EA044608B1 - Fibronectin structural domain-based proteins that bind PCSK9 - Google Patents

Description

Field of technology

This invention relates to subtilisin/kexin type 9 (PCSK9) convertase precursor protein binding proteins containing a fibronectin structural domain. This invention also relates to the use of these original proteins in therapeutic applications for the treatment of atherosclerosis, hypercholesterolemia and other diseases associated with high cholesterol levels. This invention also relates to cells containing such proteins, polynucleotides encoding such proteins, or fragments thereof, and vectors containing polynucleotides encoding these original proteins.

Introduction

Atherosclerosis is a disease of the arteries responsible for the development of coronary heart disease (CHD), which causes the majority of deaths in industrialized countries (Lusis (2000)). Currently, some risk factors for the development of coronary artery disease are well known: dyslipidemia, arterial hypertension, diabetes mellitus, smoking, poor diet, lack of exercise and stress. The most clinically significant and common dyslipidemias are characterized by increased levels of low- and very low-density lipoproteins (LDL and VLDL) together with hypercholesterolemia in the absence or presence of hypertriglyceridemia (Fredrickson et al. (1967)). A separate increase in LDL cholesterol levels is one of the most common risk factors for the development of coronary artery disease. The PCSK9 protein (also known as HCHOLA3, NARC-1 or FH3) is a protease and a subfamily of the proteinase K family of secretory subtilases (Naureckiene et al., Arch. Biochem. Biophy., 420:55-57 (2003)). PCSK9 has been shown to be a key regulator of cholesterol homeostasis and circulating low-density lipoprotein levels. The circulating protein PCSK9 controls LDL metabolism by directly binding to the LDL receptor and mediating its degradation in hepatocytes. PCSK9-mediated reduction in LDL receptor abundance and activity results in decreased LDL clearance from the circulation and increased LDL levels. Several mutant forms of PCSK9, including S127R, N157K, F216L, R218S, and D374Y, with S127R, F216L, and D374Y, are known to be associated with autosomal dominant hypercholesterolemia (ADH). Wild-type PCSK9 is thought to increase LDL receptor turnover and cause lower LDL clearance (Maxwell et al., Proc. Natl. Acad. Sci., 102(6):2069-2074 (2005), whereas mutations leading to loss of PCSK9 functionality results in increased low-density lipoprotein receptor (LDL) levels, increased clearance of circulating LDL, and a corresponding decrease in plasma cholesterol (Rashid et al., Proc. Natl. Acad. Sci., 102(15):5374-5379 (2005) Thus, PCSK9 is a potential target for the development of treatments for atherosclerosis, hypercholesterolemia and other diseases associated with high cholesterol levels.

Fibronectin sequence-based structures are a family of proteins that can change their structure to bind any target substance. Proteins of this type, whose structures are usually formed on the basis of fibronectin type III domain (Fn3) or Fn3-like domain, have similar functional properties to those of natural or recombinant antibodies (polyclonal, monoclonal or single-chain), and also have structural advantages. In particular, the structure of such antibody mimetics is designed for optimal spatial folding, stability and solubility even under conditions that would normally result in loss of antibody structure and function. An example of structural proteins based on fibronectin are adnectins (Adnexus, Bristol-Myers Squibb R&D Company).

Fibronectin type III domains (Fn3) contain the following regions (in order from N-terminus to C-terminus): beta or beta-like region A, loop AB, beta or beta-like region B, beta or beta-like region C, loop CD, beta or beta-like region D, loop DE, beta or beta-like region E; loop EF, beta or beta-like region F; the FG loop and the beta or beta-like region of G. Any or all of the AB, BC, CD, DE, EF, and FG loops may be involved in binding the target molecule. The BC, DE and FG loops are structural and functional analogues of immunoglobulin complementarity determining regions (CDR regions). US Patent Application No. 7,115,396 describes Fn3 domain-containing proteins in which changes in the BC, DE and FG loops result in high affinity for TNF-α binding. US Patent Application No. 2007/0148126 describes Fn3 domain-containing proteins in which changes in the BC, DE and FG loops result in high binding affinity for VEGFR2.

Preparation of optimized structural proteins containing fibronectin-type domains that bind PCSK9 for the therapeutic use of such proteins in the treatment of atherosclerosis, hypercholesterolemia and other diseases associated with high cholesterol levels.

The essence of the invention

The present application provides adnectins against human PCSK9. One object of the present invention is polypeptides containing an Fn3 domain in which one or more solvent accessible loops have a random or mutated primary sequence. In some embodiments, the Fn3 domain is a modified wild-type Fn3 domain derived from the tenth module of the human type III fibronectin domain ( ^Δ10 Fn3). In some embodiments, such a ¹⁰ Fn3 polypeptide described herein is at least 40, 50, 60, 65, 70, 75, 80, 85, or 90% identical to the human ¹⁰ Fn3 domain.

In some embodiments, one or more loops from the group BC, DE, and FG may be longer or shorter than the corresponding human fibronectin loop.

In some embodiments, the polypeptides described in this invention contain a tenth fibronectin type III domain ( ¹⁰ Fn3), in which ^{the 10} Fn3 domain contains loops AB, BC, CD, DE, EF and FG and at least one loop from the group BC, DE and FG with a change in the primary sequence relative to the sequence in the corresponding loop of human Fn3 domain ¹⁰ .

In some embodiments, the polypeptide described in this invention contains an Fn3 domain containing an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to the non-loop region sequence.

In some embodiments, the BC loop of a protein described in the present invention contains an amino acid sequence selected from the group of sequences SEQ ID NO: 2-17, 106-135 and 301-303. In certain embodiments, the BC loop of the protein described in the present invention contains darkened regions of any of the sequences of SEQ ID NO: 2-17, 106-135 and 301-303, as shown in table. 3. For example, in one embodiment, the BC loop contains the sequence PPPSHGYG (residues 3-10 of SEQ ID NO: 2), DAPAHAYG (residues 3-10 of SEQ ID NO: 5), EPFSRLPGGGE (residues 3-13 of SEQ ID NO: 5) NO: 106) or DAPADGGYG (residues 3-11 of SEQ ID NO: 107).

In some embodiments, the DE loop of a protein of the present invention comprises an amino acid sequence selected from the following group: SEQ ID NOs: 18-27 and 136-141. In certain embodiments, the DE loop of a protein of the present invention comprises the shaded portions of any of SEQ ID NOs: 18-27 and 136-141, as shown in Table 1. 3. For example, in one embodiment, the DE loop contains the sequence PGKG (residues 2-5 of SEQ ID NO: 18), VGVG (residues 2-5 of SEQ ID NO: 27) or VSKS (residues 2-5 of SEQ ID NO: 27) : 137).

In some embodiments, the FG loop of a protein of the present invention comprises an amino acid sequence selected from the following group of sequences: SEQ ID NOs: 28-38 and 142-172. In certain embodiments, the DE loop of a protein of the present invention comprises the shaded portions of any of SEQ ID NOs: 28-38 and 142-172, as shown in Table 1. 3. For example, in one embodiment, the FG loop contains the sequence EYPYKHSGYYHR (residues 1-12 of SEQ ID NO: 28), EYPYDYSGYYHR (residues 1-12 of SEQ ID NO: 142), or EFDFVGAGYYHR (residues 1-12 of SEQ ID NO: 142) : 167).

In some embodiments, Fn3 domain ¹⁰ may contain altered amino acid residues at the beginning and/or end of the sequence, as well as insertions or deletions.

In some embodiments, the protein described in this invention contains one BC loop SEQ ID NO: 2-17, 106-135 and 301-303; one of the loop sequences DE SEQ ID NO: 18-27 and 136-141; one FG loop sequence SEQ ID NO: 28-38 and 142-172. In certain embodiments, the protein described in this invention contains a single BC loop sequence including the shaded portion of any of the sequences SEQ ID NO: 2-17, 106-135 and 301-303 shown in table. 3; one DE loop sequence including the shaded portion of any of the sequences SEQ ID NOs: 18-27 and 136-141 shown in table. 3; and one FG loop sequence including the shaded portion of any of SEQ ID NOs: 28-38 and 142-172 shown in table. 3.

In some embodiments, the protein described in this invention contains amino acid sequences of the BC, DE and FG loops that match at least 70, 75, 80, 85, 90, 95, 98, 99 or 100% with any of the SEQ ID sequences NO: 2-38, 106-172 and 301-303. In certain embodiments, the protein described in this invention contains amino acid sequences of the BC, DE and FG loops that match at least 70, 75, 80, 85, 90, 95, 98, 99 or 100% with any of the shaded portions of the sequences loops BC, DE and FG shown in table. 3, as described previously.

In some embodiments, anti-PCSK9 adnectin comprises the amino acid sequence of any of SEQ ID NOs: 39-76, 173-290, and 304-309.

In some embodiments, the anti-PCSK9 adnectin sequence comprises positions 3 through 96 of the Fn3 domain amino acid sequence of any of SEQ ID NOs: 39-76, 173-290, and 304-309.

One of the objects protected by the present invention is an anti-PCSK9 adnectin containing a BC loop with the sequence SW(X1) _z X ₂ G (SEQ ID NO: 323), in which X1 is any amino acid, Z is a number from 6 to 9 and X ₂ is Y or H.

One of the objects protected by the present invention is an anti-PCSK9 adnectin containing a DE loop with the sequence PX1X1X1X3T, (SEQ ID NO: 324), in which X1 is any amino acid and X3 is either G or S.

One of the objects protected by the present invention is an anti-PCSK9 adnectin containing an FG loop with the sequence EX4X1X5X1X1X6GYX4HRP (SEQ ID NO: 325), in which X1 is any amino acid, _X4 is Y or F, X5 is Y, F or W , X6 is either S or A. In certain embodiments, _X4 and X5 are aromatic moieties, each independently being Y, F, W or H.

One of the objects protected by the present invention is an anti-PCSK9 adnectin containing a BC loop with the sequence SW(X1) _z X ₂ G (SEQ ID NO: 323), a DE loop with the sequence PX1X1X1X3T (SEQ ID NO: 324) and a FG loop with the sequence EX4X1X5X1X1X6GYX4HRP (SEQ ID NO: 325) as defined in this application.

One of the objects protected by the present invention is an anti-PCSK9 adnectin containing a BC loop with the sequence SWEPFSRLPGGGE (SEQ ID NO: 106), a DE loop with the sequence PX1X1X1X3T (SEQ ID NO: 324) and an FG loop with the sequence EX4X1X5X1X1X6GYX4HRP (SEQ ID NO : 325) as defined herein.

