, Neurturin, NOV / CCN3, NFAM1, NRAGE, NF-H, NrCAM, NFkB1, NRL, NFkB2, NT-3, NF-L, NT-4, NF-M, NTB-A / SLAMF6, NG2 / MCSP, NTH1, NGF R / TNFRSF16, nuclear stem cell factor, β-NGF, Nurr-1 / NR4A2, NGFI-Bα / NR4A1, OAS2, orexin B, OBCAM, OSCAR, OCAM, OSF-2 / periostin, OCIL / CLEC2d, oncostatin M / OSM, OCILRP2 / CLEC21, OSM Rβ, Oct-3 / 4, osteoactivin / GPNMB, OGG1, osteoadhesin, Olig 1, 2, 3, osteocalcin, Olig1, osteotactin, Olig2, osteopontin, Olig3, osteoprotegerin / TNFRSF1 IB,Oligodendrocyte marker 01, Otx2, Oligodendrocyte marker O4, OV-6, OMgp, OX40 / TNFRSF4, Oculoplasmic neoglycoprotein, OX40 ligand / TNFSF4, Orexin A, OAS2, Orexin B, OBCAM, OSCAR, OCAM, OSF-2 / periostin, OCIL / CLEC2d, Oncostatin M / OSM, OCILRP2 / CLEC2i, OSM Rβ, Oct-3 / 4, osteoactivin / GPNMB, OGG1, osteoadhesin, Olig1, 2, 3, osteocalcin, Olig1, osteotactin, Olig2, osteopontin, Olig3, osteoprotegerin / TNFRSF1IB, oligodendrocyte marker 01, Otx2, oligodendrocyte marker 04, OV-6, OMgp, OX40 / TNFRSF4, oculomotor glycoprotein, OX40 ligand / TNFSF4, orexin A, RACK1, Ret, Rad1, REV-ERBα / NR1 D1, Rad17, REV-ERBβ / NR1D2, Rad51, Rex-1, Rae-1, RGM-A, Rae-1α, RGM-B, Rae-1β, RGM-C, Rae-1δ, Rheb, Rae-1ε, ribosomal protein S6, Rae-1γ, RIP1, Raf-1, ROBO1, RAGE, ROBO2, Ra1A / Ra1B, R0B03, RaIA, ROBO4, RaIB, R0R / NR1F1-3(pan), RANK / TNFRSF1 1A, RORα / NR1F1, CCL5 / RANTES, RORγ / NR1F3, Rap1A / B, RTK-like orphan receptor 1 / ROR1, RARα / NR1B1, RTK-like orphan receptor 2 / ROR2, RARβ / NR1B2, RP105, RARγ / NR1B3, RPA2, Ras, RSK(pan), RBP4, RSK1 / RSK2, RECK, RSK1, Reg 2 / PAP, RSK2, Reg I, RSK3, Reg II, RSK4, Reg III, R-spondin 1, Reg I1ia, R-spondin 2, Reg IV, R-spondin 3, relaxin-1, RUNX1 / CBFA2, relaxin-2, RUNX2 / CBFA1, relaxin-3, RUNX3 / CBFA3, RELMα, RXRα / NR2B1, RELMβ, RXRβ / NR2B2, RELT / TNFRSF19L, RXRγ / NR2B3, resistin, S1OOAlO, SLITRK5, S100A8, SLPI, S100A9, SMAC / Diablo, S1OOB, Smad1, S1OOP, Smad2, SALL1, Smad3, delta-sarcoglycan,Smad4, Sca-1 / Ly6, Smad5, SCD-I, Smad7, SCF, Smad8, SCF R / c-kit, SMC1, SCGF, α-smooth muscle actin, SCL / Tall, SMUG1, SCP3 / SYCP3, Snail, CXCL12 / SDF-1, sodium calcium exchanger 1, SDNSF / MCFD2, Soggy-1, α-secretase, sonic hedgehog, γ-secretase, S or CS1, β-secretase, S or CS3, E-selectin, sortilin, L-selectin, SOST, P-selectin, SOX1, armplate protein 3A, SOX2, armplate protein 3C, SOX3, armplate protein 3E, SOX7, armplate protein 3F, SOX9, armplate protein 6A, SOX1O, armplate protein 6B, SOX 17, Armplate protein 6C, SOX21 Armplate protein 6D, SPARC, Armplate protein 7A, SPARC-like 1, Separase, SP-D, Serine / threonine phosphatase substrate I, Spondin, Serpin A1, F-spondin, Serpin A3, SR-AI / MSR, Serpin A4 / kallistatin, Src, Serpin A5 / protein C inhibitor, SREC-I / SR-F1, Serpin A8 / angiotensinogen, SREC-II, Serpin B5, SSEA-I, Serpin C1 / Anti-thrombin-III, SSEA-3, Serpin D1 / heparin cofactor II, SSEA-4, Serpin E1 / PAI-1, ST7 / LRP12, Serpin E2, Stabilin-1, Serpin F1, Stabilin- 2, Serpin F2, stanniocalcin 1, Serpin G1 / C1 inhibitor, stanniocalcin 2, Serpin 12, STAT1, serum amyloid A1, STAT2, SF-1 / NR5A1, STAT3, SGK, STAT4, SHBG, STAT5a / b, SHIP, STAT5a, SHP / NR0B2, STAT5b, SHP-I, STATE, SHP-2, VE-statin, SIG IRR, Stella / Dppa3, Sialic acid-binding immunoglobulin-like lectin-2 / CD22, STRO-I, Sialic acid-binding immunoglobulin-like lectin-3 / CD33, Substance P, Sialic acid-binding immunoglobulin-like lectin-5, Sulfamidase / SGSH, Sialic acid-binding immunoglobulin-like lectin-6, Sulfatase modification factor 1 / SUMF1, Sialic acid-binding immunoglobulin-like lectin-7, Sulfatase modification factor 2 / SUMF2,Sialic acid binding immunoglobulin-like lectin-9, SUMO1, Sialic acid binding immunoglobulin-like lectin-10, SUMO2 / 3 / 4, Sialic acid binding immunoglobulin-like lectin-11, SUMO3, Sialic acid binding immunoglobulin-like lectin-F, superoxide dismutase, SIGNR1 / CD209, superoxide dismutase-1 / Cu

[0099] --Zn SOD, SIGNR4, superoxide dismutase-2 / Mn-SOD, SIRPβ1, superoxide dismutase-3 / EC-SOD, SKI, survivin, SLAM / CD150, synapsin I, Sleeping Beauty Transposase, syndecan-I / CD 138, Slit3, Syndecan-2, SLITRK1, Syndecan-3, SLITRK2, Syndecan-4, SLITRK4, TACI / TNFRSF13B, TMEFF 1 / Thrombomodulin-1, TAO2, TMEFF2, TAPP1, TNF-α / TNFSFIA, CCL17 / TARC, TNF-β / TNFSF1B, Tau, TNF R1 / TNFRSFIA, TC21 / R-Ras2, TNF RII / TNFRSF1B, TCAM-I, TOR, TCCR / WSX-1, TP-I, TC-PTP, TP63 / TP73L, TDG, TR, CCL25 / TECK, TRα / NR1A1, tenascin C, TRβ1 / NR1A2, tenascin R, TR2 / NR2C1, TER-119, TR4 / NR2C2, TERT, TRA-1-85, testis proteoglycan 1 / SPOCK1, TRADD, testis proteoglycan 2 / SPOCK2, TRAF-1, testis proteoglycan 3 / SPOCK3, TRAF-2, TFPI, TRAF-3, TFPI-2, TRAF-4, TGF-α, TRAF-6, TGF-β, TRAIL / TNFSF10, TGF-β1, TRAIL R1 / TNFRSFIOA, LAP (TGF-β1), TRAIL R2 / TNFRSF10B, latent TGF-β1, TRAIL R3 / TNFRSF10C, TGF-β1.2, TRAIL R4 / TNFRSF10D, TGF-β2, TRANCE / TNFSF1 1, TGF-β3, TfR (transferrin R), TGF-β5, apotransferrin, latent TGF-βby 1, holotransferrin, latent TGF-βbp2, trap protein (Trappin)-2 / endogenous polypeptide (Elafin), latent TGF-βbp4, TREM-1, TGF-βR1 / ALK-5, TREM-2,TGF-βR11, TREM-3, TGF-βRIIb, TREML1 / TLT-1, TGF-βRIII, TRF-I, thermolysin, TRF-2, thioredoxin-1, TRH-degrading exoenzyme / TRHDE, thioredoxin-2, TRIMS, thioredoxin-80, tripeptidyl-peptidase I, thioredoxin-like 5 / TRP14, TrkA, THOP1, TrkB, thrombomodulin / CD141, TrkC, thrombopoietin, TROP-2, thrombopoietin R, troponin I peptide 3, thrombospondin-1, troponin T, thrombospondin-2, TROY / TNFRSF 19. Thrombospondin-4, trypsin 1, thymopoietin, trypsin 2 / PRSS2, thymic chemokine-1, trypsin 3 / PRSS3, Tie-1, trypsin-5 / Prss32, Tie-2, trypsin α / TPS1, TIM-I / KIM-I / HAVCR, trypsin β-1 / MCPT-7, TIM-2, trypsin β-2 / TPSB2, TIM-3, trypsin ε / BSSP-4, TIM-4, trypsin γ-1 / TPSG1, TIM-5, tryptophan hydroxylase, TIM-6, TSC22, TIMP-I, TSG, TIMP-2, TSG-6, TIMP-3, TSK, TIMP-4, TSLP, TL1A / TNFSF15, TSLP R, TLR1, TSP50, TLR2, β-III tubulin, TLR3, TWEAK / TNFSF12, TLR4, TWEAK R / TNFRSF 12, TLR5, Tyk2, TLR6, phosphotyrosine, TLR9, tyrosine hydroxylase, TLX / NR2E1, tyrosine phosphatase substrate I, ubiquitin, UNC5H3, Ugi, UNC5H4, UGRP1, UNG, ULBP-I, uPA, ULBP-2, uPAR, ULBP-3, URB, UNC5H1, UVDE, UNC5H2, capsaicin R1, VEGF R, VASA, VEGF R1 / Flt-1, vasohibin, VEGF R2 / KDR / Flk-1, vasorin, VEGF R3 / FU-4, angiostatic factor, versican, Vav-1, VG5Q, VCAM-1, VHR, VDR / NR1I1, vimentin, VEGF, vitronectin, VEGF-B, VLDLR, VEGF-C, vWF-A2, VEGF-D, synuclein-α, Ku70, WASP, Wnt-7b, WIF-I, Wnt-8aWISP-1 / CCN4, Wnt-8b, WNK1, Wnt-9a, Wnt-1, Wnt-9b,Wnt-3a、Wnt-10a、Wnt-4、Wnt-10b、Wnt-5a、Wnt-11、Wnt-5b、wnvNS3、Wnt7a、XCR1、XPE / DDB1、XEDAR、XPE / DDB2、Xg、XPF、XIAP、XPG、XPA、XPV、XPD、XRCC1、Yes、YY1、EphA4。、