One of the objects protected by the present invention is an anti-PCSK9 adnectin containing a BC loop with the sequence (X1) _z X ₂ G (SEQ ID NO: 449), in which X1 is any amino acid, Z is a number in the range 6-9, and X ₂ represents Y or H.

One of the objects protected by the present invention is an anti-PCSK9 adnectin containing a DE loop with the sequence X1X1X1X3 (SEQ ID NO: 450), in which X1 is any amino acid, _X3 is G or S.

One of the objects protected by the present invention is an anti-PCSK9 adnectin containing an FG loop with the sequence EX4X1X5X1X1X6GYX4HR (SEQ ID NO: 451), in which X1 is any amino acid, _X4 is Y or F, _X5 is Y, F or W, X ₆ is S or A. In certain embodiments, X ₄ and X ₅ are aromatic amino acid residues, each of which independently can be Y, F, W or H.

One of the objects protected by the present invention is an anti-PCSK9 adnectin containing a BC loop with the sequence (X1) _z X ₂ G (SEQ ID NO: 449), a DE loop with the sequence X1X1X1X3 (SEQ ID NO: 450) and an FG loop with sequence EX4X1X5X1X1X6GYX4HR (SEQ ID NO: 451), as defined in this application.

In some embodiments, there is at least one amino acid deletion at the N-terminus of the adnectin PCSK9 sequence.

In some embodiments, there is at least one amino acid deletion, insertion, or substitution at the C-terminus of the adnectin PCSK9 sequence.

In some embodiments, a linker sequence is added to the C-terminus of the adnectin PCSK9 sequence.

In some embodiments, PCSK9 adnectin may be conjugated to a molecule other than ¹⁰ Fn3, such as human serum albumin (HSA), as described in patent applications WO 2009/133208 and WO 2009/083804.

In some embodiments, PCSK9 adnectin may contain mutations in the amino acid sequences of the AB, CD and EF loops, as described in patent applications WO 2009/133208 and WO 2009/083804.

One of the objects of the invention is PCSK9 adnectin containing a pharmacokinetic group (PK). In one embodiment, the pharmacokinetic group contains polyethylene glycol (PEG). In some embodiments, the pharmacokinetic group contains an Fc fragment. In some embodiments, the pharmacokinetic group contains one or more adnectins that bind serum albumin. Examples of anti-PCSK9 adnectin-Fc fusion proteins are shown in Table. 1. Examples of anti-PCSK9 adnectins that bind serum albumin contain the sequences of SEQ ID NO: 618 or 619.

In certain embodiments, the PK group-containing anti-PCSK9 adnectins comprise the sequence set forth in SEQ ID NO: 322.

Another object of the invention is anti-PCSK9 adnectin that does not contain any PK group (ie, free anti-PCSK9 adnectin). In certain embodiments, free anti-PCSK9 Adnectin may be administered at a frequency sufficient to achieve the desired therapeutic effect. In another embodiment, free anti-PCSK9 adnectin can be administered in a sustained release dosage form (eg, subcutaneous dosage form). In some embodiments, the extended release dosage form prolongs the absorption phase or prolongs the pharmacodynamic effect, or both. As an example, the extended release dosage form contains a solution of propylene glycol in phosphate buffer (PBS).

One of the objects of the present invention is the use of anti-PCSK9 adnectin for the treatment of atherosclerosis, hypercholesterolemia and other diseases associated with high cholesterol levels.

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One aspect of the present invention is a fusion polypeptide containing serum albumin binding domain ten of fibronectin type III ( ¹⁰ Fn3), and anti-PCSK9 adnectin, in which serum albumin binding domain ¹⁰ Fn3 binds to serum albumin, for example HSA, with a Kd of 1 μM or less. In certain embodiments, Fn3 serum albumin binding domain ¹⁰ comprises an amino acid sequence that is at least 70% identical to SEQ ID NO: 330. In one embodiment, Fn3 serum albumin binding domain ¹⁰ comprises a BC loop with the amino acid sequence set forth in SEQ ID NO: 331, a DE loop having the amino acid sequence set forth in SEQ ID NO: 332, an FG loop having the amino acid sequence set forth in SEQ ID NO: 333. In another embodiment, the serum albumin binding domain ¹⁰ of Fn3 comprises one or more loops BC with the amino acid sequence shown in the sequence SEQ ID NO: 331, DE loops with the amino acid sequence shown in the sequence SEQ ID NO: 332, FG loops with the amino acid sequence shown in the sequence SEQ ID NO: 333.

In one embodiment, a given Fn3 serum albumin binding domain ¹⁰ of the fusion polypeptide is also capable of binding one or more of the albumins rhesus monkey serum albumin (RhSA), cynomolgus serum albumin (CySA), or mouse serum albumin (MuSA). In other embodiments, a given serum albumin binding domain ¹⁰ of Fn3 does not cross-react with one or more proteins among RhSA, CySA, or MuSA.

In certain embodiments, Fn3 serum albumin binding domain ¹⁰ of the fusion polypeptide binds HSA with a Kd of 1 μM or less. In some embodiments, the serum albumin binding domain ¹⁰ of Fn3 binds HSA with a Kd of 500 nM or less. In other embodiments, the serum albumin binding domain ¹⁰ of Fn3 binds HSA with a Kd of at least 200, 100, 50, 20, 10, or 5 nM.

In other embodiments, the Fn3 serum albumin binding domain ¹⁰ of the fusion polypeptide binds to HSA domains I or II. In one embodiment, serum albumin binding domain ¹⁰ of Fn3 binds both HSA domains (I and II). In some embodiments, serum albumin binding domain ¹⁰ of Fn3 binds HSA in the pH range of 5.5 to 7.4. In other embodiments, serum albumin binding domain ¹⁰ of Fn3 binds HSA with a Kd of 200 nM or less at pH 5.5. In other embodiments, serum albumin binding domain ¹⁰ of Fn3 binds HSA with a Kd of at least 500, 200, 100, 50, 20, 10, or 5 nM over a pH range of 5.5 to 7.4. In one embodiment, serum albumin binding domain ¹⁰ of Fn3 binds HSA with a Kd of at least 500, 200, 100, 50, 20, 10, or 5 nM at pH 5.5.

In some embodiments, the plasma half-life of the fusion polypeptide in the presence of serum albumin is at least 5 times greater than the half-life of the polypeptide in the absence of serum albumin. In certain embodiments, the plasma half-life of the fusion polypeptide in the presence of serum albumin is at least 2, 5, 7, 10, 12, 15, 20, 22, 25, 27, or 30 times greater than the half-life of the polypeptide in the absence of serum albumin. albumin. In some embodiments, the serum albumin is one of HSA, RhSA, CySA, or MuSA.

In certain embodiments, the plasma half-life of the fusion polypeptide in the presence of serum albumin is at least 20 hours. In certain embodiments, the plasma half-life of the fusion polypeptide in the presence of serum albumin is at least 10, 12, 15, 20, 25, 30 , 40, 50, 75, 90, 100, 110, 120, 130, 150, 170, or 200 hours. In some embodiments, the half-life of the fusion polypeptide is determined in primates (eg, human or monkey) or mice.

In any of the following aspects or embodiments, serum albumin binding domain ¹⁰ of Fn3 comprises a sequence selected from the group of sequences SEQ ID NOs: 334, 338, 342, 346 and 348-370.

Brief description of drawings

In fig. Figure 1 shows an alignment of an example amino acid sequence of anti-PCSK9 adnectin. The amino acid sequences of the BC, DE, and FG loops are indicated in underline, italic, and underline, or in bold and underline, respectively.

In fig. 2 depicts a schematic of the PCSK9:epidermal growth factor precursor homologous domain (EGFA domain) fluorescence resonance energy transfer (FRET) assay that was used to measure the ability of PCSK9 adnectins to inhibit the PCSK9:LDL interaction as described in Example 2.

In fig. Figure 3 shows the curve obtained by FRET analysis when measuring the inhibition of human PCSK9:EGFA interaction by clones 1459D05, 1784F03, 1813E02, 1922G04 and 1923B02 (panel A) and clones 1459D05, 2012A04, 2011H05 and 2013E 01 (panel B) PCSK9 adnectin as described in example 2.

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In fig. Figure 4 shows the curve obtained by FRET analysis when measuring the inhibition of the interaction of human PCSK9:ATI000972 by clones 1459D05, 1784F03, 1813E02, 1922G04 and 1923B02 (panel A) and clones 2012A04, 2011H05 and 2013E01 ( panel B) PCSK9 adnectin as described in the example 2.

In fig. Figure 5 shows the activity of clones 1459D05, 1784F03, 1813E02, 1922G04 and 1923B02 (panel A) and clones 2012A04, 2011H05 and 2013E01 (panel B) of PCSK9 adnectin in a direct FRET binding assay of human PCSK9, as described in the example 2.

In fig. Figure 6 shows the inhibition of PCSK9 activity in HepG2 cells measured using the DiI-LDL cell uptake method as described in Example 2.

In fig. Figure 7 shows inhibition of PCSK9-induced reduction in the amount of LDLR on the surface of HepG2 cells by PCSK9 adnectin expressed by clones 1459D05, 1784F03, 2012A04 and 2011H05 (panel A) and 2011H05, 2012A04 and 2013E01 (panel B), as described in the example 2. Indicator EC ₅₀ (nM) for clones 1784F03, 2012A04, 2011H05 and 2013E01 are 15.98, 7.78, 8.85 and 12.41, respectively. The percentage level of PCSK9 inhibition at a concentration of 75 nM PCSK9 adnectin clones 1459D05, 1784F03, 2012A04, 2011H05 and 2013E01 is 66.8, 150.2, 190.1, 177.4 and 152.2, respectively.

In fig. Figure 8 demonstrates the effect of administration of PCSK9 adnectin ATI000959 (100 mg/kg) on plasma cholesterol levels (panel A) and on plasma unbound hPCSK9 levels (panel B) in transgenic mice expressing high levels of hPCSK9, as described in Example 3. The ATI000959 variant contains a 40 kDa branched PEG (NOF).

In fig. Figure 9 demonstrates the effect of administration of PCSK9 adnectin ATI001114 (10 or 60 mg/kg) on plasma cholesterol levels (panel A) and on plasma unbound hPCSK9 levels (panel B) in transgenic mice expressing high levels of hPCSK9, as described in the example. 3.