[0224] Various human ion channels are targets of particular interest. Non-limiting examples include serotonin 3 receptor B subunit, serotonin 3 receptor precursor, serotonin receptor 3 subunit C, AAD 14 proteins, acetylcholine receptor protein, α subunit precursor, acetylcholine receptor protein, β subunit precursor, acetylcholine receptor protein, δ subunit precursor, acetylcholine receptor protein, ε subunit precursor, acetylcholine receptor protein, γ subunit precursor, acid sensing ion channel 3 splice variant b, acid sensing ion channel 3 splice variant c, acid sensing ion channel 4, ADP ribose pyrophosphatase, mitochondrial precursor, Α1A voltage-dependent calcium channel, amiloride-sensitive cation channel 1, neuronal amiloride-sensitive cation channel 2, neuronal amiloride-sensitive cation channel 4 isoform 2, amiloride-sensitive sodium channel, amiloride-sensitive sodium channel α subunit, amiloride-sensitive sodium channel β subunit, amiloride-sensitive sodium channel δ subunit, amiloride-sensitive sodium channel γ subunit, annexin A7, apical-like protein protein), ATP-sensitive inward rectifier potassium channel 1, ATP-sensitive inward rectifier potassium channel 10, ATP-sensitive inward rectifier potassium channel 11, ATP-sensitive inward rectifier potassium channel 14, ATP-sensitive inward rectifier potassium channel 15, ATP-sensitive inward rectifier potassium channel 8, calcium channel α12.2 subunit, calcium channel α12.2 subunit, calcium channel α1E subunit, δ19δ40δ46 splice variant, calcium-activated potassium channel α subunit 1, calcium-activated potassium channel β subunit 1, calcium-activated potassium channel β subunit 2, calcium-activated potassium channel β subunit 3, calcium-dependent chloride channel 1, cation channel TRPM4B, CDNA FLJ90453 fis, clone NT2RP3001542 (highly similar to potassium channel tetramerization domain 6), CDNA FLJ90663 fis, clone PLACE1005031 (highly similar to chloride intracellular channel protein 5), cGMP-gated cation channel beta subunit, chloride channel protein, chloride channel protein 2, chloride channel protein 3, chloride channel protein 4, chloride channel protein 5, chloride channel protein 6, chloride channel protein C1C-Ka, chloride channel protein C1C-Kb, chloride channel protein, skeletal muscle chloride intracellular channel 6, chloride intracellular channel protein 3, chloride intracellular channel protein 4, chloride intracellular channel protein 5, CHRNA3 protein, Clcn3e protein, CLCNKB protein, CNGA4 protein, stagnation protein-5, cyclic GMP-gated potassium channel, cyclic nucleotide-gated cation channel 4, cyclic nucleotide-gated cation channel α3, cyclic nucleotide-gated cation channel β3, cyclic nucleotide-gated olfactory channel, cystic fibrosis transmembrane conductance regulator, cytochrome B-245 heavy chain, dihydropyridine-sensitive L-type calcium channel α-2 / δ subunit precursor, FXYD domain-containing ion transport regulator 3 precursor, FXYD domain-containing ion transport regulator 5 precursor,FXYD domain-containing ion transport regulatory factor 6 precursor, FXYD domain-containing ion transport regulatory factor 7, FXYD domain-containing ion transport regulatory factor 8 precursor, G protein-activated inward rectifier potassium channel 1, G protein-activated inward rectifier potassium channel 2, G protein-activated inward rectifier potassium channel 3, G protein-activated inward rectifier potassium channel 4, γ-aminobutyric acid receptor α-1 subunit precursor, γ-aminobutyric acid receptor α-2 subunit precursor, γ-aminobutyric acid receptor α-3 subunit precursor, γ-aminobutyric acid receptor α-4 subunit precursor, γ-aminobutyric acid receptor α-5 subunit precursor, γ-aminobutyric acid receptor α-6 subunit precursor, γ-aminobutyric acid receptor β-1 subunit precursor, γ-aminobutyric acid receptor β-2 subunit precursor, γ-aminobutyric acid receptor GluR6 kainate receptor, glutamate receptor 1 precursor, glutamate receptor 2 precursor, glutamate receptor 3 precursor, glutamate receptor 4 precursor, glutamate receptor 7, glutamate receptor B, glutamate receptor δ-1 subunit precursor, glutamate receptor, ionotropic kainate 1 precursor, glutamate receptor, ionotropic kainate 2 precursor, glutamate receptor , ionotropic kainate 3 precursor, glutamate receptor, ionotropic kainate 4 precursor, glutamate receptor, ionotropic kainate 5 precursor, glutamate [NMDA] receptor subunit 3A precursor, glutamate [NMDA] receptor subunit 3B precursor, glutamate [NMDA] receptor subunit ε1 precursor, glutamate [NMDA] receptor subunit ε2 precursor, glutamate [NMDA] receptor subunit ε4 precursor, glutamate [NMDA] receptor subunit ζ1 precursor, glycine receptor alpha-1 chain precursor, glycine receptor alpha-2 chain precursor, glycine receptor alpha-3 chain precursor, glycine receptor beta chain precursor, H / ACA ribonucleoprotein complex subunit 1, high affinity immunoglobulin epsilon receptor beta subunit, hypothetical protein DKFZp31 310334, hypothetical protein DKFZp761M1724, hypothetical protein FLJ12242, hypothetical protein FLJ14389, hypothetical protein FLJ14798, hypothetical protein FLJ14995, hypothetical protein FLJ16180, hypothetical protein FLJ16802, hypothetical protein FLJ32069, hypothetical protein FLJ37401, hypothetical protein FLJ38750, hypothetical protein FLJ40162, hypothetical protein FLJ41415, hypothetical protein FLJ90576, hypothetical protein FLJ90590, hypothetical protein FLJ90622, hypothetical protein KCTD15, hypothetical protein MGC15619, inositol 1,4,5-trisphosphate receptor type 1,Inositol 1,4,5-triphosphate receptor type 2, inositol 1,4,5-triphosphate receptor type 3, medium conductance calcium-activated potassium channel protein 4, inward rectifier potassium channel 13, inward rectifier potassium channel 16, inward rectifier potassium channel 4, inward rectifier K(+) channel negative regulator Kir2.2v, kainate receptor subunit KA2a, KCNH5 protein, KCTD 17 protein, KCTD2 protein, keratinocyte-associated transmembrane protein 1, Kv channel interacting protein 4, melanostanin 1, membrane protein MLC1, MGC15619 protein, mucolipin-1, mucolipin-2, mucolipin-3, multidrug resistance-associated protein 4, N-methyl-D-aspartate receptor 2C subunit precursor, NADPH oxidase homolog 1Nav1.5, neuronal acetylcholine receptor protein, alpha-10 subunit precursor, neuronal acetylcholine receptor protein, alpha-2 subunit precursor, neuronal acetylcholine receptor protein, alpha-3 subunit precursor, neuronal acetylcholine receptor protein, alpha-4 subunit precursor, Neuronal acetylcholine receptor protein, alpha-5 subunit precursor, neuronal acetylcholine receptor protein, alpha-6 subunit precursor, neuronal acetylcholine receptor protein, alpha-7 subunit precursor, neuronal acetylcholine receptor protein, alpha-9 subunit precursor, neuronal acetylcholine receptor protein, beta-2 subunit precursor, neuronal acetylcholine receptor protein, beta-3 subunit precursor, neuronal acetylcholine receptor protein, beta-4 subunit precursor, neuronal voltage-dependent calcium channel alpha 2D subunit, P2X purinergic receptor 1, P2X purinergic receptor 2, P2X purinergic receptor 3, P2X purinergic receptor 4, P2X purinergic receptor 5, P2X purinergic receptor 6, P2X purinergic Receptor 7, pancreatic potassium channel TALK-Ib, pancreatic potassium channel TALK-Ic, pancreatic potassium channel TALK-Id, phospho-neural membrane protein precursor, plasma lipoprotein, polycystic kidney disease 2-related protein, polycystic kidney disease 2-like 1 protein, polycystic kidney disease 2-like 2 protein, receptor for polycystic kidney disease and egg-related protein precursor, polycystic kidney protein (Polycystin)-2, potassium channel regulatory factor, potassium channel subfamily K member 1, potassium channel subfamily K member 10, potassium channel subfamily K member 12, potassium channel subfamily K member 13, potassium channel subfamily K member 15, potassium channel subfamily K member 16, potassium channel subfamily K member 17 , potassium channel subfamily K member 2, potassium channel subfamily K member 3, potassium channel subfamily K member 4, potassium channel subfamily K member 5, potassium channel subfamily K member 6, potassium channel subfamily K member 7, potassium channel subfamily K member 9, potassium channel tetramerization domain-containing 3, potassium channel tetramerization domain-containing protein 12, potassium channel tetramerization domain-containing protein 14, potassium channel tetramerization domain-containing protein 2, potassium channel tetramerization domain-containing protein 4, potassium channel tetramerization domain-containing protein 5, potassium channel tetramerization domain-containing 10, potassium channel tetramerization domain-containing protein 13, potassium channel tetramerization domain-containing 1, potassium voltage-gated channel subfamily A member 1,Potassium voltage-gated channel subfamily A member 2, potassium voltage-gated channel subfamily A member 4, potassium voltage-gated channel subfamily A member 5, potassium voltage-gated channel subfamily A member 6, potassium voltage-gated channel subfamily B member 1, potassium voltage-gated channel subfamily B member 2, potassium voltage-gated channel subfamily C member 1, potassium voltage-gated channel subfamily C member 3, potassium voltage-gated channel subfamily C member 4, potassium voltage-gated channel subfamily D member 1, potassium voltage-gated channel subfamily D member 2, potassium voltage-gated channel subfamily D member 3, potassium voltage-gated channel subfamily E member 1, potassium voltage-gated channel subfamily E member 2, potassium voltage-gated channel Member 3 of potassium voltage-gated channel subfamily E, member 4 of potassium voltage-gated channel subfamily E, member 1 of potassium voltage-gated channel subfamily F, member 1 of potassium voltage-gated channel subfamily G, member 2 of potassium voltage-gated channel subfamily G, member 3 of potassium voltage-gated channel subfamily G, member 4 of potassium voltage-gated channel subfamily G, member 1 of potassium voltage-gated channel subfamily H, member 2 of potassium voltage-gated channel subfamily H, member 3 of potassium voltage-gated channel subfamily H, member 4 of potassium voltage-gated channel subfamily H, member 5 of potassium voltage-gated channel subfamily H, member 6 of potassium voltage-gated channel subfamily H, member 7 of potassium voltage-gated channel subfamily H Member 8, potassium voltage-gated channel subfamily KQT member 1, potassium voltage-gated channel subfamily KQT member 2, potassium voltage-gated channel subfamily KQT member 3, potassium voltage-gated channel subfamily KQT member 4, potassium voltage-gated channel subfamily KQT member 5, potassium voltage-gated channel subfamily S member 1, potassium voltage-gated channel subfamily S member 2, potassium voltage-gated channel subfamily S member 3, potassium voltage-gated channel subfamily V member 2, potassium voltage-gated channel subfamily H member 7 isoform 2, potassium / sodium hyperpolarization-activated cyclic nucleotide gated channel 1, potassium / sodium hyperpolarization-activated cyclic nucleotide gated channel 2, potassium / sodium hyperpolarization-activated cyclic nucleotide gate channel 3, potassium / sodium hyperpolarization-activated cyclic nucleotide-gated channel 4, possible mitochondrial import receptor subunit TOM40 homolog, purinergic receptor P2X5 isoform A, putative 4-repeat voltage-gated ion channel, putative chloride channel protein 7, putative GluR6 kainate receptor, putative ion channel protein CATSPER2 variant 1, putative ion channel protein CATSPER2 variant 2, putative ion channel protein CATSPER2 variant 3, putative regulator of potassium channel protein variant 1, putative tyrosine-protein phosphatase TPTE, ryanodine receptor 1, ryanodine receptor 2, ryanodine receptor 3, SH3 KBP1 binding protein 1, short transient receptor potential channel 1, short transient receptor potential channel 4, short transient receptor potential channel 5, short transient receptor potential channel 6, short transient receptor potential channel 7, small conductance calcium-activated potassium channel protein 1, small conductance calcium-activated potassium channel protein 2 isoform b, small conductance calcium-activated potassium channel protein 3 isoform b, small conductance calcium-activated potassium channel SK2,Small conductance calcium-activated potassium channel SK3, sodium channel, sodium channel β-1 subunit precursor, type II sodium channel protein α subunit, type III sodium channel protein α subunit, type IV sodium channel protein α subunit, type IX sodium channel protein α subunit, type V sodium channel protein α subunit, type VII sodium channel protein α subunit, type VIII sodium channel protein α subunit, type X sodium channel protein α subunit, type XI sodium channel protein α subunit, sodium and chloride activated ATP-sensitive potassium channel, ATPase γ chain transporting sodium / potassium, sperm-associated cation channel 1, sperm-associated cation channel 2 isoform 4, syntaxin-1B1, transient receptor potential cation channel subfamily A member 1, transient receptor potential cation channel subfamily M member 2, transient Receptor potential cation channel subfamily M member 3, transient receptor potential cation channel subfamily M member 6, transient receptor potential cation channel subfamily M member 7, transient receptor potential cation channel subfamily V member 1, transient receptor potential cation channel subfamily V member 2, transient receptor potential cation channel subfamily V member 3, transient receptor potential cation channel subfamily V member 4, transient receptor potential cation channel subfamily V member 5, transient receptor potential cation channel subfamily V member 6, transient receptor potential channel 4ε splice variant, transient receptor potential channel 4ζ splice variant, transient receptor potential channel 7γ splice variant, tumor necrosis factor, α-inducible protein 1, endothelial cell two-pore calcium channel protein 2, VD AC4 protein, voltage-gated potassium channel Kv3.2b, voltage-gated sodium channel β1B subunit, voltage-dependent anion channel, voltage-dependent anion channel 2, voltage-dependent anion selective channel protein 1, voltage-dependent anion selective channel protein 2, voltage-dependent anion selective channel protein 3, voltage-dependent calcium channel γ-1 subunit, voltage-dependent calcium channel γ-2 subunit, voltage-dependent calcium channel γ-3 subunit, voltage-dependent calcium channel γ-4 subunit, voltage-dependent calcium channel γ-5 subunit, voltage-dependent calcium channel γ-6 subunit, voltage-dependent calcium channel γ-7 subunit, voltage-dependent calcium channel γ-8 subunit, voltage-dependent L-type calcium channel α-1C subunit, voltage-dependent L-type calcium channel α-1D subunit of voltage-dependent L-type calcium channel, α-IS subunit of voltage-dependent L-type calcium channel, β-1 subunit of voltage-dependent L-type calcium channel, β-2 subunit of voltage-dependent L-type calcium channel, β-3 subunit of voltage-dependent L-type calcium channel, β-4 subunit of voltage-dependent L-type calcium channel, α-1B subunit of voltage-dependent N-type calcium channel, α-1A subunit of voltage-dependent P / Q-type calcium channel, α-1E subunit of voltage-dependent R-type calcium channel, α-1G subunit of voltage-dependent T-type calcium channel, α-1H subunit of voltage-dependent T-type calcium channel, α-1I subunit of voltage-dependent T-type calcium channel, α-1 subunit of voltage-gated L-type calcium channel, β-1 subunit of voltage-gated potassium channel, β-2 subunit of voltage-gated potassium channel, β-3 subunit of voltage-gated potassium channel,Voltage-gated potassium channel KCNA7. The Nav1.x family of human voltage-gated sodium channels is also a particularly promising target. This family includes, for example, channels Nav1.6 and Nav1.8.

[0225] In certain embodiments, the therapeutic protein can be a G protein coupled receptor (GPCR). Exemplary GPCRs include, but are not limited to, class A rhodopsin-like receptors, such as vertebrate type 1 muscarinic acetylcholine, vertebrate type 2 muscarinic acetylcholine, vertebrate type 3 muscarinic acetylcholine, vertebrate type 4 muscarinic acetylcholine; adrenergic receptors (alpha adrenergic receptor type 1, alpha adrenergic receptor type 2, beta adrenergic receptor type 1, beta adrenergic receptor type 2, beta adrenergic receptor type 3, vertebrate type 1 dopamine, vertebrate type 2 dopamine, vertebrate type 3 dopamine, vertebrate type 4 dopamine, histamine type 1, histamine type 2, histamine type 3, histamine type 4, serotonin type 1, serotonin type 2, serotonin type 3, serotonin type 4, serotonin type 5, serotonin type 6, Serotonin type 7, Serotonin type 8, Other serotonin types, Trace amines, Angiotensin type 1, Angiotensin type 2, Bombesin, Bradykinin, C5a anaphylatoxin, Fmet-leu-phe, APJ-like, Interleukin-8 type A, Interleukin-8 type B, Other interleukin-8 types, C--C chemokines type 1 to 11 and other types, C--X--C chemokines (types 2 to 6 and other types), C--X3-C chemokines, Cholecystokinin CCK, CCK type A, CCK type B, Other CCK, Endothelin, Melanocortins (melanocyte stimulating hormone, adrenocorticotropic hormone, melanocortin hormone), Duffy antigen (Duffy antigen), prolactin releasing peptide (GPR10), neuropeptide Y (types 1 to 7), neuropeptide Y, other neuropeptides Y, neurotensin, opioids (types D, K, M, X), somatostatin (types 1 to 5), tachykinins (substance P (NK1), substance K (NK2), neuromodulatory peptide K (NK3), tachykinin-like 1, tachykinin-like 2, vasopressin / oxytocin (types 1 to 2), oxytocin, oxytocin / middle oxytocin, conopressin, galanin-like, protease-activated, orexin and neuropeptide FF.QRFP, chemokine receptor-like, neuromodulatory peptide U-like (neuromodulatory peptide U, PRXamide), kinesin Oxytocin (follicle stimulating hormone, luteinizing-chorionic gonadotropin, thyrotropin, type I gonadotropin, type II gonadotropin), (rhodopsin) opsin, vertebrate rhodopsin (types 1-5), vertebrate type 5 rhodopsin, arthropod rhodopsin, arthropod type 1 rhodopsin, arthropod type 2 rhodopsin, arthropod type 3 rhodopsin, mollusk rhodopsin, rhodopsin, olfactory (olfactory II family 1 to 13), prostaglandins (prostaglandin E2 subtype EP1, prostaglandin E2 / D2 subtype EP2, prostaglandin E2 subtype EP3, prostaglandin E2 subtype EP4, prostaglandin F2-α, prostacyclin), thromboxane, adenosine types 1 to 3, purinergic receptors, purinergic receptors P2RY1-4,6,1 1GPR91, purinergic receptor P2RY5,8,9,10GPR35,92,174, purinergic receptor P2RY12-14 GPR87 (UDP-glucose), cannabinoids, platelet-activating factor, gonadotropin-releasing hormone, type I gonadotropin-releasing hormone, type II gonadotropin-releasing hormone, fat-mobilizing hormone-like, melanization-inducing hormone, thyrotropin-releasing hormone and secretagogue, thyrotropin-releasing hormone, growth hormone secretagogue, growth hormone secretagogue-like, ecdysone (ETHR), melatonin, lyso-sphingolipids and LPA (EDG), sphingosine 1-phosphate Edg-1, lysophosphatidic acid Edg-2, sphingosine 1-phosphate Edg-3, lysophosphatidic acid Edg-4, sphingosine 1-phosphate Edg-5, sphingosine 1-phosphate Edg-6, lysophosphatidic acid Edg-7, sphingosine 1-phosphate Edg-8, other Edg leukotriene B4 receptors, leukotrienes B4 receptor BLT1, leukotriene B4 receptor BLT2, class A orphan / other, putative neurotransmitter, SREB, Mas proto-oncogene and Mas-related (MRGs), GPR45-like, cysteinyl leukotrienes, G protein-coupled bile acid receptor, free fatty acid receptor (GP40, GP41, GP43), class B secretin-like, calcitonin, adrenocorticotropin-releasing factor, gastric inhibitory peptide, glucagon, growth hormone-releasing hormone, parathyroid hormone, PACAP, secretin, vasoactive intestinal polypeptide, latrophilin, latrophilin receptor type 1, latrophilin receptor type 2, latrophilin receptor type 3, ETL receptor, brain-specific angiogenesis inhibitor (BAI), Methuselah-like protein (Methuselah-like protein, MTH), cadherin EGF LAG (CELSR), maximal G protein-coupled receptor, C class metabotropic glutamate / pheromone, group I to III metabotropic glutamate, calcium sensing-like, extracellular calcium sensing, pheromone, other calcium sensing-like, putative pheromone receptor, GABA-B, GABA-B subtype 1, GABA-B subtype 2, GABA-B-like, orphan GPRC5, orphan GPCR6, Bride of sevenless protein (BOSS), taste receptor (T1R), class D fungal pheromone, fungal pheromone A-factor-like (STE2.STE3), fungal pheromone B-like (BAR, BBR, RCB, PRA), class E cAMP receptor, ocular albinism protein, frizzled protein / smoothened family, group A frizzled proteins (Fz 1 and 2 and 4 and 5 and 7-9), group B frizzled proteins (Fz 3 and 6), group C frizzled proteins (others), vomeronasal receptors, nematode chemoreceptors, insect odor receptors, and class Z archaeal / bacterial / fungal opsins.