In fig. Figure 10 demonstrates the effect of administration of the PCSK9 adnectins ATI000959 (panel A) or ATI001114 (panel B), administered in an amount of 5 mg/kg intraperitoneally (i.p.), in a single dose, on the level of free hPCSK9 in the serum of mice of a transgenic line with a normal level of hPCSK9 expression (average value±SD), as described in example 3.

In fig. Figure 11 shows the dose dependence of the effect of PCSK9 adnectin ATI001114 on the level of free hPCSK9 in mice transgenic for hPCSK9 with a normal expression level, as described in example 3.

In fig. 12 shows the effect of a single administration of PCSK9 adnectin ATI001114 (5 mg/kg i.v.) on reducing LDL-C in cynomolgus monkeys (mean±SEM, n=3), as described in Example 3.

In fig. 13 shows the binding affinity and stoichiometry of PCSK9 adnectin to hPCSK9 determined by ITC. PCSK9 adnectins bind to hPCSK9 in a 1:1 ratio. The left side of the figure shows data for PCSK9 adnectin ATI001081, and the right side shows data for PCSK9 adnectin ATI001174.

In fig. 14 shows the inhibition of PCSK9:EGFA (left panel) and PCSK9:ATI-972 (right panel) signal by PCSK9 adnectins when analyzed by FRET.

In fig. Figure 15 shows inhibition of the PCSK9-induced decrease in the amount of RLGGNP on the surface of HepG2 cells under the influence of anti-PCSK9 adnectins.

In fig. 16 shows the inhibition of entry of PCSK9-AF647 into HepG2 cells.

In fig. 17 shows the plasma level of free hPCSK9 in transgenic mice administered PRD460 (administered i.p.).

In fig. 18 shows the effect of PRD460 administration (15 mg/kg i.v.) on LDL-C and free PCSK9 levels in cynomolgus monkeys (mean±SEM, n=3).

In fig. 19 shows the effect of ATI-1081 (also designated ATI001081) on free PCSK9 levels in cynomolgus monkeys.

In fig. 20 shows the effect of ATI-1081 on LDL-C levels in cynomolgus monkeys.

In fig. 21 shows the effect of ATI-1081 in PBS when administered to transgenic mice. This figure shows plasma levels of free PCSK9 in transgenic mice.

In fig. 22 shows the effect of subcutaneous administration of ATI-1081 in polyethylene glycol to transgenic mice. This figure shows data on the level of free hPCSK9 in transgenic mice.

In fig. 23 shows the half-life of HSA in mice (in vivo). HSA was administered to nude mice at doses of 20 mg/kg (left panel) and 50 mg/kg (right panel).

In fig. 24 shows the determination of the half-lives of SABA1.1 (panel A), SABA2.1 (panel B), SABA3.1 (panel C) and SABA4.1 (panel D) when administered to mice.

In fig. 25 is a graph showing the increase in half-life of SABA1-4 when co-administered with HSA to mice.

In fig. 26 shows the determination of the plasma half-lives of SABA1.1 (panel A) and SABA5.1 (panel B) in cynomolgus monkeys.

In fig. 27 shows SABA1.2 binding to human, mouse and rat albumins by direct ELISA.

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In fig. Figure 28 shows the determination of the stoichiometric binding parameters of SABA1.1 and HSA.

The stoichiometry of this binding is 1:1.

In fig. 29 shows an analysis of SABA1.2 binding to recombinant HSA domain fragments using the BIACORE® system.

In fig. 30 shows the pharmacokinetic profile of SABA1.2 in cynomolgus monkeys when administered at dosages of 1 and 10 mg/kg.

In fig. 31 shows the pharmacokinetic profile of SABA1.2 in monkeys when administered intravenously or subcutaneously at a dosage of 1 mg/kg.

Detailed Description of the Invention

Definitions.

The term polypeptide refers to any sequence of two or more amino acids, regardless of length, post-translational modifications or function. The terms polypeptide, peptide and protein are used interchangeably throughout this document. Polypeptides may include both natural and unnatural amino acids, such as those described in the U.S. application. patent No. 6559126 and incorporated into this invention by reference. Polypeptides can also be modified by any of a variety of standard chemical methods (e.g., an amino acid can be modified with a protecting group, a C-terminal carboxyl can be transformed into an amide group, an N-terminal amino acid residue can be modified with various groups to enhance lipophilicity, or the polypeptide may be chemically glycosylated or otherwise modified to increase stability and half-life in vivo). Modifications of polypeptides may include the addition of other structures, such as cyclic compounds or other molecules, to the polypeptide. Also, polypeptides may contain one or more amino acid residues with a changed configuration (for example, R, or S, or L, or D). The peptides described in the present invention are modified derivatives of the tenth domain III of fibronectin modified to specifically bind PCSK9. These peptides are designated herein by the following names: anti-PCSK9 adnectin or PCSK9 adnectin.

The term PK is an acronym for the term pharmacokinetic and refers to various properties of a chemical, including, for example, absorption, distribution, metabolism and excretion by a subject. The terms PK modulating protein or PK group refer to any protein, peptide or agent that affects the pharmacokinetic properties of a biologically active molecule when fused together or co-administered into the body with a biologically active molecule. Examples of PK modulating proteins or PK groups are PEGs, human serum albumin (HSA) binders (as described in U.S. Patent Applications Publication No. 2005/0287153 and 2007/0003549, PCT Publication No. WO 2009/083804 and WO 2009/133208, and SABA molecules as described herein), human serum albumin, Fc moiety or portions and variants thereof, and sugars (eg, sialic acids).

Percentage (%) amino acid sequence identity in this application is defined as the percentage of amino acid residues of a candidate sequence that are identical to amino acid residues of the selected sequence, after aligning the sequences and introducing gaps where necessary to achieve the maximum percentage of sequence identity, and considering any conservative substitutions as different parts of the compared sequences. Alignments to determine percent amino acid sequence identity can be generated by various methods known to those skilled in the art, for example, using publicly available computer programs such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR®). Those skilled in the art can determine appropriate parameters for evaluating the alignment, including any algorithms necessary to achieve maximum alignment over the entire length of the sequences being compared.

The term isolated is used for a polypeptide that has been identified and isolated from its natural environment. Contaminants in a peptide's natural environment are substances that may interfere with the diagnostic or therapeutic applications of the polypeptide and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In preferred embodiments, the polypeptide will be purified (1) to greater than 95% by weight as determined by the Lowry method and in most preferred embodiments to greater than 99% by weight; (2) to a degree of purity sufficient to detect at least the N-terminal or internal amino acid sequence using a rotating beaker sequencer; or (3) until homogeneous as determined by SDS-PAGE under reducing or non-reducing conditions with Coomassie blue or preferably silver staining. The term isolated polypeptide includes the polypeptide in recombinant cells in situ, since at least one component of the polypeptide's natural environment is absent. Typically, however, the isolated polypeptide is obtained using at least one purification step.

The symbol mpk, mg/kg or mg per kg is used to indicate the dosage of a substance per kg of weight. All of these conventions are used equally throughout this document.

The term half-life of an amino acid sequence or compound can be generally defined as the time required to reduce the serum concentration of a polypeptide by 50% in vivo, for example, due to degradation of the sequence or compound and/or clearance or isolation of the sequence or compound by natural mechanisms. The half-life can be determined by any known method, for example, by pharmacokinetic analysis. Suitable methods are known to one skilled in the art and may, for example, generally comprise the steps of: administering to primates in a suitable manner suitable doses of the amino acid sequence or compound of interest described in the present invention; routine collection of blood or other specimens from specified primates; determining the level or concentration of the administered amino acid sequence or compound in said blood samples and calculating, based on the data so obtained, a time sufficient for the level or concentration of the amino acid sequence or compound described in this invention to be reduced by 50% compared to with the initial level upon administration. For examples, see standard manuals such as Kenneth, A. et al., Chemical Stability of Pharmaceuticals: A Handbook for Pharmacists; and Peters et al., Pharmacokinete Analysis: A Practical Approach (1996). Also see Gibaldi, M. et al., Pharmacokinetics, 2nd rev. edition, Marcel Dekker (1982).

Half-life can be expressed using parameters such as t _1/2 -a, t _1/2 -e, HLLambdaz and area under the curve (AUC). As used herein, an increase in half-life refers to an increase in any of these parameters, any two of these parameters, any three of these parameters, or all four parameters. An increase in half-life, in particular, means an increase in t _1/2 -e and/or HLLambdaz, either together or not with an increase in t _{1/2 -a} and/or AUC.

Review.

This application describes adnectins against human PCSK9. To identify specific antagonists for PCSK9, PCSK9 molecules were screened against a large synthetic adnectin library. PCSK9-bound adnectins were characterized for PCSK9 binding, biophysical properties, and PCSK9 inhibitory activity. Selected pools of adnectins were subjected to mutagenesis and subsequent rounds of selection, reducing the concentration of the binding agent and isolating adnectins with a low dissociation constant. After implementing the described optimization process, a group of adnectins that specifically inhibit PCSK9 were selected with the desired biochemical and biophysical properties.

Structures based on fibronectin.

One of the objects of this application are polypeptides containing an Fn3 domain in which the sequences of one or more solvent accessible loops are subjected to random or site-directed mutagenesis. In some embodiments, the Fn3 domain is an Fn3 domain derived from the wild-type sequence of the tenth human fibronectin type III domain ( ¹⁰ Fn3):

VSDVPRDLEVVAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVOEFTVPGSK

STATISGLKPGVDYTITVYAVTGRGDSPASSKPISINYRT (SEQ Ш NO: 1).

In the indicated sequence ¹⁰ Fn3 the loops BC, DE and FG are underlined.

As described herein, the ¹⁰ Fn3 sequences that do not bind ligands, i.e. being structural regions ^{of 10} Fn3, can be modified to such an extent that the ¹⁰ Fn3 domain retains ligand binding functions and/or structural stability. A large number of different mutant variants of structural regions ¹⁰ of Fn3 have been described. In one aspect of the present invention, one or more amino acid residues from Asp 7, Glu 9 and Asp 23 are replaced with other amino acid residues, for example non-negatively charged ones (for example, Asn, Lys, etc.). Such substitutions have been shown to increase the stability of ¹⁰ Fn3 at neutral pH compared to the wild type ¹⁰ Fn3 domain (see PCT Publication No. WO 02/04523). A large number of additional structural changes in domain ¹⁰ of Fn3 have been described, which have either beneficial or neutral effects. See, for example, Baton et al., Protein Eng., 15(12):1015-1020 (Dec. 2002); Koide et al., Biochemistry, 40(34):10326-10333 (Aug. 28, 2001).