[0226] In certain embodiments, the serum albumin binding Fn3 fusions described herein may comprise any of the following active polypeptides: BOTOX, Myobloc, Neurobloc, Dysport (or other serotypes of botulinum neurotoxin), alglucosidase alfa, daptomycin, YH-16, chorionic gonadotropin alfa, filgrastim, cetrorelix, interleukin-2, aldesleukin, tesileukin, denileukin diftitox, interferon alpha-n3 (injection), interferon alpha-n1, DL-8234, interferon, Suntory (gamma-Ia), interferon gamma, thymosin alpha 1, tasonamine, DigiFab, ViperaTAb, EchiTAb, CroFab, nesiritide, abatacept, alefacept, Rebif, eptotermin alfa (eptotermin alfa), tasonamine, ... alfa), teriparatide (osteoporosis), injectable calcitonin (bone disease), calcitonin (nasal, osteoporosis), etanercept, glutamer 250 (cattle), drotrecogin alfa, collagenase, carperitide, recombinant human epidermal growth factor (topical gel, wound healing), DWP-401, darbepoetin alfa, epoetin omega, epoetin beta, epoetin alpha, desirudin, lepirudin, bivalirudin, nonacog alpha, factor IX powder injection (mononine), eptacog alpha (eptacog alfa (activated), recombinant factor VIII + VWF, concentrated recombinant antihemophilic factor (Recombinate), recombinant factor VIII, factor VIII (recombinant), blood-derived coagulation factor (Alphanate), octocog alfa, factor VIII, palifermin, indikinase, tenecteplase, alteplase, pamiplase, reteplase, natplase, monteplase, follitropin alfa (follitropinalfa), rFSH, hpFSH, micafungin, pegfilgrastim, lenograstim, nartograstim, sermorelin, glucagon, exenatide, pramlintide, imiglucerase, galsulfase, leucotropin, moramistim, triptorelin acetate, histrelin (subcutaneous implant,Hydron), deslorelin, histrelin, nafarelin, leuprolide extended-release reservoir (ATRIGEL), leuprolide implant (DUROS), goserelin, somatropin, Eutropin, KP-102 procedure, somatropin, somatropin, mecasermin (growth failure), enfuvirtide, Org-33408, insulin glargine, insulin glulisine, insulin (inhaled), insulin lispro, insulin detemir, insulin (buccal, RapidMist), mecaserminrinfabate, anakinra, simulleukin, 99mTc-apcitide injection, myelopid, Betaseron, glatiramer acetate acetate), Gepon, Sargramostim, Oprelvekin, Human Leukocyte Derived Interferon Alpha, Bilive, Insulin (Recombinant), Recombinant Human Insulin, Insulin Aspart, Mecasermin, Interferon-A, Interferon-Alpha 2, Alfaferone, Interferon Alfacon-1, Interferon Alpha, Avonex Recombinant Human Luteinizing Hormone, Dornase Alfa, Trifermin, Ziconotide, Tatirelin, Diboterminalfa, Atosiban, Becaplermin, Eptibatide, Zemaira, CTC-111, Shanvac-B, HPV Vaccine (Quadrivalent), NOV-002, Octreotide, Lanreotide, Ancistimin, Agalsidase Beta, Agalsidase Alfa, Laronease, Prezatide copperacetate (topical gel), rasburicase, ranibizumab, Actimmune, PEG-Intron, Tricomin, recombinant house dust mite allergy desensitization injection, recombinant human parathyroid hormone (PTH) 1-84 (sc, osteoporosis), epoetin delta, transgenic antithrombin III, granditropin, hyaluronidase (Vitrase), recombinant insulin, interferon-alpha (oral tablet), GEM-2IS, vapreotide, idursulfase, omapatrilat, recombinant serum albumin, certolizumab pegol, glutaric acid, human recombinant C1 esterase inhibitor (angioedema), lanoteplase, recombinant human growth hormone, enfuvirtide (needle-free injection,Biojector 2000), VGV-I, interferon (α), lucinatan, actidil (inhaled, lung disease), icatibant, ecallantide, omiganan, Aurograb, pexiganan acetate, ADI-PEG-20, LDI-200, degarelix, cintredekin besudotox), FavId, MDX-1379, ISAtx-247, liraglutide, teriparatide (osteoporosis), tefacillin, AA-4500, T4N5 liposome wash, catumaxomab, DWP-413, ART-123, Chrysalin, desmoteplase, andeplase, corifollitropin alpha, TH-9507, teduglutide, Diamyd, DWP-412, growth hormone (sustained-release injection), recombinant G-CSF, insulin (inhaled, AIR), insulin (inhaled, Technosphere), insulin (inhaled, AERx), RGN-303, DiaPep277, interferon beta (hepatitis C virus infection (HCV)), interferon alpha-n3 (oral), belatacept (bel atacept), transdermal insulin patch, AMG-531, MBP-8298, Xerecept, Opebakan, AIDSVAX, GV-1001, LymphoScan, leuprorecept, Lipoxysan, lusupeptide, MP52 (β-tricalcium phosphate carrier, bone regeneration), melanoma vaccine, sipuleucel-T, CTP-37, Insegia, Vitspon itespen), human thrombin (frozen, surgical blood draw), thrombin, TransMID, alfimeprase, puricase, terlipressin (intravenous, hepatorenal syndrome), EUR-1008M, recombinant FGF-I (injectable, vascular disease), BDM-E, rotigaptide, ETC-216, P-113, MBI-594AN, duramycin (inhaled,cystic fibrosis), SCV-07, OPI-45, endostatin, angiostatin, ABT-510, Bowman Birk inhibitor concentrate, XMP-629, 99mTc-hydrazide nicotinamide-annexin V, kahalalide F, CTCE-9908, tevrelix (extended release), ozarelix, romidepsin, BAY-50-4798, interleukin-4, PRX-321, Pepscan, iboctadekin, rh lactoferrin (rh lactoferrin), TRU-015, IL-21, ATN-161, cilengitide, Albuferon, Biphasix, IRX-2, omega interferon, PCK-3145, CAP-232, pasireotide, huN901-DM1, ovarian cancer immunotherapy vaccine, SB-249553, Oncovax-CL, OncoVax-P, BLP-25, CerVax-16, multi-epitope peptide melanoma vaccine (MART-I, gp100, tyrosinase), nemifitide, rAAT (inhaled), rAAT (skin disease), CGRP (inhaled, asthma), penacept (p egsunercept), thymosin beta-4, plitidepsin, GTP-200, ramoplanin, GRASPA, OBI-I, AC-100, salmon calcitonin (oral, eligen), calcitonin (oral, osteoporosis), esarelin, caprorelin, Cardeva, velafermin, 131I-TM-601, KK-220, TP-10, ularitide, depelestat, hematide, crisarin (topical), rNAPc2, recombinant factor VIII (PEGylated liposomal), bFGF, PEGylated recombinant staphylokinase variant, V-10153, SonoLysis Prolyse, NeuroVax, CZEN-002, islet cell regeneration therapy, rGLP-1, BIM-51077, LY-548806, exenatide (controlled release, Medisorb), AVE-0010, GA-GCB, avorelin, AOD-9604, linaclotide acetate, CETi-I, Hemospan, VAL (injectable), rapid-acting insulin (injectable,Viadel), intranasal insulin, insulin (inhaled), insulin (oral, eligen), recombinant human leptin, pitrakinra (subcutaneous injection, eczema), pitrakinra (inhaled dry powder, asthma), Multikine, RG-1068, MM-093, NBI-6024, AT-001, PI-0824, Org-39141, Cpn1O (autoimmune disease / inflammation), talactoferrin (topical), rEV-131 (ophthalmic), rEV-131 (respiratory disease), oral recombinant human insulin (diabetes), RPI-78M, oprelvekin (oral), C YT-99007CTLA4-Ig, DTY-001, valategrast, interferon α-n3 (topical), IRX-3, RDP-58, Tauferon, bile salt-stimulated lipase, merispase, alkaline phosphatase, EP-2104R, Melanotan-II, bremelanotide, ATL-104, recombinant human microplasmin, AX-200, SEMAX, ACV-I, Xen-2174, CJC-1008, dynorphin A, SI-6603, LAB GHRH, AER-002, BGC-728, Malaria vaccine (Virosome, PeviPRO), ALTU-135, Parvovirus B19 vaccine, Influenza vaccine (recombinant neuraminidase), Malaria / HBV vaccine, Anthrax vaccine, Vacc-5q, Vacc-4x, HIV vaccine (oral), HPV vaccine, Tat toxoid, YSPSL, CHS-13340, PTH (1-34) liposomal cream (Novasome), Ostabolin-C, PTH analogs (topical,psoriasis), MBRI-93.02, MTB72F vaccine (tuberculosis), MVA-Ag85 A vaccine (tuberculosis), FAR-404, BA-210, recombinant plague F1V vaccine, AG-702, OxSODro1, rBetV1, Der-p1 / Der-p2 / Der-p7 allergen targeted vaccine (dust mite allergy), PR1 peptide antigen (leukemia), mutant ras vaccine, HPV-16E7 lipopeptide vaccine, labyrinth protein vaccine (adenocarcinoma), CML vaccine, WT1-peptide vaccine (cancer), IDD-5, CDX-110, Pentrys, Norelin, CytoFab, P-9808, VT-111, icrocaptide, telbermi n) (skin disease, diabetic foot ulcer), rupintrivir, reticulose, rGRF, P1A, α-galactosidase A, ACE-011, ALTU-140, CGX-1160, angiotensin therapeutic vaccine, D-4F, ETC-642, APP-018, rhMBL, SCV-07 (oral, tuberculosis), DRF-7295, ABT-828, ErbB2-specific immunotoxin (anticancer agent), DT388IL-3, TST-10088, PRO-1762, Combotox, cholecystokinin-B / gastrin receptor binding peptide, 1 1 1ln-hEGF, AE-37, trastuzumab-DM1, antagonist G, IL-12 (recombinant), PM-02734, IMP-321, rhIGF-BP3, BLX-883, CUV-1647 (topical), L-19-based radioimmunotherapy (cancer), Re-188-P-2045, AMG-386, DC / I540 / KLH vaccine (cancer), VX-001, AVE-9633, AC-9301, NY-ESO-I vaccine (peptide), NA17.A2 peptide, melanoma vaccine (pulse antigen therapy), prostate cancer vaccine, CBP-501, recombinant human lactoferrin (dry eye disease), FX-06, AP-214, WAP-8294A2 (injectable), ACP-HIP, SUN-11031, peptide YY [3-36] (obesity, intranasal), FGLL, atacicept, BR3-Fc, BN-003, BA-058, human parathyroid hormone 1-34 (intranasal, osteoporosis), F-18-CCR1, AT-1001 (celiac disease / diabetes), JPD-003, PTH (7-34) liposome cream (Novasome), duramycin (ophthalmic,dry eye disease), CAB-2, CTCE-0214, glycopegylated erythropoietin, EPO-Fc, CNTO-528, AMG-114, JR-013, factor XIII, aminocandin, PN-951, 716155, SUN-E7001, TH-0318, BAY-73-7977, tevirek (immediate release), EP-51216, hGH (controlled release, Biosphere), OGP-I, sifuvirtide, TV-4710, ALG-889, Org-41259, rhCCI0, F-991, thymopentin (lung disease), r(m)CRP, liver-selective insulin, subalin, L 19-IL-2 fusion peptide, endogenous peptide (elafin), NMK-150, ALTU-139, EN-122004, rhTP0, thrombopoietin receptor agonist (thrombocytopenia), AL-108, AL-208, nerve growth factor antagonist (pain), SLV-317, CGX-1007, INNO-105, oral teriparatide (eligen), GEM-OS1, AC-162352, PRX-302, LFn-p24 fusion vaccine (Therapore), EP-1043, S.pneumoniae pediatric vaccine, malaria vaccine, Neisseria meningitidis serogroup B (Neisseria meningitidis vaccine, neonatal group B streptococcal vaccine), anthrax vaccine, HCV vaccine (gpE1+gpE2+MF-59), otitis media therapy, HCV vaccine (core antigen + ISCOMATRIX), hPTH (l-34) (transdermal, ViaDerm), 768974, SYN-101, PGN-0052, aviscumine, BIM-23190, tuberculosis vaccine, multi-epitope tyrosinase peptide, cancer vaccine, enkastim, APC-8024, G1-5005, ACC-001, TTS_CD3, vascular targeted TNF (solid tumor), desmopressin (buccal controlled release), onercept, TP-9201. ,

[0227] Other Modifications

[0228] In certain embodiments, serum albumin binders and fusions thereof may further comprise post-translational modifications. Exemplary post-translational protein modifications include phosphorylation, acetylation, methylation, ADP ribosylation, ubiquitination, glycosylation, carbonylation, sumoylation, biotinylation, or addition of polypeptide side chains or hydrophobic groups. As a result, modified serum albumin binders and their fusions may contain non-amino acid components, such as lipids, polysaccharides or monosaccharides, and phosphates. A preferred form of glycosylation is sialylation, which couples one or more sialic acid moieties to the polypeptide. Sialic acid moieties can increase solubility and serum half-life while also reducing the potential immunogenicity of the protein. See, e.g., Raju et al., Biochemistry. 2001 Jul. 31; 40(30): 8868-76. The ability of serum albumin binders to bind to a specific serum albumin (e.g., HSA or RhSA) and / or to be expressed in the fusion environment by a specific non- 10 The functional role conferred by the Fn3 moiety was examined to test the effect of such non-amino acid components on the functionality of serum albumin binders or their fusions.

[0229] F. Nucleic acid-protein fusion technology

[0230] In one aspect, the invention provides fibronectin-based scaffold proteins comprising a fibronectin type III domain that binds to HSA. One way to rapidly manufacture and test Fn3 domains with specific binding properties is the nucleic acid-protein fusion technology of Adnexus (a Bristol-Myers Squibb company). This in vitro expression and labeling technology (called PROfusion) using nucleic acid-protein fusion (RNA- and DNA-protein fusion) can identify new polypeptides and amino acid motifs that are important for binding to proteins. Nucleic acid-protein fusion technology is a technology that covalently couples proteins to their encoded genetic information. For a detailed description of RNA-protein fusion technology and fibronectin-based scaffold protein library screening methods, see Szostak et al., U.S. Patents 6,258,558; 6,261,804; 6,214,553; 6,281,344; 6,207,446; 6,518,018; PCT Publication WO00 / 34784; WO01 / 64942; WO02 / 032925; and Roberts and Szostak, Proc Natl. Acad. Sci. 94:12297-12302, 1997, which are incorporated herein by reference.

[0231] G. Vector and Polynucleotide Embodiments

[0232] The present disclosure also includes nucleic acid sequences encoding any of the proteins described herein. As will be appreciated by those skilled in the art, 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. In addition, minor base pair changes may result in conservative substitutions in the encoded amino acid sequence, but are not expected to substantially alter the biological activity of the gene product. Therefore, the nucleic acid sequences encoding the proteins described herein may be slightly modified in sequence while still encoding their corresponding gene products. Certain exemplary nucleic acids encoding the serum albumin binders and fusions thereof described herein include nucleic acids having the sequences shown in SEQ ID NOs: 126-151. The invention also encompasses nucleic acid sequences that are at least 50%, such as at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NOs: 126-151, and preferably encode a protein that binds to serum albumin, and nucleic acids encoding tandem PCSK9-PKE2 Adnectins that preferably bind to serum albumin and PCSK9. In some embodiments, nucleotide substitutions are introduced so as not to alter the translated amino acid sequence.

[0233] Nucleic acids encoding any protein or polypeptide disclosed herein can be chemically synthesized. Codon usage can be selected to improve expression in cells. Such codon usage will depend on the cell type selected. Codon usage patterns specifically for E. coli and other bacteria, as well as mammalian cells, plant cells, yeast cells, and insect cells have been developed. See, for example, Mayfield et al., Proc Natl Acad Sci USA. 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. 19917(7):657-78.

[0234] The general techniques of nucleic acid manipulation are within the capabilities of those skilled in the art, and are described in, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Vols. 1-3, Cold Spring Harbor Laboratory Press, 2nd edition, 1989, or F. Ausubel et al., Current Protocols in Molecular Biology (Green Publishing and Wiley-Interscience: New York, 1987) and regular updates, which are incorporated herein by reference. The DNA encoding the protein is operably linked to a transcription or translation regulatory element suitable for deriving from a mammalian, viral, or insect gene. Such regulatory elements include a transcription promoter, an optional operating sequence for controlling transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence for controlling the termination of transcription and translation. In addition, the ability to replicate in a host, which is often conferred by a replication origin, and a selection gene that is convenient for identifying transformants are incorporated. Suitable regulatory elements are well known in the art.

[0235] The proteins and fusion proteins described herein can be produced in the form of fusion proteins with heterologous polypeptides, which are preferably signal sequences, or other polypeptides having specific cleavage sites at the N-terminus of mature proteins or polypeptides. The heterologous signal sequence preferably selected is a signal sequence that can be recognized and processed by the host cell (i.e., cut by a signal peptidase). For prokaryotic host cells that do not recognize and process natural signal sequences, the signal sequence is replaced with a prokaryotic signal sequence selected from the group consisting of, for example, alkaline phosphatase, penicillinase, Ipp, or heat-stable enterotoxin II leader sequence. For yeast secretion, the natural signal sequence can be replaced with, for example, a yeast invertase leader sequence, an α-factor leader sequence (including yeast and Kluyveromyces α-factor leaders), or an acid phosphatase leader sequence, a Candida albicans glucoamylase leader sequence, or a signal described in PCT Publication No. WO 90 / 13646. In mammalian cell expression, mammalian signal sequences and viral secretory leaders, such as herpes simplex gD signals, can be used. The DNA in such a precursor region can be connected to the DNA encoding the protein in a reading frame.

[0236] 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 the mRNA. Such sequences are commonly available from the 5' and sometimes 3' untranslated regions of eukaryotic or viral DNA or cDNA. These regions contain nucleotide segments that are transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding the multivalent antibody. A useful transcription termination component is the bovine growth hormone polyadenylation region. See PCT Publication WO 94 / 11026 and the expression vectors disclosed therein.

[0237] The recombinant DNA may also include any type of protein tag sequence that can be used to purify the protein. 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 use in 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.

[0238] The expression construct is introduced into the host cell using a method appropriate for the host cell, which is readily apparent to those skilled in the art. Various methods for introducing nucleic acids into host cells are known in the art, including, but not limited to, electroporation; transfection using calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran or other substances; microprojectile bombardment; liposome transfection; and infection (where the vector is the infectious agent).

[0239] Suitable host cells include prokaryotes, yeast, mammalian cells, or bacterial cells. Suitable bacteria include gram-negative or gram-positive organisms, for example, Escherichia coli or Bacillus species. Yeast, preferably yeast from Saccharomyces species such as Saccharomyces cerevisiae, can also be used to produce polypeptides. Various mammalian or insect cell culture systems can also be used to express recombinant proteins. The baculovirus system for producing heterologous proteins in insect cells is reviewed by Luckow and Summers (Bio / Technology, 6:47, 1988). In some cases, such as for glycosylation, it is desirable to produce proteins in vertebrate cells, and breeding 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 large size of the protein multimers described herein makes E. coli the preferred expression method.

[0240] H. Protein production

[0241] Host cells are transformed with the expression or cloning vectors described herein for protein production and cultured in conventional nutrient media modified as necessary as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.

[0242] Host cells for producing fibronectin-based scaffold proteins or fusions thereof can be cultured in a variety of culture media. Commercially available culture media such as Ham's F10 (Sigma), Minimum Essential Medium ((MEM), (Sigma)), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), (Sigma)) are suitable for culturing host cells. In addition, any culture medium described in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal. Biochem. 102:255 (1980), U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO90 / 03430; WO87 / 00195; or U.S. Pat. No. Re. 30,985 can be used as a culture medium for host cells. Any of these media may be supplemented as needed with hormones and / or other growth factors (e.g., insulin, transferrin, or epidermal growth factor), salts (e.g., sodium chloride, calcium, magnesium, and phosphate), buffers (e.g., HEPES), nucleotides (e.g., adenosine and thymidine), antibiotics (e.g., GENTAMYCIN TM Drugs), trace elements (defined as inorganic compounds, usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements known to those skilled in the art may also be included at appropriate concentrations. Culture conditions such as temperature, pH, etc. are those previously used for the host cell selected for expression and will be apparent to the skilled artisan.

[0243] Cell-free translation systems can also be used to produce the fibronectin-based scaffold proteins or fusions thereof disclosed herein. For such purposes, nucleic acids encoding fibronectin-based scaffold proteins must be modified to allow in vitro transcription to produce mRNA and allow mRNA to be cell-free translated in the specific cell-free system used (eukaryotic cell-free translation systems such as mammalian or yeast cell-free translation systems, prokaryotic cell-free translation systems such as bacterial cell-free translation systems).

[0244] Fibronectin-based scaffold proteins or fusions thereof can also be produced by chemical synthesis (eg, by the methods described in Solid Phase Peptide Synthesis, 2nd Edition, 1984, The Pierce Chemical Co., Rockford, IL). Modifications of fibronectin-based scaffold proteins can also be produced by chemical synthesis.

[0245] The scaffold protein based on fibronectin disclosed herein or its fusion can be purified by the separation / purification method for protein commonly known in the field of protein chemistry. Non-limiting examples include extraction, recrystallization, salting out (for example, using ammonium sulfate or sodium sulfate), centrifugation, dialysis, ultrafiltration, adsorption chromatography, ion exchange chromatography, hydrophobic chromatography, normal phase chromatography, reverse phase chromatography, gel filtration, gel permeation chromatography, affinity chromatography, electrophoresis, countercurrent distribution method or any combination of these methods. After purification, the scaffold protein based on fibronectin can be exchanged into different buffers and / or concentrated by any method in a variety of methods known in the art, including but not limited to, filtration and dialysis.

[0246] The purified fibronectin-based scaffold protein is preferably at least 85% pure, more preferably at least 95% pure, and most preferably at least 98% pure. Regardless of the exact numerical value of purity, the fibronectin-based scaffold protein is sufficiently pure for use as a pharmaceutical product.

[0247] I. Imaging, Diagnosis, and Other Applications

[0248] Based on binding to serum albumin 10 The identity of the heterologous molecule fused to the Fn3 domain, the serum albumin binding protein provided herein 10 Fn3 fusions can be used to treat a variety of diseases and conditions. 10 The use of Fn3 fusions can be determined by a skilled artisan based on the knowledge in the art and the information provided herein. Various serum albumin binding proteins are described in detail herein. 10 Uses of Fn3 fusion proteins. Binding to serum albumin 10 The Fn3 fusions can be administered to any mammalian subject or patient (including humans and non-human organisms).

[0249] The serum albumin binders and fusion molecules described herein can be detectably labeled and contacted with cells expressing, for example, a protein bound by the fusion molecule for imaging or diagnostic applications. Any method known in the art for conjugating a protein to a detectable moiety can be used, 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).

[0250] In certain embodiments, the serum albumin binders and fusion molecules described herein are further attached to a label that can be detected (e.g., the label can be a radioactive isotope, a fluorescent compound, an enzyme, or an enzyme cofactor). The label can be a radioactive substance, such as a radioactive heavy metal, such as an iron chelate, a radioactive chelate of gadolinium or manganese, a positron emitter of oxygen, nitrogen, iron, carbon, or gallium, 43 K. 52 Fe, 57 Co. 67 Cu, 67 Ga, 68 Ga, 123 I. 125 I. 131 I. 132 I. or 99 Tc. In certain embodiments, the label may be a fluorescent or chemiluminescent compound, such as fluorescein isothiocyanate, rhodamine, or luciferin; or an enzyme, such as alkaline phosphatase, β-galactosidase, or horseradish peroxidase. The serum albumin conjugate or fusion molecule attached to such a portion may be used as an imaging agent and administered in an amount effective for diagnostic use in a mammal, such as a human, and the localization and accumulation of the imaging agent is then detected. The localization and accumulation of the imaging agent may be detected by radioscintigraphy, magnetic resonance imaging, computed tomography, or positron emission tomography. It is clear to the skilled person that the amount of radioisotope administered depends on the radioisotope. Based on the specific activity and energy of a given radionuclide used as the active portion, it is easy for a person of ordinary skill in the art to formulate the amount of the imaging agent administered.

[0251] Serum albumin binders and fusion molecules can also be used as affinity purification agents. In this method, the protein is immobilized on a suitable carrier (such as Sephadex resin or filter paper) using methods well known in the art. The protein can be used in any known assay method, 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)).

[0252] J. Biophysical and Biochemical Characterization

[0253] The equilibrium constant (e.g., dissociation, K D ) and kinetic constants (e.g., the association rate constant k on and the dissociation rate constant k off ) is used to assess the binding of serum albumin binding Adnectins described herein to serum albumin (e.g., HSA). Serum albumin binding Adnectins (e.g., PKE2-monomer or tandem-Adnectin) will typically have a K of less than 500 nM, 100 nM, 10 nM, 1 nM, 500 pM, 200 pM, or 100 pM. D Binds to the target molecule, but at k off Low enough or k on High enough occasions, higher K D Values ​​are also allowed.

[0254] In vitro determination of binding affinity

[0255] Various in vitro assays can be used to identify PKE2-Adnectins that bind to serum albumin (e.g., HSA). In certain embodiments, the assay is a high-throughput assay that allows for the simultaneous screening of multiple candidate Adnectins.

[0256] Exemplary assays for determining the binding affinity of an Adnectin to its target include, but are not limited to, solution phase methods such as kinetic exclusion assay (KinExA) (Blake et al., JBC 1996; 271: 27677-85; Drake et al., Anal Biochem 2004; 328: 35-43), surface plasmon resonance (SPR) using a Biacore system (Uppsala, Sweden) (Welford et al., Opt. Quant. Elect 1991; 23: 1; Morton and Myszka, Methods in Enzymology 1998; 295: 268), and homogeneous time-resolved fluorescence (HTRF) assays (Newton et al., J Biomol Screen 2008; 13: 674-82; Patel et al., Assay Drug Dev Technol 2008; 6: 55-68).

[0257] In certain embodiments, the interaction of biomolecules can be monitored in real time in a Biacore system, which uses SPR to detect the change in the resonance angle of light from the surface of a thin gold film on a glass support to 300 nm away due to a change in the refractive index. Biacore analysis (e.g., as described in Example 2) generates association rate constants, dissociation rate constants, equilibrium dissociation constants, and affinity constants. Binding affinity is obtained by evaluating the association rate constant and the dissociation rate constant using a Biacore surface plasmon resonance system (Biacore, Inc.). The biosensor chip is activated to covalently couple the target. The target is then diluted and injected over the chip to obtain a signal expressed in reaction units of the fixed material. Since the signal expressed in resonance units (RU) is proportional to the mass of the fixed material, this represents the range of the density of the fixed target on the matrix. The binding data and the dissociation data are fitted simultaneously in a global analysis to solve for the net rate expression of a 1:1 bimolecular interaction, thereby generating k on , k off和 R max The best fit value for (maximum response at saturation). The equilibrium dissociation constant K for binding was calculated from the SPR measurements. D , denoted as k off / k on .

[0258] It should be understood that the assays described above are exemplary and that any method known in the art for determining binding affinity between proteins (e.g., fluorescence resonance energy transfer (FRET), enzyme-linked immunosorbent assay, and competitive binding assay (e.g., radioimmunoassay)) can be used to assess the binding affinity between the PKE2-Adnectins described herein.