Both mutant and wild type Fn3 domain ¹⁰ have a similar structure, described as 7 beta regions, designated A to G, and 6 loop regions (loops AB, BC, CD, DE and FG) connecting these beta regions. The beta regions located closest to the N and C termini of the molecule can adopt a beta-like conformation in solution. In the sequence SEQ ID NO:1, loop AB corresponds to residues 15-16, loop BC corresponds to residues 21-30. the CD loop corresponds to residues 39-45, the DE loop corresponds to residues 51-56, the EF loop corresponds to residues 60-66, the FG loop corresponds to residues 76-87 (Xu et al., Chemistry & Biology, 9:933-942 (2002)) .

In some embodiments, the sequence of the Fn3 ¹⁰ polypeptide may be at least 40, 50, 60, 65, 70, 75, 80, 85, or 90% identical to the human Fn3 ¹⁰ domain sequence set forth in SEQ ID NO:1. Most of the variability will typically be located in one or more loops. The sequences of each of the beta and beta-like regions of Fn3 polypeptide ¹⁰ may consist almost entirely of an amino acid sequence that is at least 80, 85, 90, 95 or 100% identical to the sequences of the corresponding beta or beta-like regions of SEQ ID NO:1 , such that the differences do not impair the stability of the polypeptide under physiological conditions.

In some embodiments, the present invention includes polypeptides comprising a tenth fibronectin type III domain ( ¹⁰ Fn3), wherein said ¹⁰ Fn3 domain comprises an AB loop, a BC loop, a CD loop, a DE loop, an EF loop, and an FG loop, wherein the sequence is at least at least one of the loops BC, DE and FG is changed relative to the sequence of the corresponding loop of human Fn3 domain ¹⁰ . In some embodiments, the sequences of loops BC and FG are changed; in some embodiments, the sequences of loops BC, DE and FG are changed, i.e. the Fn3 domain contains loops whose structure differs from that of the natural domain. In some embodiments, the AB, CD and/or EF loop sequences have been changed. A change is defined as the presence of one or more differences between a given amino acid sequence and a reference sequence (the corresponding domain of human fibronectin). The change includes insertions, deletions and substitutions of amino acid residues. Amino acid sequence changes can be achieved through the introduction of targeted, undirected or spontaneous sequence changes, typically at the level of the coding sequence, and can be accomplished by any method, such as PCR, error PCR or chemical DNA synthesis.

In some embodiments, the length of one or more loops from the group BC, DE and FG may differ from the sequence of the corresponding human fibronectin loop to a greater or lesser extent. In some embodiments, the length of the loop can be increased by 2-25 amino acid residues. In some embodiments, the length of the loop may be reduced by 1-11 amino acid residues. Thus, to optimize antigen binding, any loop of Fn3 domain ¹⁰ can be varied in length as well as amino acid sequence to achieve the best possible flexibility and affinity for antigen binding.

In some embodiments, the polypeptide comprises an Fn3 domain containing an amino acid sequence that is at least 80, 85, 90, 95, 98, 99 or 100% identical to the non-loop regions of SEQ ID NO: 1, in which at least one loop of groups BC, DE and FG is modified. In some embodiments, the altered BC loop sequence contains up to 10 amino acid substitutions, up to 4 deletions and up to 10 insertions of amino acid residues, or combinations of these changes. In some embodiments, the altered DE loop sequence contains up to 6 amino acid substitutions, up to 4 deletions, and up to 13 amino acid residue insertions, or combinations of these changes. In some embodiments, the altered FG loop sequence contains up to 12 amino acid substitutions, up to 11 deletions, and up to 25 amino acid residue insertions, or combinations of these changes.

As described above, the amino acid residues corresponding to residues at positions 21-30, 51-56 and 76-87 of SEQ ID NO:1 correspond to the BC, DE and FG loops, respectively. However, it should be understood that not every amino acid residue in the loop region must be changed in order to obtain high affinity of ¹⁰ Fn3 for the selected target (eg, PCSK9).

For example, it is not necessary to modify residues 21 (S) and 22 (W) of the BC loop, as indicated in SEQ ID NO: 1, for binding to PCSK9. Those. Fn3 domains ¹⁰ with high binding affinity for PCSK9 can be obtained by changing residues only in positions 23-30 of the BC loop, as shown in the sequence SEQ ID NO: 1. This is clearly shown in the examples of BC loop sequences given in table. 3, where it is shown that only amino acid residues in the shaded positions of the given sequences have been changed. Thus, in some embodiments, the BC loop, in accordance with this designation, contains a darkened portion of any of the sequences SEQ ID NO: 2-17, 106-135 and 301-303, as reflected in table. 3. For example, in one embodiment, the BC loop may contain the sequence PPPSHGYG (residues 3-10 of SEQ ID NO: 2), DAPAHAYG (residues 3-10 of SEQ ID NO: 5), EPFSRLPGGGE (residues 3-13 of SEQ ID NO: 106) or DAPADGGYG (residues 3-11 of SEQ ID NO: 107).

Similarly, there is no need to change the amino acid residues at positions 51 (P) and 56 (T) of the DE loop of SEQ ID NO: 1 for binding to PCSK9. Those. Fn3 domains ¹⁰ with high binding affinity for PCSK9 can be obtained by changing residues only in positions 52-55 of the DE loop, as shown in the sequence SEQ ID NO: 1. This is clearly shown in the example DE loop sequences given in table. 3, where it is shown that only amino acid residues in the shaded positions of the given sequences have been changed. Thus, in some embodiments, the DE loop, in accordance with this designation, contains the darkened portion of any of the sequences SEQ ID NO: 18-27 and 136-141, as reflected in table. 3. For example, in one embodiment, the DE loop may contain the sequence PGKG (residues 2-5 of SEQ ID NO: 18), VGVG (residues 2-5 of SEQ ID NO: 27) or VSKS (residues 2-5 of SEQ ID NO: 27). 8044608 sequence SEQ ID NO: 137).

Similarly, there is no need to change the amino acid residues at position 87 (P) of the FG loop of SEQ ID NO:1 for binding to PCSK9. Those. Fn3 high affinity PCSK9 binding domains ¹⁰ can be obtained by altering residues only at positions 76-86 of the FG loop as shown in SEQ ID NO:1. This is clearly shown in the examples of FG loop sequences given in Table. 3, where it is shown that only amino acid residues in the shaded positions of the given sequences have been changed. Thus, in some embodiments, the FG loop, in accordance with this designation, contains the darkened portion of any of the sequences SEQ ID NO: 28-38 and 142-172, as reflected in table. 3. For example, in one embodiment, the FG loop may comprise the sequence EYPYKHSGYYHR (residues 1-12 of SEQ ID NO: 28), EYPYDYSGYYIIR (residues 1-12 of SEQ ID NO: 142), or EFDFVGAGYYHR (residues 1-12 of SEQ ID NO: 167).

In some embodiments, the present application demonstrates that the BC, DE, and FG loops can be generally described by their respective consensus sequences. For example, a BC loop can be generally described using the consensus sequence SW(X1) _z X2G (SEQ ID NO: 323), in which X1 is any amino acid, Z is a number from 6 to 9, X2 is Y or H This consensus sequence is presented in the form of examples by sequences of BC loops in Table. 3 except for the BC loop defined by SEQ ID NO: 106. In other embodiments, Z is a number from 2 to 5. In certain embodiments, X ₂ is any aromatic amino acid residue (i.e., Y, F, W, or H) .

In another embodiment, the DE loop may be generally expressed using the consensus sequence PX1X1X1X3T (SEQ ID NO: 324), in which X1 is any amino acid, _X3 is G or S. This consensus sequence is exemplified by loop sequences DE in table 3.

In another embodiment, the FG loop can be generally expressed using the consensus sequence EX4X1X5X1X1X6GYX4HRP (SEQ ID NO: 325), in which X1 is any amino acid, _X4 is Y or F, X5 is Y, F or W, X6 is S or A. In certain embodiments, _X4 and X5 are aromatic amino acid residues, each of which independently may be Y, F, W or H. This consensus sequence is exemplified by the FG loop sequences in Table 1. 3.

Accordingly, in certain embodiments, the present application discloses a PCSK9 binding adnectin comprising a BC loop with the sequence SW(X1) _Z X ₂ G (SEQ ID NO: 323), a DE loop with the sequence PX1X1X1X3T (SEQ ID NO: 324) and a FG loop with the sequence EX4X1X5X1X1X6GYX4HRP (SEQ ID NO: 325), as described above.

In other embodiments, the present application discloses a PCSK9 binding adnectin comprising a BC loop with the sequence SWEPFSRLPGGGE (SEQ ID NO: 106), a DE loop with the sequence PX1X1X1X3T (SEQ ID NO: 324) and an FG loop with the sequence EX4X1X5X1X1X6GYX4HRP (SEQ ID NO: 325) , as described above.

In certain embodiments, the BC loop sequence may be generally expressed by the consensus sequence (X1) _Z X ₂ G (SEQ ID NO: 449), wherein X1 is any amino acid, Z is any number from 6 to 9, X ₂ represents Y or H. This consensus sequence is exemplified by the BC loop sequences in Table. 3, except for loop BC, defined by SEQ ID NO: 106. In other embodiments, Z is any number from 2 to 5. In certain embodiments, Z is any number from 10 to 15. In certain embodiments, X ₂ is the remainder of any aromatic amino acid (i.e. Y, F, W or H).

In certain embodiments, the DE loop can be generally expressed using the consensus sequence X1X1X1X3, (SEQ ID NO: 450), in which X1 is any amino acid, X ₃ is G or S. This consensus sequence is represented by example sequences loops DE in table. 3.

In certain embodiments, the FG loop may be generally expressed using the consensus sequence EX4X1X5X1X1X6GYX4HR (SEQ ID NO: 451), wherein X1 is any amino acid, _X4 is Y or F, _X5 is Y, F or W X ₆ is S or A. In certain embodiments, X ₄ and X ₅ are aromatic amino acid residues, each of which independently can be Y, F, W or H. This consensus sequence is exemplified by the FG loop sequences in Table 1. 3.

Accordingly, in one embodiment, the present application discloses a PCSK9 binding adnectin comprising a BC loop with the sequence (X ₁ ) _Z X ₂ G (SEQ ID NO: 449), a DE loop with the sequence X ₁ X ₁ X ₁ X ₃ (SEQ ID NO: 450) and an FG loop with the sequence EX4X1X5X1X1X6GYX4HR (SEQ ID NO: 451), as described above.