[0259] In certain embodiments, the melting temperature (T) of a serum albumin binding Adnectin described herein, or a fusion protein comprising such an Adnectin, when measured using, for example, differential scanning calorimetry (DSC) or thermal scanning fluorescence (TSF), e.g., as described in the Examples. m ) is at least 50°C, such as at least 51°C, at least 52°C, at least 53°C, at least 54°C, at least 55°C, at least 56°C, at least 57°C, at least 58°C, at least 59°C, at least 60°C, at least 61°C, at least 62°C, at least 63°C, at least 64°C, at least 65°C, at least 66°C, at least 67°C, at least 68°C, at least 69°C, at least 70°C, at least 71°C, at least 72°C, at least 73°C, at least 74°C, or at least 75°C. In certain embodiments, the serum albumin binding Adnectins described herein, or fusion proteins comprising the same, have a melting temperature (T) when measured using, for example, differential scanning calorimetry (DSC) or thermal scanning fluorescence (TSF), e.g., as described in the Examples. m ) is 50-75°C, such as 51-75°C, 52-75°C, 53-75°C, 54-75°C, 55-75°C, 56-75°C, 57-75°C, 58-75°C, 59-75°C, 60-75°C, 61-75°C, 62-75°C, 63-75°C, 64-75°C, 65-75°C, 66-75°C, 67-75°C, 68-75°C, 69-75°C, 70-75°C, 50-74°C, 50-73°C , 50-72°C, 50-71°C, 50-70°C, 50-69°C, 50-68°C, 50-67°C, 50-66°C, 50-65°C, 50-64°C, 50-63°C, 50-62°C, 50-61°C, 50-60°C, 50-59°C, 50-58°C, 50-57°C, 50-56°C, 50-55°C, 51-74°C, 52-73°C, 53-71°C, 54-70°C, or 55-65°C.

[0260] K. In vivo therapeutic use

[0261] Provided herein are fibronectin-based scaffold proteins useful in treating conditions. In the case of fusion proteins comprising serum albumin binding Adnectins, the disease or condition that can be treated will be determined by the binding specificity of the moiety (e.g., a second Adnectin) attached to the Adnectin. As described herein, fibronectin-based scaffold proteins can be designed to bind to any target of interest. In one embodiment, the target is PCSK9. Fibronectin-based scaffold proteins that bind to PSCK9, and fusion proteins comprising the scaffold proteins, can be used to treat atherosclerosis, hypercholesterolemia, and other cholesterol-related diseases.

[0262] The present application also provides a method of administering a fibronectin-based scaffold protein to a subject. In some embodiments, the subject is a human. In some embodiments, the fibronectin-based scaffold protein is pharmaceutically acceptable to mammals, especially humans. A "pharmaceutically acceptable" composition refers to a composition that is administered to an animal without significant adverse medical consequences. Examples of pharmaceutically acceptable compositions include: 10 Fn3 domains, and compositions that are substantially free of endotoxins or pyrogens or have very low levels of endotoxins or pyrogens.

[0263] L. Preparation and Administration

[0264] The present application provides a method for administering and binding serum albumin 10 The method comprises a method for preparing a therapeutic moiety fused to an Fn3 domain, wherein the half-life of the therapeutic moiety is greater than that of a therapeutic moiety that binds serum albumin. 10 The technique and dosage of administering the fusion construct will depend on the amount of protein that binds to serum albumin. 10 The amount of pyrogen required for use as a therapeutic agent varies depending on the type of therapeutic moiety to which the Fn3 domain is fused and the specific condition being treated, but can be readily determined by a skilled artisan. In general, regulatory agencies require that protein agents to be used as therapeutic agents be formulated with the goal of acceptably low levels of pyrogens. Thus, therapeutic formulations are typically distinguished from other formulations in that they are substantially free of pyrogens, or contain at least no more than acceptable levels of pyrogens, as determined by the appropriate regulatory agency (e.g., FDA). In certain embodiments, a serum albumin-binding 10 Pharmaceutical formulations of Fn3 domains and their fusion molecules contain, for example, 1-20 mM succinic acid, 2-10% sorbitol, and 1-10% glycine (pH 4.0-7.0). 10 Pharmaceutical formulations of Fn3 domains and their fusion molecules contain, for example, 10 mM succinic acid, 8% sorbitol, and 5% glycine (pH 6.0).

[0265] In some embodiments, the serum albumin-binding 10 The Fn3 domain and its fusions are pharmaceutically acceptable to mammals, especially humans. A "pharmaceutically acceptable" polypeptide is a polypeptide that can be administered to an animal without significant adverse medical consequences. 10 Examples of Fn3 domains and fusions thereof include: 10 Fn3 domain; and serum albumin binding 10 Fn3 domain or serum albumin binding 10 Fn3 domain fusions that are substantially free of endotoxin or have very low endotoxin levels.

[0266] The therapeutic composition can be administered in a unit dosage form with a pharmaceutically acceptable diluent, carrier, or excipient. As non-limiting examples, it can be administered parenterally (e.g., intravenously, subcutaneously), orally, or topically. The composition can be in the form of a pill, tablet, capsule, liquid, or sustained-release tablet for oral administration; a liquid for intravenous, subcutaneous, or parenteral administration; or a gel, lotion, ointment, cream, or polymer or other sustained-release vehicle for topical administration.

[0267] Methods for making preparations well known in the art are found in, for example, "Remington: The Science and Practice of Pharmacy" (20th ed., ed. A R Gennaro A R., 2000, Lippincott Williams & Wilkins, Philadelphia, Pa.). Preparations for parenteral administration may, for example, contain excipients, sterile water, saline, polyalkylene glycols (such as polyethylene glycol), oils of plant origin, or hydrogenated naphthalene. Biocompatibility, biodegradable lactide polymers, lactide / glycolide copolymers, or polyoxyethylene-polyoxypropylene copolymers can be used to control the release of the compound. Nanoparticle preparations (such as 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 preparation varies depending on many factors, including the dose and route of administration of the drug to be administered.

[0268] The polypeptide is optionally administered in the form of a pharmaceutically acceptable salt, a non-toxic acid addition salt or a metal complex commonly used in the pharmaceutical industry. Examples of acid addition salts include organic acids such as acetic acid, lactic acid, pamoic acid, maleic acid, citric acid, malic acid, ascorbic acid, succinic acid, benzoic acid, palmitic acid, suberic acid, salicylic acid, tartaric acid, methanesulfonic acid, toluenesulfonic acid, or trifluoroacetic acid, etc.; polymeric acids such as tannic acid, carboxymethyl cellulose, etc.; and inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, etc. Metal complexes include zinc, iron, etc. In one example, the polypeptide is formulated in the presence of sodium acetate to increase thermal stability.

[0269] Formulations suitable for oral use include tablets containing a mixture of the active ingredient and non-toxic pharmaceutically acceptable excipients. These excipients may be, for example, inert diluents or fillers (e.g., sucrose and sorbitol), lubricants, glidants, and anti-adhesives (e.g., magnesium stearate, zinc stearate, stearic acid, silicon dioxide, hydrogenated vegetable oils, or talc).

[0270] Formulations 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 water or an oil medium.

[0271] A therapeutically effective dose refers to a dose that produces the therapeutic effect sought by the administration. The exact dose will depend on the condition to be treated and can be determined by one skilled in the art using known techniques. Generally, about 0.01 μg / kg to about 50 mg / kg per day, preferably 0.01 mg / kg to about 30 mg / kg per day, and most preferably 0.1 mg / kg to about 20 mg / kg per day of serum albumin-bound is administered. 10 Fn3 domain or serum albumin binding 10 Fn3 domain fusions. The polypeptides may be administered daily (e.g., once, twice, three times, or four times a day) or less frequently (e.g., once every other day, once or twice a week, or once a month). In addition, as is known in the art, adjustments may need to be made for age and weight, general health, sex, diet, dosing time, drug interactions, and disease severity, and may be determined by those skilled in the art using routine experiments.

[0272] ************************

[0273] The contents of all figures and all references, Genbank sequences, patents and published patent applications cited throughout this application are expressly incorporated herein by reference. Specifically, the disclosure of U.S. Provisional Patent Application No. 61 / 968,181 (filed on March 20, 2014) is expressly incorporated herein by reference.

[0274] The above disclosure generally describes the present disclosure, which is further illustrated by the following examples. These specific embodiments are described only for illustrative purposes and are not intended to limit the scope of the present disclosure. Although specific targets, terms, and numerical values ​​are employed herein, such targets, terms, and numerical values ​​are also to be understood as being exemplary and non-restrictive for the scope of the present disclosure. Example

[0275] High Throughput Protein Production (HTPP)

[0276] Clone the selected binders into the pET9d vector HIS 6 The upstream of the tag was transformed into E. coli BL21DE3plysS cells and incubated in 5 ml of LB medium containing 50 μg / mL kanamycin in a 24-well format and cultured overnight at 37°C. 200 μl was aspirated from the overnight culture and distributed to the appropriate wells, and a fresh 5 ml LB medium (50 μg / mL kanamycin) culture was prepared for inducing expression. The culture was grown at 37°C until A 600 After induction with 1 mM isopropyl-β-thiogalactoside (IPTG), the culture was expressed at 30°C for 6 hours and harvested by centrifugation at 2750 g at 4°C for 10 minutes.

[0277] The cells were lysed by adding 450 μl of lysis buffer (50 mM NaH 2 PO 4 , 0.5M NaCl, 1x Complete TM The cell pellet (24-well format) was resuspended in EDTA-free complete protease inhibitor cocktail (Roche), 1 mM PMSF, 10 mM CHAPS, 40 mM imidazole, 1 mg / ml lysozyme, 30 μg / ml DNase, 2 μg / ml aprotinin, pH 8.0) and shaken at room temperature for 1-3 hours. The lysate was clarified and re-racked into a 96-well format by transferring to a 96-well Whatman GF / DUnifilter equipped with a 96-well 1.2 ml catch plate and filtered under positive pressure. The clarified lysate was transferred to a 96-well equilibration buffer (50 mM NaH 2 PO 4 , 0.5 M NaCl, 40 mM imidazole, pH 8.0) and incubated for 5 minutes. Unbound material was removed with the aid of positive pressure. Wash buffer #1 (50 mM NaH 2 PO 4The resin was washed twice with 0.3 ml / well (PBS + 20 mM EDTA, 0.5 M NaCl, 5 mM CHAPS, 40 mM imidazole, pH 8.0). Each wash was removed with positive pressure. Before elution, each well was washed with 50 μl of elution buffer (PBS + 20 mM EDTA), incubated for 5 minutes, and the wash was discarded with positive pressure. An additional 100 μl of elution buffer was applied to each well to elute the protein. After incubation at room temperature for 30 minutes, the plate was centrifuged at 200 g for 5 minutes and the eluted protein was collected in a 96-well capture plate to which 5 μl of 0.5 M MgCl was added to the bottom before elution. 2 Using total protein assay, wild-type 10 The Fn3 domain was used as a protein standard to quantify the eluted proteins.

[0278] Mid-scale expression and purification of insoluble conjugates of fibronectin-based scaffold proteins

[0279] For expression of insoluble clones, clones were cloned with the following HIS 6 The tag was cloned into the pET9d (EMDBioscience, San Diego, CA) vector and expressed in E. coli HMS174 cells. A 20 ml inoculum culture (generated from a single colony plated) was used to inoculate 1 liter of LB medium containing 50 μg / ml carbenicillin and 34 μg / ml chloramphenicol. The culture was grown at 37°C until A 600 The pH value was 0.6-1.0. After induction with 1 mM isopropyl-β-thiogalactoside (IPTG), the culture was grown at 30°C for 4 hours and harvested by centrifugation at >10,000 g at 4°C for 30 minutes. The cell pellet was frozen at -80°C. Use on ice The cell pellet was resuspended in 25 ml of lysis buffer (20 mM NaH 2 PO 4 , 0.5M NaCl, 1x Complete TM EDTA-free protease inhibitor cocktail (Roche), 1 mM PMSF, pH 7.4). (Microfluidics) high pressure homogenization (> 18,000psi) to achieve cell lysis. Insoluble parts were separated by centrifugation at 23,300g for 30 minutes at 4 ° C. The insoluble centrifugal precipitate recovered from the lysate was washed with 20mM sodium phosphate / 500mM NaCl (pH 7.4). The precipitate was redissolved in 6.0M guanidine hydrochloride in 20mM sodium phosphate / 500mM NaCl (pH 7.4) with ultrasonic treatment, and then incubated for 1-2 hours at 37 degrees. The redissolved precipitate was filtered to 0.45 μm and loaded onto a Histrap column equilibrated with 20mM sodium phosphate / 500M NaCl / 6.0M guanidine (pH 7.4) buffer. After loading, the column was washed with another 25CV of the same buffer. Bound protein was eluted with 50 mM imidazole in 20 mM sodium phosphate / 500 mM NaCl / 6.0 M guanidine-HCl (pH 7.4). The purified protein was refolded by dialyzing against 50 mM sodium acetate / 150 mM NaCl (pH 4.5).

[0280] Mid-scale expression and purification of soluble conjugates of fibronectin-based scaffold proteins

[0281] For expression of soluble clones, clones were cloned with a following HIS 6 The tag was cloned into the pET9d (EMDBioscience, San Diego, CA) vector and expressed in E. coli HMS174 cells. A 20 ml inoculum culture (generated from a single colony plated) was used to inoculate 1 liter of LB medium containing 50 μg / ml carbenicillin and 34 μg / ml chloramphenicol. The culture was grown at 37°C until A 600 The pH value was 0.6-1.0. After induction with 1 mM isopropyl-β-thiogalactoside (IPTG), the culture was grown at 30°C for 4 hours and harvested by centrifugation at >10,000 g at 4°C for 30 minutes. The cell pellet was frozen at -80°C. The cell pellet was resuspended in 25 ml lysis buffer (20 mM NaH 2 PO 2 , 0.5M NaCl, 1x Complete TM EDTA-free protease inhibitor cocktail (Roche) (Roche), 1 mM PMSF, pH 7.4). (Microfluidics) carries out high pressure homogenization (>18,000psi) to realize cell lysis.Soluble part is separated by centrifugation at 23,300g for 30 minutes at 4 ℃.Supernatant is clarified via 0.45 μm filter.The clarified lysate is loaded onto a Histrap column (GE) pre-equilibrated with 20mM sodium phosphate / 500M NaCl (pH 7.4).Then the column is washed with 25 column volumes of the same buffer, 20 column volumes of 20mM sodium phosphate / 500mM NaCl / 25mM imidazole (pH 7.4) and 35 column volumes of 20mM sodium phosphate / 500M NaCl / 40mM imidazole (pH 7.4) afterwards. The protein was eluted with 15 column volumes of 20 mM sodium phosphate / 500 mM NaCl / 500 mM imidazole (pH 7.4), and the fractions were pooled according to absorbance at A280 and dialyzed against 1 x PBS, 50 mM Tris, 150 mM NaCl (pH 8.5) or 50 mM NaOAc, 150 mM NaCl (pH 4.5). Any precipitate was removed by filtration at 0.22 μm.

[0282] Example 1: Screening of binders based on the parent southern loop (CD loop) that binds to serum albumin

[0283] The first generation of Arctic-based serum albumin-binding Adnectins (SABAs) do not bind to mouse and rat serum albumin, do not have high affinity for serum albumin across species, and are not available in multivalent-based 10 To improve upon the Fn3 platform, which is not always compatible, second generation Antarctic-based serum albumin binding Adnectins (PKE2 Adnectins) with modified CD loop sequences were screened using mRNA display as described below.