In certain embodiments, immunoglobulin-like proteins based on structural moiety ¹⁰ of Fn3 may be generally defined by the following sequence:

- 9 044608

EWAAT(X) _a SLLI(X)xYYRITYGE(X)z,QEFTV(X)j,ATI(X) _with DYTITVYAV(X)JSINY

RT (SEQ ID NO:328)

In the sequence SEQ ID NO: 328, loop AB is represented by X _a , loop CD is represented by X _b , loop EF is represented by Xc, loop BC is represented by X _x , loop DE is represented by X _y , loop FG is represented by Xz. X denotes any amino acid, the subscript X denotes an integer number of amino acid residues. In particular, a anywhere can be 1-15, 2-15, 1-10, 2-10, 1-8, 2-8, 1-5, 2-5, 1-4, 2-4, 1 -3, 2-3 or 1-2 amino acid residues; b, c, x, y and z, each independently anywhere can be 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 acid residues. In preferred embodiments, a is 2 amino acid residues, b is 7 amino acid residues, c is 7 amino acid residues, x is 9 amino acid residues, y is 6 amino acid residues, z is 12 amino acid residues. The sequences of these beta regions can have anywhere from 0 to 10, 0 to 8, 0 to 6, 0 to 5, 0 to 4, 0 to 3, 0 to 2, 0 to 1 amino acid substitutions , deletions or insertions throughout all 7 structural regions relative to the corresponding amino acid residues specified in the sequence SEQ ID NO: 1. In certain embodiments, the amino acid residues constituting the core portion are fixed and any substitutions, conservative substitutions, deletions or insertions are made with any other amino acid residues.

In exemplary embodiments, the BC, DE and FG loops, represented by the sequences (X) _x , (X) _y and (X) _z , respectively, are replaced by polypeptides containing the sequences of the BC, DE and FG loops of any variants of the PCSK9 binding proteins presented in table. 3, or the shaded regions of these variants, or consensus sequences 323-325 or 449-451.

In certain embodiments, immunoglobulin-like proteins based on the Fn3 structure ¹⁰ can be generally defined by the following sequence:

EVVAATPTSLLI(X) _x YYRITYGETGGNSPVQEFTV(X) _y ATISGLKPGVDYTITVYA

V(X)zISINYRT (SEQ Ш NO:329)

In the sequence SEQ ID NO: 329, loop BC is represented by X _x , loop DE is represented by X _y , loop FG is represented by X _z . X denotes any amino acid, the subscript X denotes an integer number of amino acid residues. Specifically, x, y and z each independently anywhere can be 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 acid residues. In preferred embodiments, x is 9 amino acid residues, y is 6 amino acid residues, z is 12 amino acid residues. The sequences of these beta regions can have anywhere from 0 to 10, 0 to 8, 0 to 6, 0 to 5, 0 to 4, 0 to 3, 0 to 2, 0 to 1 amino acid substitutions , deletions or insertions throughout all 7 structural regions relative to the corresponding amino acid residues indicated in the sequence SEQ ID NO: 1. In embodiments of the invention, the sequences of these beta regions may be anywhere from 0 to 10, from 0 to 8, from 0 to 6, 0 to 5, 0 to 4, 0 to 3, 0 to 2, 0 to 1 conservative amino acid substitutions throughout all 7 structural regions relative to the corresponding amino acid residues indicated in the sequence SEQ ID NO: 1. B In certain embodiments, the core amino acid residues are fixed and any substitutions, conservative substitutions, deletions or insertions are made with any other amino acid residues. In exemplary embodiments, the BC, DE and FG loops, represented by the sequences (X) _x , (X) _y and (X) _z , respectively, are replaced by polypeptides containing the sequences of the BC, DE and FG loops of any variants of the PCSK9 binding proteins presented in table. 3, or the shaded regions of these variants, or consensus sequences 323-325 or 449-451.

In certain embodiments, the anti-PCSK9 adnectin described herein may comprise a sequence as set forth in SEQ ID NO: 328 or 329, in which the BC, DE, and FG loops represented by the sequences (X) _x , (X) _y , and ( X) _z , respectively, are replaced by the corresponding set of specific sequences of loops BC, DE and FG from any of the clones in table. 3, or sequences at least 75, 80, 85, 90, 95, 97, 98 or 99% identical to the sequences of the BC, DE and FG loops of the clones given in table. 3. In embodiments of the invention, the anti-PCSK9 adnectin described in this application is determined by the sequence SEQ ID NO: 329 and contains the corresponding set of BC, DE and FG loop sequences from any of the clones shown in table. 3. For example, clone 1459D05 in table. 3 contains the same BC, DE and FG loop sequences as in SEQ ID NOs: 2, 18 and 28, respectively. Thus, an anti-PCSK9 adnectin based on these loops may contain the sequence of SEQ ID NO: 328 or 329, in which (X) _x contains the sequence of SEQ ID NO: 2, (X) _y contains the sequence of SEQ ID NO: 18 and (X ) _z contains the sequence SEQ ID NO: 28. Similar designs can be implemented using a set of loops BC, DE and FG of other clones listed in table. 3, or consensus sequences 323-325 or 449-451. The structural parts of such anti-PCSK9 adnectin variants may contain any part from 0 to 20, 0 to 15, 0 to 10, 0 to 8, 0 to 6, 0 to 5, 0 to 4, 0 up to 3, 0 to 2, 0 to 1 amino acid substitution, conservative amino acid substitution, deletion or insertion

- 10 044608 relative to the amino acid residues of the structural part indicated in the sequence SEQ ID

NO: 1. Such structural modifications can be carried out as long as anti-PCSK9 adnectin is able to bind PCSK9 to the desired _KD .

In some embodiments, the BC loop of a protein described in this invention contains an amino acid sequence selected from the following group:

SWPPPSHGYG (SEQ Ш NO: 2), SWRPPIHAYG (SEQ Ш NO: 3), SWDAPIHAYG (SEQ Ш NO:4), SWDAPAHAYG (SEQ Ш NO:5) and SWDAPAVTYG (SEQ Ш NO:6), SWSPPANGYG (SEQ Ш NO :7), SWTPPPKGYG (SEQ ID NO:8), SWRPPSHAYG (SEQ ID NO:9), SWDPPSHAYG (SEQ ID NO:10), SWEPPSHAYG (SEQ ID NO:11), SWSPPSHAYG (SEQ ID NO: 12), SWRPPSNGHG (SEQ ID NO: 13), SWVPPSDDYG (SEQ ID NO: 14), SWVPSSHAYG (SEQ ID NO: 15), SWDPSSHAYG (SEQ ID NO: 16) and SWEPSSHAYG (SEQ ID NO: 17).

In subsequent embodiments, the BC loop of the protein described in this invention contains an amino acid sequence selected from the sequences of SEQ ID NO: 106-135 and 301-303. In other embodiments, the BC loop of the protein described in the invention contains the darkened portion of any of the sequences SEQ ID NO: 2-17, 106-135 and 301-303, as shown in table. 3. For example, in one embodiment, the BC loop contains the sequence PPPSHGYG (residues 3-10 of SEQ ID NO: 2), DAPAHAYG (residues 3-10 of SEQ ID NO: 5), EPFSRLPGGGE (residues 3-13 of SEQ ID NO: 5 NO: 106) or DAPADGGYG (residues 3-11 of SEQ ID NO: 107).

In some embodiments, the DE loop of a protein of the present invention comprises an amino acid sequence selected from the following group:

PPGKGT (SEQ Ш NO: 18), PIVEGT (SEQ Ш NO: 19), PGSEGT (SEQ Ш NO:20),

PGSKGT (SEQ ID NO:21), PGSKST (SEQ ID NO:22), PVGRGT (SEQ ID NO:23), PVGEGT (SEQ ID NO:24), PIGKGT (SEQ ID NO:25), PVNEGT (SEQ ID NO :26) and PVGVGT (SEQ ID NO:27).

In subsequent embodiments, the DE loop of the protein described in the present invention contains an amino acid sequence selected from the sequences of SEQ ID NO: 136-141. In other embodiments, the DE loop of a protein of the present invention comprises a shaded portion of any of SEQ ID NOs: 18-27 and 136-141, as shown in Table 1. 3. For example, in one embodiment, the DE loop contains the sequence PGKG (residues 2-5 of SEQ ID NO: 18), VGVG (residues 2-5 of SEQ ID NO: 27) or VSKS (residues 2-5 of SEQ ID NO: 27) NO: 137).

In some embodiments, the FG loop of a protein described in the present invention contains an amino acid sequence selected from the following group:

EYPYKHSGYYHRP (SEQ Ш NO:28), EYTFKHSGYYHRP (SEQ ID NO:29),

EYTYKGSGYYHRP (SEQ ID NO:30), EYTYNGAGYYHRP (SEQ ID NO:31),

EYTYIGAGYYHRP (SEQ ID NO:32), EYTYEGAGYYHRP (SEQ ID NO:33),

EYAYNGAGYYHRP (SEQ ID NO:34), EYPWKGSGYYHRP (SEQ ID NO:35),

EFPFKWSGYYHRP (SEQ ID NO:36), EFPWPHAGYYHRP (SEQ ID NO:37) and EYAEEGAGYYHRP (SEQ ID NO:38).

In subsequent embodiments, the FG loop of the protein described in the present invention contains an amino acid sequence selected from the sequences of SEQ ID NO: 142-172. In other embodiments, the FG loop of the protein described in the present invention contains the darkened portion of any of the sequences SEQ ID NO: 28-38 and 142-172, as shown in table. 3. For example, in one embodiment, the FG loop contains the sequence EYPYKHSGYYHR (residues 1-12 of SEQ ID NO: 28), EYPYDYSGYYHR (residues 1-12 of SEQ ID NO: 142), or EFDFVGAGYYHR (residues 1-12 of SEQ ID NO: 142) NO: 167).

In some embodiments, the protein described herein comprises a single BC loop sequence selected from the group of BC sequences comprising SEQ ID NOs: 2-17, 106-135 and 301-303, or the shading portion of any one of the SEQ ID sequences NO: 2-17, 106-135 and 301-303, as shown in table. 3; one DE loop sequence selected from the group of DE loop sequences comprising the sequences SEQ ID NOs: 18-27 and 136-141, or the shaded portion of any one of the sequences SEQ ID NOs: 18-27 and 136-141 as shown in table. 3; and one FG loop sequence selected from the group of FG sequences comprising the sequences SEQ ID NOs: 28-38 and 142-172, or the shaded portion of any one of the sequences SEQ ID NOs: 28-38 and 142-172 as shown in table. 3. In some embodiments, the protein described in the present invention contains amino acid sequences of the BC, DE and FG loops that are at least 70, 75, 80, 85, 90, 95, 98, 99 or 100% identical any one of the sequences SEQ ID NO: 2-38, 106-172, 301-303. In other embodiments, the protein described in the present invention contains amino acid sequences of the BC, DE and FG loops that are at least 70, 75, 80, 85, 90, 95, 98, 99 or 100% identical to the shaded portion of any of the sequences of SEQ ID NO: 2-38, 106-172, 301-303 as shown in table. 3.

In some embodiments, the amino acid sequence of anti-PSCK9 adnectin comprises any of SEQ ID NOs: 39-76, 173-290, and 304-309. In some embodiments, anti-PSCK9 adnectin contains a fragment of the amino acid sequence of the Fn3 domain from positions 3 to 96 of any of SEQ ID NOs: 39-76, 173-290 and 304-309. In some embodiments, anti-PSCK9 adnectin contains an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identical to any of SEQ ID NOs: 39-76, 173-290, and 304-309.

Fibronectin binds to various types of integrins through the arginine-glycine-aspartic acid (RGD) integrin-binding motif. In some embodiments, the protein described in this invention contains Fn3 domain ¹⁰ without an interin binding (RGD) motif. The integrin binding motif can be removed by modifying the RGD sequence through amino acid substitutions, deletions or insertions.

In certain embodiments, the anti-PSCK9 adnectin molecules described in the present invention can be modified by extending the N- or C-terminal portions. For example, the MG sequence may be added to the N-terminus of the Fn3 domain ¹⁰ sequence defined by SEQ ID NO: 1. The additional amino acid M is usually cleaved off, leaving a G amino acid residue at the N-terminus of the protein. Otherwise, the first 10 amino acid residues of the anti- PSCK9 adnectin, given in table. 4 may be replaced by another N-terminal sequence, referred to herein as an N-terminal extension, as shown in Table. 6 (eg SEQ ID NO: 371-379). In addition, M, G or MG can be placed at the N-terminus of any N-terminal extension, SEQ ID NO: 371-379. The Ahtu-PSCK9 adnectins described herein may also contain various C-terminal sequences, referred to herein as C-terminal extensions. For example, the anti-PSCK9 adnectin sequence shown in Table. 4 may be truncated after the threonine corresponding to the threonine at position 94 of SEQ ID NO: 1 (ie, truncated after the sequence 1NYRT (SEQ ID NO: 636)). Such a truncated version may be used as a therapeutic molecule in a truncated form, or another C-terminal sequence may be added after the threonine residue. Examples of C-terminal extension sequences are given in table. 6 as SEQ ID NO: 380-395. Examples of anti-PSCK9 adnectin sequences containing C-terminal extensions are given in Table. 4. For example, the sequence SEQ ID NO: 49 (clone 1813E02) contains the natural C-terminal extension sequence EIDKPSQ (SEQ ID NO: 380) followed by a His6 tag sequence (SEQ ID NO: 637). However, it is worth understanding that the His6 tag sequence is completely optional.

In certain embodiments, the C-terminal extension sequences (also called tails) contain residues E and D and can be 8 to 50, 10 to 30, 10 to 20, 5 to 10, and 2 to 4 amino acid residues long . In some embodiments, the tail sequences include ED-based linkers in which the sequence consists of tandem ED repeats. In exemplary embodiments, the tails contain 2-10, 2-7, 2-5, 3-10, 3-7, 3-5, 3, 4 or 5 ED repeats. In certain embodiments, ED-based tails may also include additional amino acid residues, such as, for example: EI (SEQ ID NO: 385), EG, ES, EC, EGS and EGC. These sequences are similar to known adnectin tail sequences, such as EIDKPSQ (SEQ ID NO: 380), in which the D and K residues are deleted. In exemplary embodiments, such ED tails contain E, I, or EI residues (SEQ ID NO: 385 ) before ED repeats.

In other embodiments, the N- and C-terminal sequences may be combined with other known linker sequences (eg, SEQ ID NOs: 396-419 in Table 6) if desired in the construction of anti-PSCK9 adnectin fusions. Examples of anti-PSCK9 adnectin sequences containing linker sequences are given in table. 4 (eg SEQ ID NO: 53, 55 and 57). In some embodiments, a C-terminal sequence may be added to the Fn3 domain ¹⁰ sequence to facilitate attachment of a pharmacokinetic group. For example, a cysteine-containing linker sequence such as GSGC (SEQ ID NO: 77) can be added to the C-terminus for site-directed pegylation of a given cysteine residue.

Pharmacokinetic parts.

One of the objects described in this application for invention is anti-PCSK9 adnectin containing a pharmacokinetic (PK) part. Improved pharmacokinetics may be desirable according to the therapeutic need. It is often desirable to increase bioavailability and/or increase the time between dosing of the drug, which is possible by increasing the period

- 12 044608 time when the protein remains available in the blood serum after administration of the drug. In some cases, it is desirable to maintain a constant plasma protein concentration (eg, reduce the difference between the serum protein concentration shortly after administration and shortly before the next administration). Ahtu-PCSK9 adnectin may be attached to a specific moiety that reduces the rate of clearance of the polypeptide in mammals (eg, mice, rats, or humans) by more than three times compared to unmodified anti-PCSK9 adnectin. Other indicators of improved pharmacokinetics include plasma half-life, which is often divided into alpha and beta phases. The performance of each or both phases can be significantly improved by adding the appropriate RK group.

Molecules that slow protein clearance from the blood are referred to herein as PK groups and include polyoxoalkylene groups, e.g., polyethylene glycol, sugars (e.g., sialic acid), and well-tolerated protein groups (e.g., Fc moiety and parts thereof and variants thereof, transferrin , serum albumin). Ahtu-PCSK9 adnectin can be fused to albumin or a fragment thereof or one of the albumin variants as described in U.S. Publication No. 2007/0048282. In some embodiments, the anti-PCSK9 adnectin can be fused to one or more serum albumin binding adnectins, as described herein.

In some embodiments, the PK group is a protein that binds serum albumin, such as proteins such as those described in U.S. publications No. 2007/0178082 and 2007/0269422.

In some embodiments, the PK group is a whey protein that binds immunoglobulins, such as proteins such as those described in U.S. publications No. 2007/0178082.

In some embodiments, the anti-PCSK9 adnectin contains polyethylene glycol (PEG). By reactions with amines, thiols, or other suitable reactive groups, one or more PEG molecules can be coupled to anti-PCSK9 adnectin at various positions. For this purpose, a primary amino group at the N-terminus of the protein or side groups of amino acid residues such as lysine or arginine can be used. In some embodiments, the PEG is attached at one of the positions selected from the following groups: a) N-terminus; b) between the N-terminus and the beta or beta-like region closest to the N-terminus; c) on a loop located on the reverse surface of the polypeptide with respect to the binding site; d) between the C-terminus and the beta or beta-like region closest to the C-terminus; and e) at the C-terminus of the polypeptide.

PEGylation can be accomplished by site-directed PEGylation, in which a suitable reactive group is introduced into the protein to create a site at which PEGylation will preferentially occur. In some embodiments, the protein sequence is modified by introducing a cysteine residue at the required position, which ensures targeted PEGylation at this cysteine residue. PEG varies widely in molecular weight and can be branched or linear. In one embodiment, the PEG has two chains. In another embodiment, the PEG has 4 chains.

In some embodiments, the anti-PCSK9 adnectin is fused to an immunoglobulin Fc fragment, or a portion thereof, or a variant of the fragment or portion. In an exemplary embodiment of the invention, the Fc fragment of immunoglobulins of the IgG1 subclass is used, however, Fc fragments of other immunoglobulin subclasses (for example, IgG2, IgG3 and IgG4) can also be used. Below is the sequence of the Fc region of human immunoglobulin IgG1, with the relative positions of each region marked according to European Union (EU) nomenclature:

ASTKGPSVFPLAPSSKSTSGGTAALGCL VKDYFPEPVTVSWNSGALTSGVHTFPA VLQ

SSGLYSLSSWTVPSSSLGTQΊΎICNVNHKPSNTKVDKKVEPKSCΌ^USHCPUSSHΈML·GGY SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP QVYTLPP SRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ IDNO: 315)

The core sequence of the hinge region is underlined, the CH1 region is in italics, and the CH2 and CH3 regions are in normal font. It should be understood that the C-terminal lysine residue is optional.

Said fusion protein can be produced by attaching an antiPCSK9 adnectin sequence to either end of the Fc domain sequence, i.e. Fc-anti-PCSK9 adnectin or anti-PCSK9 adnectin-Fc fusion proteins can be produced. In certain embodiments, the Fc moiety and anti-PCSK9 adnectin are joined by a linker sequence. Examples of linker sequences include

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AGGGGSG (SEQ GO NO: 310),

GSGSGSGSGSGS (SEQ ID NO: 311), QPDEPGGS (SEQ ID NO: 312), ELQLEESAAEAQDGELD (SEQ ID NO: 313), TVAAPS (SEQ ID NO: 314), KAGGGGSG (SEQ ID NO: 620), KGSGSGSGSGSGS (SEQ ID NO : 621), KQPDEPGGS (SEQ ID NO: 622), KELQLEESAAEAQDGELD (SEQ ID NO: 623), KTVAAPS (SEQ ID NO: 624), KAGGGGSGG (SEQ ID NO: 625), KGSGSGSGSGSGSG (SEQ ID NO: 626), KQPDEPGGSG (SEQ ID NO: 627), KELQLEESAAEAQDGELDG (SEQ ID NO: 628), KTVAAPSG (SEQ ID NO: 629) AGGGGSGG (SEQ ID NO: 630), GSGSGSGSGSGSG (SEQ ID NO: 631), QPDEPGGSG (SEQ ID NO: 632 ), ELQLEESAAEAQDGELDG (SEQ ID NO: 633) and TVAAPSG (SEQ ID NO: 634).

In some embodiments, the Fc domain included in the aHTU-PCSK9 adnectin fusion protein contains an Fc domain hinge region sequence. When used in this application, the term hinge region includes the core amino acid residues of the hinge region of the IgG1 molecule at positions 104-119 of the sequence SEQ ID NO: 315 (DKTHTCPPCPAPELLG; SEQ ID NO: 316) corresponding to the region 221-236 according to the European Union nomenclature. In certain embodiments, the anti-PCSK9 adnectin-Fc fusion protein multimerizes (eg, forms a dimer), in part through cysteine residues within the hinge region at positions 109 and 112 of SEQ ID NO: 315 (EU positions 226 and 229, respectively). In other embodiments, the hinge region as described herein may further include the amino acid residues of the CH1 and CH2 regions flanking the core sequence of the hinge region as shown in SEQ ID NO: 315.