[0284] The mRNA display method (Xu et al., Chem Biol 2002; 9:933-42) was used to screen for genes containing the modified 10 The ability of a library of Fn3 domain-based CD loop binder peptides to bind to human serum albumin (HSA). CD loop binders were designed with different CD loop lengths, up to +7 amino acids, and 10 The rest of the Fn3 sequence remained wild type.Target binding was detected by qPCR and when a specific binding signal was observed populations were cloned and expressed in E. coli.

[0285] Example 2: Identification of CD loop binders capable of binding to HSA that cross-react with Rh-SA and MSA

[0286] A direct binding ELISA format was used to identify CD loop binders generated in Example 1 that bind to HSA and cross-react with rhesus serum albumin (Rh-SA) and / or mouse serum albumin (MSA). MaxiSorp was coated with 10 μg / mL of either HSA, Rh-SA, or MSA. TM ELISA plates were used to test purified CD loop binders at 1 μM. Bound Adnectins were detected via HRP-conjugated anti-histidine mAb (R&D Systems) and TMB detection reagent (BD Biosciences). ELISA results were confirmed using Biacore as described below. For CD loop binders identified as cross-reactive with Rh-SA and / or MSA (>2X background) in the ELISA experiments, their aggregation was analyzed by SEC to demonstrate that the binding was due to monomers - as expected for a stable, well-folded protein. The stability of the protein was confirmed by differential scanning calorimetry (DSC) as described below.

[0287] One clone identified, referred to herein as 2270_C01, had the following amino acid sequence:

[0288] MASTSGVPRDLEVVAATPTSLLISWDAPAVTVRYYRITY GWQVQMYSDWGPLYIYKE FTVPGSKSTATISGLKPGVDYTITVYAVTGSGESPASSKPISINYRTEGDKPSQHHHHHH(2270_C01; SEQ ID NO:23)

[0289] The CD loop is underlined. The AB, BC, DE, EF, and FG loops have similar 10 The same sequence as the Fn3 domain (SEQ ID NO: 1). Size exclusion chromatography and DSC analysis were performed on a mid-scale 2270_C01 to confirm monomericity and determine thermal stability.

[0290] Standard size exclusion chromatography (SEC) is carried out on 2270_C01 generated from medium-scale process. SEC of medium-scale material is carried out on Superdex 200 10 / 30 or on Superdex 7510 / 30 posts (GE Healthcare) on Agilent 1100 or 1200HPLC systems, UV detection is carried out at A214 nm and A280 nm, and fluorescence detection (excitation=280nM, emission=350nm) is carried out. SEC posts adopt 100mM sodium sulfate, 100mM sodium phosphate, 150mM sodium chloride buffer of pH 6.8 of appropriate flow rate. Gel filtration standards (Bio-Rad Laboratories, Hercules, CA) are used for molecular weight calibration. As shown in Table 2, 2270_C01 is mainly monomer (98% monomer).

[0291] Differential scanning calorimetry (DSC) analysis of mid-scale Adnectins was performed to determine their corresponding T m . A 0.5 mg / ml solution was scanned at 70 p.si in a VP-capillary differential scanning calorimeter (GE Microcal) with the temperature gradually increased from 15°C to 110°C at a rate of 1 degree per minute. The data were analyzed using Origin Software (OriginLab Corp) using best fit with appropriate buffer control runs as a comparison. As shown in Table 2, the Tm of 2270_C01 was 64°C.

[0292] To determine the binding kinetics to human, rhesus, and mouse serum albumin, and to determine whether binding is maintained at physiological and endosomal pH, the corresponding serum albumin was immobilized on a Biacore CM5 chip using NHS / EDC coupling to a surface density of approximately 1200 RU. A range of concentrations (0.25 nM–5 μM) of 2270_C01 was applied to the immobilized albumin in either HBS-P+ (0.01 M HEPES pH 7.4, 0.15 M NaCl, 0.05% v / v surfactant P-20) or acetate (0.02 mM sodium acetate pH 5.5, 0.15 M NaCl, 0.05% v / v surfactant P-20) running buffer. Kinetic measurements used a 3-minute association phase and a 6-10-minute dissociation phase. The kinetic trajectories of the reference-subtracted sensorgrams were fitted to a 1:1 binding model using Biaevaluation software. As shown in Table 1, 2270_C01 bound to each species of albumin with equivalent affinity at neutral pH and low pH, however the affinity for mouse albumin was approximately 1 / 10 of the binding affinity to human or rhesus albumin.

[0293] Table 1: 2270_C01 has slightly faster on-rates and significantly faster off-rates to MuSA at pH 7.4 and 5.5 compared to HuSA and RhSA

[0294] Buffer Combined with ka(l / Ms) kd(l / s) kD(nM) Rmax(RU) HBS-P, pH 7.4 HuSA 6.59E+04 3.68E-04 5.58 121.6 RhA 8.27E+04 5.77E-04 6.98 103.3 MuSA 1.34E+05 9.09E-03 67.67 77.64 Acetate, pH 5.5 HuSA 1.02E+05 8.98E-04 8.82 111.9 RhA 5.96E+04 1.05E-03 17.55 85.5 MuSA 7.59E+04 1.46E-02 ～192.4 57.91

[0295] In order to improve the properties of 2270_C01, namely the in silico predicted immunogenicity, 2270_C01 sequence optimization was performed by mRNA display. The Adnectin generated from this optimization is referred to herein as PKE2 Adnectin.

[0296] Example 3: Generation of 2270_C01 progeny Adnectins with further modified CD loops: PKE2 Adnectin

[0297] Using a custom designed library, the 2270_C01 sequence was optimized by mRNA display to reduce potential immunogenicity and the sequence was screened for binding to both human and mouse serum albumin during mRNA display to obtain progeny molecules that retained cross-species albumin binding and were less immunogenic. The generated sequences were evaluated for their in silico predicted immunogenicity and only clones with in silico immunogenicity scores below a predetermined cutoff were further used for protein production by HTPP. The generated Adnectins were purified by HTPP and screened by direct binding ELISA and SEC-HPLC as described above.

[0298] Of the 308 PKE2 Adnectins obtained and tested in the screening of the 2270_C01 progeny, the following 25 were the best performing molecules in terms of in silico predicted immunogenicity, monomericity determined by SEC, and binding to serum albumin from various species determined by direct binding ELISA. The affinity determination results of the best candidates were analyzed by SPR as described above.

[0299]

[0300]

[0301]

[0302] Example 4: Biophysical Properties of PKE2 Adnectins

[0303] Two PKE2 Adnectins, 2629_E06 and 2630_D02, identified as performing well in the screen in Example 3, were subjected to size exclusion chromatography (SEC) as described above. As shown in Table 2, both PKE2 molecules were mostly monomeric.

[0304] Differential scanning calorimetry (DSC) analysis of the two PKE2 Adnectins was performed as described above to determine their corresponding T m As shown in Table 2, the T M 56℃ and 57℃ respectively.

[0305] Table 2

[0306]

[0307] Example 5: Characterization of PKE2 Adnectin Binding to Serum Albumin from Various Species

[0308] The binding kinetics of 2629_E06 and 2630_D02, as well as the first generation Arctic-based serum albumin binding Adnectin, 1318_H04, to serum albumin were determined as described above. In addition, binding to albumin was performed under different pH conditions ranging from pH 5.5 to pH 7.4. Neither 2629_E06 nor 2630_D02 showed pH-dependent binding to human, rhesus monkey, or mouse serum albumin, suggesting that they remain bound in endosomes. As shown in Table 3, 1318_H04 has a lower affinity to human, cynomolgus monkey, and rhesus serum albumin relative to 2629_E06 and 2630_D02, and does not bind to mouse or rat serum albumin. Moreover, relative to human serum albumin, 1318_H04 exhibits one-tenth the affinity for rhesus serum albumin, while the affinity of PKE2 Adnectin for different albumin species is relatively equivalent.

[0309] Relative to 1318_H04, both PKE2 Adnectins 2629_E06 and 2630_D02 showed significantly higher affinities for all serum albumins tested, as discussed above, with K values ​​for human, cynomolgus monkey, rhesus monkey, and mouse serum albumin being D 2629_E06 also exhibited a low nanomolar K for rat serum albumin. D , and 2630_D02 exhibited a K of 200 nM for rat serum albumin D .

[0310] Table 3.

[0311]

[0312] Example 6: PKE2 Adnectin competes with hFcRn for binding to HSA

[0313] Considering that inhibiting the binding of HSA to the hFcRn receptor would prevent the recycling of HSA via hFcRn and shorten the long half-life of HSA, thereby potentially reducing the magnitude of pharmacokinetic enhancement, the level of competition between PKE2 Adnectin and hFcRn for HSA binding was tested using competitive α screening, as depicted in Figure 1 Adnectins were serially diluted in assay buffer (50 mM acetate / 150 mM NaCl / 0.1% Tween-20, pH 5.5, +0.005% antifoam-204) to obtain the desired final assay concentration range. A master premix of protein and AlphaScreen beads in assay buffer was prepared to obtain the following final assay concentrations: hFcRn-GST (BMS) 6.5 nM, biotinylated human serum albumin (Abcam) 30 nM, AlphaScreen streptavidin donor beads and AlphaLISA glutathione acceptor beads (Perkin Elmer) 5 μg / ml each. 10 μl / well of the serially diluted Adnectins, followed by 10 μl / well of the protein + beads solution, were added to a 384-well small volume assay plate (Greiner Bio-one). AlphaScreen beads, and all transfers to the assay plate were protected from ambient light. The assay plate was sealed with an adhesive sealing foil and incubated for 2-2.5 hours at room temperature with shaking. The plate was read in a Synergy 4 microplate reader (Biotek) with an excitation wavelength of 570 nm and an emission wavelength of 680 nm. The average signal from the control wells without Adnectin was set to 0% inhibition, and the percentage inhibition of FcRn-HSA interaction was calculated relative to this signal; the average background signal from the control wells without biotinylated HSA was subtracted from all data points.

[0314] Table 4 and Figure 2 The results of the screening are shown. Notably, 1318_H04 competed more strongly with hFcRn for binding to HSA than the second generation parental 2270_C01 Adnectin as well as the PKE2 2629_E06 and 2630_D02 Adnectins, suggesting that the PKE2 Adnectins may provide improved PK enhancement relative to 1318_H04.

[0315] Furthermore, the domains on HSA bound by 1318_H04, 2629_E06, and 2630_D02 were determined by SPR. As shown in Table 4, 1318_H04 Adnectin binds to domain I of HSA, while 2270_C01, 2629_E06, and 2630_D02 bind to domains I-II of HSA instead of just domain I, indicating that 1318_H04 and PKE2 Adnectin bind to different epitopes on HSA. None of the Adnectins in Table 4 bind to domain III of HSA, which is an important interaction site between HSA and FcRn.

[0316] Table 4.

[0317]

[0318] * The dose response was not saturated even at 2 μM, although the percent inhibition (i.e., inhibition of hFcRn binding to HSA) was approximately 80%

[0319] Example 7: In vivo half-life of candidate PKE2 Adnectins

[0320] PKE2 Adnectins 2629_E06 and 2630_D02 were prepared, purified, and endotoxin-free. 2629_E06 or 2630_D02 was injected into the tail vein of wild-type mice (n=3 / group) at 1 mg / kg, and the concentration in blood samples taken at certain time intervals after injection was determined using a quantitative ELISA-based assay (established for detecting Adnectins in plasma samples). Specifically, Adnectin drug levels in mouse plasma were measured using the Meso Scale technology platform or standard colorimetric ELISA. 2629_E06 and 2630_D02 were captured via anti-His mAb (BMS) and detected using rabbit antiserum against the Adnectin scaffold in combination with goat anti-rabbit HRP-conjugated pAb. Alternatively, they were detected via species-specific albumin bound to the Adnectin and species-specific anti-albumin secondary pAbs with sulfonic acid tags. The pharmacokinetic parameters of each Adnectin were determined using a non-compartmental model using Phoenix WinNonlin software.

[0321] like Figure 3 The pharmacokinetic profiles of 2629_E06 and 2630_D02 are compared as shown in Table 5. The half-life of 2629_E06 in mouse plasma is 33-41 hours, while the half-life of 2630_D02 is 35-39 hours.

[0322] Table 5.

[0323]

[0324] Example 8: Immunogenicity of PKE2 Adnectin

[0325] The computer prediction of HLA binding was evaluated using Epimatrix software (Epivax). The score comparison is shown in Table 6. PKE2 Adnectin 2629_E06 and 2630_D09 showed a reduced computer score relative to 2270_C01. In addition, the in vitro proliferation of CD4+T cells in response to 1318_H04, 2270_C01 and PKE2 Adnectin was evaluated as an in vitro assessment of potential human immunogenicity. Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood obtained from 40 independent donors matched to the MHC II class of the general population using the Ficoll density gradient method. After separating the cells from each donor, they were stored in liquid N2 and thawed before use. Cells from each donor were labeled with the fluorescent dye carboxyfluorescein succinimidyl ester (CFSE) and incubated at 37°C for 7 days with the Adnectin of interest. T cells were labeled with anti-CD4 antibodies, and proliferation was evaluated by flow cytometry using BD FACSCanto and FlowJo analysis software. The percentage of donors showing a significant increase in CD4+ proliferation was calculated to indicate the antigenicity of the protein.

[0326] Comparison of the parent 2270_C01 and its two progeny 2629_E06 or 2630_D02 revealed that the parent molecule has higher antigenicity ( Figure 4 and Table 6), indicating that the two progeny PKE2 Adnectins displayed reduced immunogenic potential relative to the parental molecule.

[0327] Table 6.

[0328]

[0329]

[0330] Example 9: Effect of single cysteine ​​mutants of PKE2 Adnectin on albumin binding

[0331] A single cysteine ​​residue was incorporated into the PKE-2 Adnectin at a site distinct from the HSA binding residue to facilitate chemical coupling of the therapeutic molecule of interest via standard maleimide chemistry. It is important to retain binding to serum albumin (thus enhancing PK) in the context of cysteine ​​mutations, so the effects of these mutations on binding to serum albumin from various species were tested using 2629_E06 as the basis for mutations. The dissociation rate (k) of each mutant was analyzed at 250 nM by an SPR-based assay. off ), wherein albumin was immobilized and Adnectin was used as the analyte. As shown in Table 7, the introduction of single cysteine ​​mutations in 2629_E06 showed serum albumin dissociation rates similar to the parental 2629_E06 molecule in different species, indicating that binding to serum albumin was retained in the context of these specific mutations. Therefore, any of these cysteine ​​mutants can be used as a chemical conjugation partner for therapeutic molecules of interest and provide PK enhancement.

[0332] Table 7.

[0333]

[0334] Example 10: Biophysical properties of single cysteine ​​mutants of 2629_E06

[0335] The biophysical properties of the single cysteine ​​mutants described in Example 9 were evaluated and are shown in Table 8. Each mutant produced a thermostable, monomeric protein.

[0336] Table 8.