In some embodiments, the hinge sequence may contain substitutions that impart the desired pharmacodynamic, biophysical and/or biological properties. Some examples of hinge region sequences include the following: EPKSSDKTHTCPPCPAPELLGGPS (SEQ ID NO: 317; hinge core sequence is underlined), EPKSSDKTHTCPPCPAPELLGGSS (SEQ ID NO: 318; hinge core sequence is underlined), EPKSSGSTHTCPPCPAPELLGGSS (SEQ ID NO: 319; hinge core sequence underlined), DKTHTCPPCPAPELLGGPS (SEQ ID NO: 320; hinge core sequence is underlined), and DKTHTCPPCPAPELLGGSS (SEQ ID NO: 321; hinge core sequence is underlined). In one embodiment, the P residue at position 122 (238 in EU nomenclature) of SEQ ID NO: 315 is replaced by an S residue to disrupt the effector function of the Fc fragment. An example of this substitution is given in any of the sequences SEQ ID NO: 318, 319 and 321. In another embodiment, the DK residues at positions 104-105 of the sequence SEQ ID NO: 315 (221-222 according to the European Union) are replaced with GS residues to remove potential stitching site. An example of such a substitution is shown in SEQ ID NO: 319. In another embodiment, the C residue at position 103 of SEQ ID NO: 315 (220 in EU nomenclature) is replaced by an S residue to prevent the formation of an incorrect disulfide bond in the absence of a light chain. An example of such a substitution is given in SEQ ID NO: 317-319.

In certain embodiments, the anti-PCSK9 adnectin-Fc fusion protein may have the following configurations: 1) α-anti-PCSK9 adnectin-hinge-Fc, 2) Fc-anti-PCSK9 adnectin hinge. Thus, any anti-PCSK9 adnectin variant described in the present invention can be incorporated into an Fc domain fusion protein containing a hinge region sequence according to these configurations. In some embodiments, a linker may be used to connect the anti-PCSK9 adnectin and hinge region-Fc modules. In this case, the fusion protein may have, for example, the following configuration: hinge region-anti-PCSK9 adnectin-linker-Fc. In addition, depending on the production system of the fusion proteins, a leader signal sequence may be located at the N-terminus of the fusion protein. For example, if the fusion protein is produced in mammalian cells, the following leader sequence may be introduced at the N-terminus of the fusion protein: METDTLLLWVLLLWVPGSTG (SEQ ID NO: 326).

If the fusion protein is produced in an E. coli system, a methionine residue will be introduced at the N-terminus of the fusion protein.

Example sequence of an anti-PCSK9 adnectin-hinge-Fc fusion protein produced in mammalian cells:

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METDTLLLWVLLLWVPGSTGGE5DEPRZ)ZEH^7PZ5ZZZ5P^

ITYGETGGNSPVQEFTVPIGKGTA TISGLKPGVDYTITVYA VEFPWPHAGYYHRPISINYRTEI

EPKSSGSTHTCPPCPAPELLGGSSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF

NWYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLTVLHQDWLNGI<EYI<CI<VSNI<ALPA

PIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG K (SEQ ID NO: 322).

In this example, the Fc domain contains the sequences of the CH1, CH2 and CH3 regions of human immunoglobulins G1:

VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY

NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS

RDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ Ш NO: 448) and the hinge region of the sequence SEQ ID NO: 319. In the sequence SEQ ID NO: 322, the leader peptide sequence is in bold, the anti-PCSK sequence 9 adnectin is italicized, the hinge region is underlined. It should be understood that the lysine residue at the end of SEQ ID NO: 322 is optional. The effectiveness of the fusion polypeptide shown in SEQ ID NO: 322 (also described herein as PRD460) is shown in Example 4.

Examples of PCSK9 adnectin-Fc fusion proteins are shown in Table. 1. All sequences may begin with a methionine and a mammalian cell leader signal sequence (eg, SEQ ID NO: 326).

Table 1

Example sequences of anti-PCSK9 adnectin-Fc fusion proteins

EQ ID Clone or title Description Subsequence PCSK9 Adnectin-Xi-Fc C-terminal variants 52 1459 D05-Fc fusion protein XI is optional and, if present, may be selected from the following group: E, EI, EID, EIDK (SEQ ID NO: 384), EIE and EIEK (SEQ ID NO: 635); X2 is selected from the hinge region sequences SEQ ID NO: 317-321; this Fc fragment may include a C-terminal K residue GVSDVPRDLEVVAATPTSLLISWPPPSH GYGYYRITYGETGGNSPVQEFTVPPGKGTATIS GLKPGVDYTITVYAVEYPYKHSGYYHRPISINY RT-X1-X2- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYT QKSLSLSPG 53 1784F OZ-Fc fusion protein XI is optional and, if present, may be selected from the following group: E, EI, EID, EIDK (SEQ ID NO: 384), EIE and EIEK (SEQ ID NO: 635); X2 is selected from the hinge region sequences SEQ ID NO: 317-321; this F fragment may include GVSDVPRDLEVVAATPTSLLISWRPPIH AYGYYRITYGETGGNSPVQEFTVPIVEGTATIS GLKPGVDYTITVYAVEYTFKHSGYYHRPISINY RT-X1-X2- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYT QKSLSLSPG

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C-terminal residue K 54 1784F 03-ml-Fc fusion protein XI is optional and, if present, may be selected from the following group: E, EI, EID, EIDK (SEQ ID NO: 384), EIE and EIEK (SEQ ID NO: 635); X2 is selected from the hinge region sequences SEQ ID NO: 317-321; this Fc fragment may include a C-terminal K residue GVSDVPRDLEVVAAT PT SLLIS WDAPIH AYGYYRITYGETGGNSPVQEFTVPGSEGTATIS GLKPGVDYTITVYAVEYTFKHSGYYHRPISINY RT-X1-X2- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYT QKSLSLSPG 55 1784F 03-m2-Fc fusion protein XI is optional and, if present, may be selected from the following group: E, EI, EID, EIDK (SEQ ID NO: 384), EIE and EIEK (SEQ ID NO: 635); X2 is selected from the hinge region sequences SEQ ID NO: 317-321; this Fc fragment may include a C-terminal K residue GVSDVPRDLEVVAAT PT SLLISWDAPAH AYGYYRITYGETGGNSPVQEFTVPGSKGTATIS GLKPGVDYTITVYAVEYTFKHSGYYHRPISINY RT-X1-X2- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPG 56 1784F 03-m3-Fc fusion protein XI is optional and, if present, may be selected from the following group: E, EI, EID, EIDK (SEQ ID NO: 384), EIE and EIEK (SEQ ID NO: 635); X2 is selected from GVSDVPRDLEVVAAT PT SLLISWDAPAV TYGYYRITYGETGGNSPVQEFTVPGSKSTATIS GLKPGVDYTITVYAVEYTFKHSGYYHRPISINY RT-Xx-Xg- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG

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hinge region sequences SEQ ID NO: 317-321; this Fc fragment may include a C-terminal K residue FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPG 57 1813E 02-Fc fusion protein XI is optional and, if present, may be selected from the following group: E, EI, EID, EIDK (SEQ ID NO: 384), EIE and EIEK (SEQ ID NO: 635); X2 is selected from the hinge region sequences SEQ ID NO: 317-321; this Fc fragment may include a C-terminal K residue GVSDVPRDLEVVAATPTSLLISWSPPAN GYGYYRITYGETGGNSPVQEFTVPVGRGTATIS GLKPGVDYTITVYAVEYTYKGSGYYHRPISINY RT-Xi-Хг- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYT QKSLSLSPG 58 1923B 02-Fc fusion protein XI is optional and, if present, may be selected from the following group: E, EI, EID, EIDK (SEQ ID NO: 384), EIE and EIEK (SEQ ID NO: 635); X2 is selected from the hinge region sequences SEQ ID NO: 317-321; this Fc fragment may include a C-terminal K residue GVSDVPRDLEVVAATPTSLLISWTPPPK GYGYYRITYGETGGNSPVQEFTVPVGEGTATIS GLKPGVDYTITVYAVEYTYNGAGYYHRPISINY RT-Xx-Xz- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYT QKSLSLSPGK 59 1923B 02(N82I)-Fc fusion protein XI is optional and, if present, can be selected from the following GVSDVPRDLEVVAATPTSLLISWTPPPK GYGYYRITYGETGGNSPVQEFTVPVGEGTATIS GLKPGVDYTITVYAVEYTYIGAGYYHRPISINY RT-Xx-Xz-

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groups: E, EI, EID, EIDK (SEQ ID NO: 384), EIE and EIEK (SEQ ID NO: 635); X2 is selected from the hinge region sequences SEQ ID NO: 317-321; this Fc fragment may include a C-terminal K residue VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYT QKSLSLSPG 60 1923B 02(N82E)-Fc fusion protein XI is optional and, if present, may be selected from the following group: E, EI, EID, EIDK (SEQ ID NO: 384), EIE and EIEK (SEQ ID NO: 635); X2 is selected from the hinge region sequences SEQ ID NO: 317-321; this Fc fragment may include a C-terminal K residue GVSDVPRDLEVVAATPTSLLISWTPPPK GYGYYRITYGETGGNSPVQEFTVPVGEGTATIS GLKPGVDYTITVYAVEYTYEGAGYYHRPISINY RT-Xx-Xz- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYT QKSLSLSPG 61 1923B 02(T80A)-Fc fusion protein XI is optional and, if present, may be selected from the following group: E, EI, EID, EIDK (SEQ ID NO: 384), EIE and EIEK (SEQ ID NO: 635); X2 is selected from the hinge region sequences SEQ ID NO: 317-321; this Fc fragment may include a C-terminal K residue GVSDVPRDLEVVAATPTSLLISWTPPPK GYGYYRITYGETGGNSPVQEFTVPVGEGTATIS GLKPGVDYTITVYAVEYAYNGAGYYHRPISINY RT-Χχ-Χζ- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYT QKSLSLSPG