[0337]

[0338] Example 11: Modularity of PKE2 Adnectin Tandem Molecules

[0339] One of the limitations of Arctic serum albumin-binding Adnectins is the lack of 10 Therefore, the compatibility of PKE2 Adnectin with other 10The compatibility of Fn3 proteins was tested. The biophysical behavior of PKE2 Adnectin when fused in tandem with Adnectins specific for different targets was tested. Two possible configurations of PKE2 Adnectin were tested: at the N-terminal position (PKE2-X), and at the C-terminal position (X-PKE2). Size exclusion chromatography behavior was tested using molecules obtained by the HTPP method. Fusions of the first generation Arctic-based 1318_H04 Adnectin were directly compared with fusions of PKE2 Adnectins, 2629_E06 and 2630_D02. The fusion partners tested included: myostatin-binding 10 Fn3 domain (2987_H07; see WO2014 / 043344), two PCSK9-binding 10 Fn3 domains (2013_E01 and 2382_D09), and EGFR binding 10 Fn3 domain (1312_E01). The sequences of the two PCSK9 Adnectins, 2382_D09 and 2013_E01, can be found in WO2011 / 130354, which is incorporated herein by reference. As shown in Figure 9, both PKE2 Adnectin molecules consistently retained good biophysical behavior, as reflected in the proportion of molecules with an SEC rating of A (i.e., corresponding to ≥90% monomeric Adnectin) relative to the Arctic-based 1318_H04 SABA molecule in the context of tandem Adnectins. PKE in this table refers to serum albumin binding 10 The Fn3 domain (i.e., PK-enhancing 10 Fn3 domain). The ratio represents the number of clones with SEC=A / total number of clones tested. Ungenerated tandems are indicated as "-".

[0340] Table 9.

[0341]

[0342] Figure 5 The data in Table 9 reproduce the binding of 2987_H07 myostatin 10 Fn3 domain and binding to PCSK9 10 The data for the Fn3 domain, except that the different shades of grey reflect the ability of the tandem molecules to still bind to HSA as determined in the direct binding ELISA assay described above. Figure 5 The different shades of gray in the figure correspond to different ECs that bind to HSA. 50 _Tandem Adnectin / EC 50_Ratio of single Adnectin, where darker shades represent stronger binding of the tandem molecule to HSA. Figure 5 The data in Figure 2 show that the PKE2-based tandem molecules have better monomericity (i.e., less prone to dimerization and aggregation) and HSA binding (i.e., less loss of HSA and RhSA binding) relative to the 1318_H04 Adnectin. Similar patterns were observed for the four additional target-binding Adnectins. These data indicate that the PKE2 Adnectins have a better affinity for other targets than the Arctic serum albumin-binding Adnectins. 10 The Fn3 protein provides a more stable and active binding partner.

[0343] Example 12: PCSK9-PKE2 tandem molecules exhibit good potency, low EpiMatrix scores, good biophysical properties, and cross-species albumin binding in PCSK9 biochemical assays

[0344] Based on PKE2 Adnectin 2629_E06 and PCSK9 Adnectin 2382_D09, various PCSK9-PKE2 tandem Adnectins were generated as shown in Table 10. Each tandem molecule differed only in the linker and was tested for biophysical and functional properties to ensure that they retained the activity of PKE2 binding to albumin and Adnectin binding to PCSK9. Cross-species albumin binding was determined using the above-mentioned ELISA method. Relative thermal stability was assessed by thermal scanning fluorescence (TSF). HTPP samples were normalized to 0.2 mg / ml in PBS. 1 μl of Sypro orange dye was diluted 1:40 with PBS and added to 25 μl of each sample, and the plate was sealed with a clear 96-well microplate sealant. Samples were scanned using a BioRad RT-PCR machine, with the temperature gradually increased from 25°C to 95°C at a rate of 2 degrees per minute. Data were analyzed using BioRad CFX manager 2.0 software. The values ​​obtained by TSF have been shown to correlate well with Tm values ​​obtained by DSC in the melting range of 40°C to 70°C. This is considered an acceptable working range for this technique. ND ("No Data") results are obtained when the slope of the transition curve is too small to allow its derivative peak (rate of change of fluorescence over time) to be distinguished from noise.

[0345] Using recombinant human PCSK9 expressed in baculovirus and a synthetic 40-mer EGFA peptide (biotinylated), the PCSK9:EGFA FRET assay measures the inhibition of PCSK9 binding to the low-density lipoprotein receptor (LDLR) epidermal growth factor precursor homology domain (EGFA domain). EGFA has been shown to be a key domain for the interaction of LDLR with PCSK9 (Kwon, HJ et al., Proc. Natl. Acad. Sci. USA, 105(6): 1820-1825 (2008)). This assay utilizes a mAb (mAb 4H5) that binds the PCSK9 C-terminal domain labeled with an Eu chelate to provide a FRET interaction with biotinylated EGFA via a streptavidin / allophycocyanin fluorophore complex. The PCSK9-LDLR FRET assay was run in a similar manner using the extracellular domain of LDLR instead of the EGFA peptide.

[0346] All tandem molecules had low immunogenicity (negative Epimatrix scores), high monomericity (as assessed by SEC), acceptable relative thermal stability (TSF), and favorable cross-species albumin binding as determined by ELISA. Moreover, the PCSK9-PKE2 tandem Adnectins retained good potency in the PCSK9 biochemical assay, with an IC 50 Similar to the unformatted 2382_D09 Adnectin.

[0347] Table 10.

[0348]

[0349] Example 13: Binding kinetics of PCSK9-PKE2 tandem molecules to human PCSK9

[0350] Binding of PCSK9-PKE2 tandem Adnectins to immobilized human PCSK9 in the presence or absence of HSA was measured by biolayer interferometry (Octet Red 96, using a Superstreptavidin sensor tip, ForteBio, Menlo Park CA). Association and dissociation events were captured in real time for a range of Adnectin concentrations using biotinylated full-length PCSK9 captured on the sensor tip. Binding curves were globally fitted to generate K D , k on , and k off The value of .

[0351] By incubating an excess of the tandem and running the binding assay in the presence of excess HSA, the tandem Adnectin-HSA complex is preformed. When the apparent mass of the tandem Adnectin increases in the presence of the same concentration of HSA, a complex is considered to have formed between the tandem Adnectin-HSA complex and human PSCK9 (see, e.g., Figure 6 As shown in Table 11, all tested tandem PCSK9-PKE2 molecules had similar binding kinetics and potency to PCSK9. For HSA-Adnectin complexes bound to huPCSK9, a slight decrease in association and slightly faster dissociation was seen.

[0352] Table 11.

[0353]

[0354] Example 14: Characterization of Binding of PCSK9-PKE2 Tandem Molecules to Serum Albumin from Multiple Species

[0355] The affinity of the PCSK9-PKE2 tandem molecules for serum albumin of multiple species, together with the affinity of PKE2 Adnectin 2629_E06, was assessed by Biacore analysis as described in Example 2.

[0356] As shown in Table 12, all three PCSK9-PKE2 tandem molecules showed comparable affinity to serum albumin across species, although the affinity of the tandems was 5-7 fold weaker than that of the 2629_E06 PKE2 Adnectin (with similar off-rates).

[0357] Table 12.

[0358]

[0359] Similar experiments were performed with the 4472_C06 tandem Adnectin without the 6X histidine tail (referred to as 5190_E01) (under the conditions described in Example 2). As shown in Table 13, 5190_E01 bound to human, cynomolgus monkey, and rhesus serum albumin with a K similar to that of 5190_E01. D Binds to mouse serum albumin with a K of 200 nM D Binds to rat serum albumin.

[0360] Table 13.

[0361]

[0362] The effect of pH on binding to PKE2 Adnectin 2629_E06 and PCSK9-PKE2 tandem Adnectins 4472_C06, 4427_E06, and 4472_F08 was tested. As shown in Tables 14 and 15, all tested Adnectins showed pH-insensitive binding to serum albumin from various species.

[0363] Table 14.

[0364]

[0365] Table 15.

[0366]

[0367] Example 15: Dual binding of tandem PCSK9-PKE2 Adnectin to albumin and PCSK9

[0368] The ability of the tandem PCSK9-PKE2 Adnectin to bind simultaneously to serum albumin and PCSK9 was assessed using SPR. It is likely that the tandem will spend most of its time in vivo bound to albumin, and therefore, it is essential that the PCSK9 Adnectin retains activity when bound to albumin. The tandem PCSK9-PKE2 Adnectin was tested for simultaneous binding to both targets in a dual injection mode, where the tandem was injected first onto albumin immobilized on the chip surface, followed by a second injection of human PCSK9, and the binding level was recorded after a 3-minute association phase for each injection. An increase in the SPR binding signal for PCSK9 relative to buffer after injection indicates simultaneous binding of the tandem to HSA and PCSK9, as shown in Figure 2. Figure 7 PCSK9 showed approximately 40% of the expected binding level to 500 nM or 1 μM tandem Adnectin pre-bound to HSA. As an additional control, PCSK9 showed no binding to PKE-2 alone (data not shown).

[0369] Example 16: In vivo clearance of PCSK9-PKE2 Adnectin in WT C57 BL / 6 mice

[0370] The in vivo half-life of the tandem PCSK9-PKE2 Adnectin 4772_C06 was determined in a 2-week single 2 mg / kg IV dosing study in wild-type C57 Bl / 6 mice. Tandem Adnectin plasma levels were determined using the MesoScale Discovery platform. Biotinylated human PCSK9 was used to capture the Adnectin and detected by mouse serum albumin bound to the tandem and a sulfo-tagged anti-mouse serum albumin secondary pAb. Non-compartmental analysis was performed using Phoenix WinNonlin 6.3 (Pharsight Corporation, Mountain View, CA) using a plasma model and a linear upregulation / log downregulation calculation method. As shown in Table 16 and Figure 8 As shown in , the average half-life of the 4772_C06 tandem Adnectin was 16.7 hours.

[0371] Table 16.

[0372]

[0373] Example 17: PCSK9-PKE2 tandem Adnectins exhibit strong PCSK9 target engagement in vivo

[0374] The pharmacodynamic activity of the PCSK9-PKE2 tandem Adnectin 4472_C06 was evaluated in a human PCSK9 transgenic mouse model that exhibits normal human PCSK9 levels. This model is genomic hPCSK9 transgenic (BAC-transgenic), which is regulated in the liver similarly to mouse PCSK9, and expresses hPCSK9 in plasma at levels close to those of humans. Unbound hPCSK9 was evaluated after a single IP dose of PBS vehicle or 0.5 or 2 mg / kg tandem (8 animals per group). An enzyme-linked immunosorbent assay (ELISA) was developed that is specific for free (unbound) human PCSK9 and does not detect mouse PCSK9. The assay employed streptavidin pretreated 96-well plates coated with 2 μg / mL biotinylated PCSK9-Adnectin 2013_E01 as a capture agent. Plasma samples that were frozen only once were diluted once as needed in ELISA buffer (25 mM Tris, 150 mM NaCl, pH 7.2, with 0.05% Tween-20 and 0.1% BSA), added to the wells and incubated for 1 hour at 20°C. The wells were then washed and incubated for 1 hour with 5 μg / mL of rabbit polyclonal anti-human PCSK9 IgG (BMS custom antibodies produced by Lampire BiologicalLabs, Pipersville PA), followed by processing of HRP-labeled anti-rabbit IgG with TMB by standard ELISA methods. Standard curves were generated using purified recombinant human PCSK9.

[0375] like Fig. 9 As shown in, analysis of free hPCSK9 levels indicated potent target engagement of the PCSK9-PKE2 tandem Adnectin at both doses tested. Free hPCSK9 was inhibited in a dose-dependent manner, with responses at the 2 mg / kg dose being more sustained than at the 0.5 mg / kg dose. These data confirm the in vivo activity of the PCSK9-PKE2 tandem Adnectin.

[0376] Example 18: In vivo half-life of PKE2 single Adnectin and tandem PCSK9-PKE2 Adnectin in cynomolgus monkeys

[0377] A single-dose PK / PD study was conducted in normal lean female cynomolgus monkeys comparing molar dose equivalent PKE2 mono Adnectins to PCSK9-PKE2 tandems of a PEGylated PCSK9 Adnectin reference, as indicated by the shaded regions in Table 17 below. Cynomolgus monkeys were dosed with the indicated concentrations and routes (see Tables 17 and Fig.10) PKE2 Adnectin 2629_E06 or PCSK9-PKE2 tandem 5190_E01 Adnectin, or PEGylated PCSK9 Adnectin (called ATI-1476) was administered as a reference, and plasma (K2EDTA) and serum samples were collected at certain time intervals for pharmacokinetic and pharmacodynamic evaluation. Adnectin drug levels in cynomolgus monkey plasma were measured using the Meso Scale technology platform. 2629_E06 was captured by anti-His mAb (BMS) and detected using cynomolgus monkey serum albumin bound to Adnectin and a sulfo-tagged anti-cynomolgus monkey serum albumin secondary pAb. For tandem analysis, biotinylated human PCSK9 was used to capture the Adnectin and detected by cynomolgus monkey albumin as described above. Pegylated Adnectin ATI-1476 was captured via biotinylated hPCSK9 and detected via anti-PEG mAb (Epitomics) conjugated to sulfo-tagged goat anti-rabbit pAb. Non-compartmental analysis was performed using Phoenix WinNonlin 6.3 (Pharsight Corporation, Mountain View, CA) using the plasma model and linear upregulation / log downregulation calculation method.

[0378] As shown in Table 17, the plasma half-life of 2629_E06 and ATI-1476 is the same at 112 hours. The half-life of 5190-E01 is shorter than that of the PKE2 mono-Adnectin and is in the range of 60-82 hours after intravenous administration. 5190_E01 exhibited dose-proportional exposure (AUC 2.0) between 3 and 10 mg / kg intravenous doses. 全 The ratio was 1.02). For all proteins tested, the volume of distribution was less than the plasma volume, indicating that the distribution of PCSK9-PKE2 tandem Adnectin and PEGylated Adnectin was mainly limited to the vascular space. Clearance was generally low and comparable between different doses and forms. The subcutaneous bioavailability of 5190_E01 tandem Adnectin was 41-49%.

[0379] Table 17.

[0380]

[0381] In another independent study, the pharmacokinetics of the parental 2270_C01 Adnectin in cynomolgus monkeys were also tested. Adnectin drug levels were quantified as described above for the mouse PK study. Fig.11As shown in , 2270_C01 Adnectin has a half-life of 83.5 hours following a single IV bolus dose of 1 mg / kg.

[0382] Table 18.

[0383]

[0384] Example 19: Tandem PCSK9-PKE2 Adnectin functions as a PCSK9 inhibitor in cynomolgus monkeys

[0385] The pharmacodynamic effects of inhibitory PCSK9 from the above cynomolgus monkey PK / PD study were evaluated. An enzyme-linked immunosorbent assay (ELISA) specific for cynomolgus monkey PCSK9 was established. The free (unbound) PCSK9 assay used the Meso Scale Discovery platform and incorporated a 96-well MSD plate pretreated with streptavidin coated with 2 μg / mL of biotinylated PCSK9-Adnectin 2013_E01 as a capture agent. The samples were diluted 1:4 with blocking and sulfo-tagged rabbit polyclonal anti-human PCSK9 IgG (BMS custom antibodies produced by Lampire Biological Labs, Pipersville PA), added to the wells, and incubated at room temperature for 10 minutes. The wells were then washed and read using MSD 2X read buffer. The total PCSK9 ELISA assay was performed similarly as described above, except that mAb-4H5 (BMS custom antibodies produced by Lampire Biological Labs, Pipersville PA) was used as the capture antibody and the detection step was performed separately from the capture step. mAb-4H5 binds to the C-terminal domain of PCSK9 and, when bound to a 96-well plate, efficiently captures total PCSK9 (Adnectin-PCSK9 complex plus free PCSK9). The capture and detection steps of PCSK9 were incubated for 1 hour. Standard curves were generated using purified recombinant human or cynomolgus monkey PCSK9.

[0386] Serum analytes were determined using standardized enzymatic procedures on a Siemens Advia 1800 Clinical Chemistry System. LDL-cholesterol was determined by the direct LDL method (Roche Diagnostics). Other analytes tested were: aspartate aminotransferase; alanine aminotransferase; alkaline phosphatase; gamma glutamyltransferase; total bilirubin; blood urea nitrogen; creatinine; total cholesterol; triglycerides; high-density lipoprotein; low-density lipoprotein; glucose; total protein; albumin; globulin; albumin / globulin ratio; calcium; inorganic phosphorus; sodium; potassium; chloride.

[0387] like Fig.12 As shown in, 5190_E01 triggered pharmacodynamic effects on unbound / free PCSK9, total PCSK9, and LDL-c, which have been previously observed with other PCSK9 Adnectin inhibitors. Specifically, rapid target engagement was observed, with free PCSK9 falling rapidly to undetectable levels within 1 hour of administration. As a result of PCSK9 inhibition, LDL-c dropped to approximately 50% baseline, with maximum inhibition observed within a 2-5 day time frame. In addition, total PCSK9 increased with the accumulation of the PCSK9-PKE2 Adnectin:PCSK9 complex. Once the complex dissociated and the drug was cleared, PCSK9 and LDL-c levels returned to baseline after approximately the 15th day of the study. Similar trends were observed with the PEGylated PCSK9 Adnectin reference. Fig.13 As shown in , 5190_E01 exhibited a similar strong LDL-c lowering effect at an equivalent molar dose of 10 mg / kg of the PEGylated PCSK9 Adnectin reference.

[0388] Example 20: Dose dependence of PCSK9 target engagement

[0389] A dose-dependent response of free PCSK9 inhibition was observed with 5190_E01 at 3 and 10 mg / kg doses, as Fig.14 The 10 mg / kg dose exhibited a longer duration of PCSK9 target engagement than the 3 mg / kg dose. The figure also shows that equivalent molar doses of the tandem Adnectin and the PEGylated Adnectin had equivalent PCSK9 target engagement. As expected, 2629_E06 did not modulate free PCSK9; any changes observed in free PCSK9 are likely due to circadian rhythms and baseline variability.

[0390] Fig.15The difference in the effects of tandem and PEGylated PCSK9 Adnectins on total PCSK9 is shown. Although the general trends are the same, total PCSK9 reaches peak and returns to baseline more rapidly in cynomolgus monkeys dosed with 5190_E01 relative to the PEGylated PCSK9 Adnectin reference, suggesting that the clearance mechanisms of PCSK9:Adnectin drug complexes differ depending on the PK enhancement method used (renal clearance for tandems; macrophage uptake for PEGylated Adnectins). Again, in this assay, 2629_E06 showed no pharmacodynamic effects as expected.

[0391] Example 21: The tandem format exhibits equivalent in vitro immunogenic responses relative to the individual components

[0392] The potential immunogenicity of several tandem PCSK9-PKE2 Adnectins was assessed in vitro using a T cell proliferation assay, as described in Example 8.

[0393] like Fig.16 As shown in , the percentage and magnitude of immunogenic responses to tandem Adnectins were similar to those of the single Adnectin components (i.e., PCSK9 or PKE2; Fig.16 These results suggest that tandem Adnectins have little / no additional immunogenicity risk relative to single Adnectins. In addition, differences in the proliferation response to the tandems were observed to vary depending on the linker sequence connecting the PCSK9 and PKE2 Adnectins. The 4472_C06 tandem Adnectin showed the lowest immunogenicity relative to the 4472_F08 and 4472_E06 tandem PCSK9-PKE2 Adnectins. One possible mechanism for these observed differences is that T cells respond to differences in protein processing by the linker sequence.

[0394] A summary of the properties of 4472_C06 is shown in Table 19 below.

[0395] Table 19.

[0396]

[0397] Exemplary embodiments

[0398] 1. A method comprising the tenth structural domain of fibronectin type III ( 10 Fn3), wherein 10 The Fn3 domain contains: a) AB, BC, CD, DE, EF, and FG loops, b) has 10The polypeptide comprises a CD loop having an amino acid sequence that is altered from the sequence of the corresponding CD loop of the Fn3 domain, and c) wherein the polypeptide has a K of less than 500 nM D Binds to human serum albumin.

[0399] 2. The polypeptide of embodiment 1, wherein 10 The Fn3 domain further binds to one or more of rhesus serum albumin, cynomolgus serum albumin, mouse serum albumin, and rat serum albumin.

[0400] 3. The polypeptide of embodiment 2, wherein 10 The Fn3 domain binds to rhesus serum albumin and cynomolgus serum albumin.

[0401] 4. The polypeptide of embodiment 3, wherein 10 The Fn3 domain has a K of less than 500 nM. D Binds to rhesus serum albumin and cynomolgus monkey serum albumin.

[0402] 5. The polypeptide of embodiment 4, wherein 10 The Fn3 domain has a K of less than 100 nM. D Binds to rhesus serum albumin and cynomolgus monkey serum albumin.

[0403] 6. The polypeptide of embodiment 5, wherein said 10 The Fn3 domain has a K of less than 10 nM D Binds to rhesus serum albumin and cynomolgus monkey serum albumin.

[0404] 7. The polypeptide according to any one of the above embodiments, wherein 10 The Fn3 domain binds to mouse and rat serum albumin.

[0405] 8. The polypeptide of embodiment 7, wherein said 10 The Fn3 domain has a K of less than 500 nM. D Binds to rhesus serum albumin and cynomolgus monkey serum albumin.

[0406] 9. The polypeptide of embodiment 8, wherein 10 The Fn3 domain has a K of less than 100 nM. D Binds to rhesus serum albumin and cynomolgus monkey serum albumin.

[0407] 10. The polypeptide of embodiment 9, wherein 10 The Fn3 domain has a K of less than 10 nM D Binds to rhesus serum albumin and cynomolgus monkey serum albumin.

[0408] 11. The polypeptide according to any one of the above embodiments, wherein 10 The Fn3 domain binds to serum albumin in the pH range of 5.5 to 7.4.

[0409] 12. The polypeptide according to any one of the above embodiments, wherein 10 The Fn3 domain binds to domains I-II of HSA.

[0410] 13. The polypeptide according to any one of the preceding embodiments, wherein the polypeptide has a serum half-life of at least 30 hours in the presence of human serum albumin.

[0411] 14. The polypeptide of any one of the above embodiments, wherein the CD loop comprises a 1 -X 2 -VX 3 -X 4 -X 5 -SX 6 -X 7 -GX 8 -X 9 -YX 10 -X 11 -X 12 -E amino acid sequence, wherein,

[0412] (a)X 1 Selected from R or W;

[0413] (b)X 2 Selected from H, E, D, Y, or Q;

[0414] (c)X 3 Select from Q or H;

[0415] (d)X 4 is selected from I, K, M, Q, L, or V;

[0416] (e)X 5 Selected from Y, F, or N;

[0417] (f)X 6 Selected from D, V, or E;

[0418] (g)X 7 Selected from L, W, or F;

[0419] (h)X 8 Select from P or T;

[0420] (i)X 9 Select from L or M;

[0421] (j)X 10 Selected from I or V;

[0422] (k)X 11 is selected from Y or F; and

[0423] (l)X 12 Selected from T, S, Q, N, or A.

[0424] 15. The polypeptide of embodiment 14, wherein:

[0425] (a)X 1 It is R;

[0426] (b)X 2 It is E;

[0427] (c)X 3 It is Q;

[0428] (d)X 4 It is K;

[0429] (e)X 5 It is Y;

[0430] (f)X 6 It is D;

[0431] (g)X 7 is L or W;

[0432] (h)X 8 It is P;

[0433] (i)X 9 It is L;

[0434] (j)X 10 is I;

[0435] (k)X 11 is Y; and

[0436] (l)X 12 It is Q or N.

[0437] 16. The polypeptide of embodiment 15, wherein X 10 is L and X 12 For Q.

[0438] 17. The polypeptide of embodiment 15, wherein X 10 is W and X 12 is N.

[0439] 18. The polypeptide of embodiment 14, wherein the CD loop comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 101-125.

[0440] 19. The polypeptide according to any one of the preceding embodiments, wherein the CD loop comprises the amino acid sequence shown in SEQ ID NO: 106.

[0441] 20. The polypeptide according to any one of the above embodiments, wherein the CD loop comprises the amino acid sequence shown in SEQ ID NO:113.

[0442] 21. The polypeptide of any one of the above embodiments, wherein the polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the non-CD loop region of SEQ ID NOs: 23-100, 184-209 and 235-260.

[0443] 22. The polypeptide of any one of the above embodiments, wherein the polypeptide comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of SEQ ID NOs: 23-100, 184-209 and 235-260.

[0444] 23. A method comprising fibronectin type III ten ( 10 Fn3) domain and a fusion polypeptide of a heterologous protein, wherein the 10 The Fn3 domain contains: a) AB, BC, CD, DE, EF, and FG loops, b) has 10 The polypeptide comprises a CD loop having an amino acid sequence that is altered from the sequence of the corresponding CD loop of the Fn3 domain, and c) wherein the polypeptide has a K of less than 500 nM D Binds to human serum albumin.

[0445] 24. The fusion polypeptide of embodiment 23, wherein 10 The Fn3 domain comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of SEQ ID NOs: 23-100, 184-209 and 235-260.

[0446] 25. The fusion polypeptide of embodiment 24, wherein 10 The Fn3 domain comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:55, 81, 190 or 241.

[0447] 26. The fusion polypeptide of embodiment 25, wherein 10 The Fn3 domain comprises the amino acid sequence of SEQ ID NO: 55, 81, 190 or 241.

[0448] 27. The fusion polypeptide of embodiment 24, wherein10 The Fn3 domain comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:62, 88, 197 or 248.

[0449] 28. The fusion polypeptide of embodiment 27, wherein 10 The Fn3 domain comprises the amino acid sequence of SEQ ID NO: 62, 88, 197 or 248.

[0450] 29. The fusion polypeptide of embodiment 23, wherein the heterologous protein is a therapeutic moiety.

[0451] 30. The fusion polypeptide of embodiment 23, wherein the heterologous protein comprises 10 Fn3 domain polypeptides.

[0452] 31. The fusion polypeptide of embodiment 30, wherein 10 The Fn3 domain binds to target proteins other than serum albumin.

[0453] 32. The fusion polypeptide of embodiment 31, wherein 10 The Fn3 domain binds to PCSK9.

[0454] 33. The fusion polypeptide of embodiment 32, wherein 10 The Fn3 domain comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:167.

[0455] 34. The fusion polypeptide of embodiment 33, wherein 10 The Fn3 domain comprises the amino acid sequence of SEQ ID NO:167.

[0456] 35. The fusion polypeptide of embodiment 23, wherein the fusion polypeptide comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 168, 169 or 261.

[0457] 36. The fusion polypeptide of embodiment 35, wherein the fusion polypeptide comprises the amino acid sequence shown in SEQ ID NO: 168, 169 or 261.

[0458] 37. The fusion polypeptide of any one of embodiments 23-36, wherein the polypeptide has a serum half-life of at least 10 hours in the presence of mouse serum albumin.

[0459] 38. The fusion polypeptide of any one of embodiments 23-36, wherein the polypeptide has a serum half-life of at least 50 hours in the presence of cynomolgus monkey serum albumin.

[0460] 39. A polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 23-125, 184-209 and 235-260, 168, and 169.

[0461] 40. A composition comprising the polypeptide of any one of the above embodiments and a carrier.

[0462] 41. An isolated nucleic acid molecule encoding the polypeptide of any one of embodiments 1-39.

[0463] 42. The isolated nucleic acid molecule of embodiment 41, wherein the nucleic acid molecule has a sequence selected from SEQ ID NOs: 126-151 and 172, or a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NOs: 126-151 and 172.

[0464] 43. An expression vector comprising the nucleotide sequence of embodiment 41 or 42.

[0465] 44. A cell comprising the nucleic acid molecule of embodiment 41 or 42 or the expression vector of embodiment 43.

[0466] 45. A method of producing the polypeptide of any one of embodiments 1-39, the method comprising culturing the cell of embodiment 44 under conditions suitable for expression of the polypeptide, and purifying the polypeptide.

[0467] Table 20: Sequence Summary

[0468]

[0469]

[0470]

[0471]

[0472]

[0473]

[0474]

[0475]

[0476]

[0477]

[0478]

[0479]

[0480]

[0481]

[0482]

[0483]

[0484]

[0485]

[0486]

[0487]

[0488]

[0489]

[0490]

[0491]

[0492]

[0493]

[0494]

[0495]

[0496]

[0497]

[0498]

[0499]

[0500]

[0501]

[0502]

[0503] Equivalent form

[0504] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

1. A polypeptide comprising two or more covalently linked fibronectin type III tenth domains ( 10 Fn3 domain), wherein the two or more 10 Fn3 domain include: (a) First 10 An Fn3 domain comprising AB, BC, CD, DE, EF, and FG loops, wherein the CD loop consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 101-125; and (b) Second 10 The Fn3 domain specifically binds to PCSK9.

2. The polypeptide according to claim 1, wherein the first 10 The Fn3 domain binds to serum albumin in the pH range of 5.5 to 7.

4.

3. The polypeptide according to claim 1, wherein the first 10 The Fn3 domain binds to domains I-II of HSA.

4. The polypeptide of claim 1, wherein the polypeptide has a serum half-life of at least 30 hours.

5. The polypeptide according to claim 1, wherein the first 10 The non-CD loop region of the Fn3 domain comprises an amino acid sequence as shown in SEQ ID NO: 81, 88, 23-80, 82-87, 89-100, 184-209 or 235-260.

6. The polypeptide according to claim 1, wherein the second 10 The Fn3 domain is linked to the first 10 Fn3 domain.

7. The polypeptide according to claim 1, wherein the polypeptide comprises an amino acid sequence as shown in SEQ ID NO: 23-100, 167, 168, 169, 184-209, 235-260 or 261.

8. The polypeptide according to claim 1, wherein the polypeptide comprises the amino acid sequence shown in SEQ ID NO: 168, 169 or 261.

9. A composition comprising a polypeptide according to any one of the preceding claims and a carrier.

10. An isolated nucleic acid molecule encoding the polypeptide of any one of claims 1-8.

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