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62 1922 G04-Fc fusion protein XI is optional and, if present, may be selected from the following group: E, EI, EID, EIDK (SEQ ID NO: 384), EIE and EIEK (SEQ ID NO: 635); X2 is selected from the hinge region sequences SEQ ID NO: 317-321; this Fc fragment may include a C-terminal K residue GVSDVPRDLEVVAATPTSLLISWRPPSH AYGYYRITYGETGGNSPVQEFTVPIGKGTATIS GLKPGVDYTITVYAVEYPWKGSGYYHRPISINY RT-Xx-Xz- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYT QKSLSLSPG 63 1922 G04(R25D)Fc fusion protein XI is optional and, if present, may be selected from the following group: E, EI, EID, EIDK (SEQ ID NO: 384), EIE and EIEK (SEQ ID NO: 635); X2 is selected from the hinge region sequences SEQ ID NO: 317-321; this Fc fragment may include a C-terminal K residue GVSDVPRDLEVVAATPTSLLISWDPPSH AYGYYRITYGETGGNSPVQEFTVPIGKGTATIS GLKPGVDYTITVYAVEYPWKGSGYYHRPISINY RT-Xx-Xz- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYT QKSLSLSPG 64 1922 G04(R25E)Fc fusion protein XI is optional and, if present, may be selected from the following group: E, EI, EID, EIDK (SEQ ID NO: 384), EIE and EIEK (SEQ ID NO: 635); X2 is selected from sequences GVSDVPRDLEVVAATPTSLLISWEPPSH AYGYYRITYGETGGNSPVQEFTVPIGKGTATIS GLKPGVDYTITVYAVEYPWKGSGYYHRPISINY RT-Xx-Xz- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF

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hinge region SEQ ID NO: 317-321; this Fc fragment may include a C-terminal K residue FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPG 65 1922 G04(R25S) Fc fusion protein XI is optional and, if present, may be selected from the following group: E, EI, EID, EIDK (SEQ ID NO: 384), EIE and EIEK (SEQ ID NO: 635); X2 is selected from the hinge region sequences SEQ ID NO: 317-321; this Fc fragment may include a C-terminal K residue GVSDVPRDLEVVAATPTSLLISWSPPSH AYGYYRITYGETGGNSPVQEFTVPIGKGTATIS GLKPGVDYTITVYAVEYPWKGSGYYHRPISINY RT-Xx-Xz- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYT QKSLSLSPG 66 2012 A04-Fc fusion protein XI is optional and, if present, may be selected from the following group: E, EI, EID, EIDK (SEQ ID NO: 384), EIE and EIEK (SEQ ID NO: 635); X2 is selected from the hinge region sequences SEQ ID NO: 317-321; this Fc fragment may include a C-terminal K residue GVSDVPRDLEVVAATPTSLLISWRPPSN GHGYYRITYGETGGNSPVQEFTVPVNEGTATIS GLKPGVDYTITVYAVEFPFKWSGYYHRPISINY RT-Xi-Xz- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYT QKSLSLSPG 67 2013E 01-Fc fusion protein XI is optional and, if present, can be selected from the following group: E, EI, EID, EIDK GVSDVPRDLEVVAATPTSLLISWVPPSD DYGYYRITYGETGGNSPVQEFTVPIGKGTATIS GLKPGVDYTITVYAVEFPWPHAGYYHRPISINY RT-Xx-Xz- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE

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(SEQ ID NO: 384), EIE and EIEK (SEQ ID NO: 635); X2 is selected from the hinge region sequences SEQ ID NO: 317-321; this Fc fragment may include a C-terminal K residue VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPG 68 2011 H05-Fc fusion protein XI is optional and, if present, may be selected from the following group: E, EI, EID, EIDK (SEQ ID NO: 384), EIE and EIEK (SEQ ID NO: 635); X2 is selected from the hinge region sequences SEQ ID NO: 317-321; this Fc fragment may include a C-terminal K residue GVSDVPRDLEVVAATPTSLLISWVPSSH AYGYYRITYGETGGNSPVQEFTVPVGVGTATIS GLKPGVDYTITVYAVEYAFEGAGYYHRPISINY RT-Xx-Xz- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYT QKSLSLSPG 69 2011 H05(V23D)Fc fusion protein XI is optional and, if present, may be selected from the following group: E, EI, EID, EIDK (SEQ ID NO: 384), EIE and EIEK (SEQ ID NO: 635); X2 is selected from the hinge region sequences SEQ ID NO: 317-321; this Fc fragment may include a C-terminal K residue GVSDVPRDLEVVAATPTSLLISWDPSSH AYGYYRITYGETGGNSPVQEFTVPVGVGTATIS GLKPGVDYTITVYAVEYAFEGAGYYHRPISINY RT-Χχ-Χζ- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYT QKSLSLSPG 2011 XI is GVSDVPRDLEVVAATPTSLLISWEPSSH

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70 H05(V23E) Fc fusion protein optional and, if present, may be selected from the following group: E, EI, EID, EIDK (SEQ ID NO: 384), EIE and EIEK (SEQ ID NO: 635); X2 is selected from the hinge region sequences SEQ ID NO: 317-321; this Fc fragment may include a C-terminal K residue AYGYYRITYGETGGNSPVQEFTVPVGVGTATIS GLKPGVDYTITVYAVEYAFEGAGYYHRPISINY RT-Xx-Xz- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYT QKSLSLSPG 71 2381B 02-Fc fusion protein XI is optional and, if present, may be selected from the following group: E, EI, EID, EIDK (SEQ ID NO: 384), EIE and EIEK (SEQ ID NO: 635); X2 is selected from the hinge region sequences SEQ ID NO: 317-321; this Fc fragment may include a C-terminal K residue GVSDVPRDLEVVAATPTSLLISWEPFSR LPGGGEYYRITYGETGGNSPLQQFTVPGSKGTA TISGLKPGVDYTITVYAVEYPYDYSGYYHRPIS INYRT-Xx-Xz- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYT QKSLSLSPG 72 2381B 04-Fc fusion protein XI is optional and, if present, may be selected from the following group: E, EI, EID, EIDK (SEQ ID NO: 384), EIE and EIEK (SEQ ID NO: 635); X2 is selected from the hinge region sequences SEQ ID GVSDVPRDLEVVAATPTSLLISWEPFSR LPGGGEYYRITYGETGGNSPLQQFTVPGSKGTA TISGLKPGVDYTITVYAVEYPYEHSGYYHRPIS INYRT-Xx-Xz- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYT

- 22 044608

NO: 317-321; this Fc fragment may include a C-terminal K residue QKSLSLSPG 73 2381B 06-Fc fusion protein XI is optional and, if present, may be selected from the following group: E, EI, EID, EIDK (SEQ ID NO: 384), EIE and EIEK (SEQ ID NO: 635); X2 is selected from the hinge region sequences SEQ ID NO: 317-321; this Fc fragment may include a C-terminal K residue GVSDVPRDLEVVAATPTSLLISWEPFSR LPGGGEYYRITYGETGGNSPLQQFTVPGSKGTA TISGLKPGVDYTITVYAVEYPYPHSGYYHRPIS INYRT-Xi-X ₂ - VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDW LNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPG 74 2381B 08-Fc fusion protein XI is optional and, if present, may be selected from the following group: E, EI, EID, EIDK (SEQ ID NO: 384), EIE and EIEK (SEQ ID NO: 635); X2 is selected from the hinge region sequences SEQ ID NO: 317-321; this Fc fragment may include a C-terminal K residue GVSDVPRDLEVVAAT PT SLLISWDAPAD GGYGYYRITYGETGGNSPVQEFTVPSSKGTATI SGLKPGVDYTITVYAVEYT FPGAGYYHRPISIN YRT-Хх-Хг- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYT QKSLSLSPG 75 2381 D02-Fc fusion protein XI is optional and, if present, can be selected from the following group: E, EI, EID, EIDK (SEQ ID NO: 384), EIE and GVSDVPRDLEVVAATPTSLLISWEPFSR LPGGGEYYRITYGETGGNSPLQQFTVPGSKGTA TISGLKPGVDYTITVYAVEYPYDHSGYYHRPIS INYRT-Xi-X ₂ - VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV

- 23 044608

EIEK (SEQ ID NO: 635); X2 is selected from the hinge region sequences SEQ ID NO: 317-321; this Fc fragment may include a C-terminal K residue LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPG 76 2381 D04-Fc fusion protein XI is optional and, if present, may be selected from the following group: E, EI, EID, EIDK (SEQ IDNO: 384), EIE and EIEK (SEQID NO: 635); X2 is selected from the hinge region sequences SEQ ID NO: 317-321; this Fc fragment may include a C-terminal K residue GVSDVPRDLEVVAATPTSLLISWEPFSR LPGGGEYYRITYGETGGNSPLQQFTVPGSKGTA TISGLKPGVDYTITVYAVEFPYDHSGYYHRPIS INYRT-Xi-X ₂ - VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWL NGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPG 77 2381F 1 Ι-Fc fusion protein XI is optional and, if present, may be selected from the following group: E, EI, EID, EIDK (SEQ IDNO: 384), EIE and EIEK (SEQ ID NO: 635); X2 is selected from the hinge region sequences SEQ ID NO: 317-321; this Fc fragment may include a C-terminal K residue GVSDVPRDLEVVAAT PT SLLISWDAPAD GGYGYYRITYGETGGNSPVQEFTVPVSKSTATI SGLKPGVDYTITVYAVEYT FPGAGYYHRPISIN YRT-Xx-Xz- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPG 78 2381 G03-Fc XI is optional and if GVSDVPRDLEVVAATPTSLLISWEPFSR LPGGGEYYRITYGETGGNSPLQQFTVPGSKGTA

- 24 044608

fusion protein present, can be selected from the following group: E, EI, EID, EIDK (SEQ ID NO: 384), EIE and EIEK (SEQID NO: 635); X2 is selected from the hinge region sequences SEQ ID NO: 317-321; this Fc fragment may include a C-terminal K residue TISGLKPGVDYTITVYAVEFPYAHSGYYHRPIS INYRT-X1-X2- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYT QKSLSLSPG 79 2381 G09-Fc fusion protein XI is optional and, if present, may be selected from the following group: E, EI, EID, EIDK (SEQ ID NO: 384), EIE and EIEK (SEQ ID NO: 635); X2 is selected from the hinge region sequences SEQ ID NO: 317-321; this Fc fragment may include a C-terminal K residue GVSDVPRDLEVVAAT PT SLLISWDAPAG DGYGYYRITYGETGGNSPVQEFTVPVSKGTATI SGLKPGVDYTITVYAVE FT FPGAGYYHRPISIN YRT-Хх-Хг- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYT QKSLSLSPG 80 2381 NOZ-Fc fusion protein XI is optional and, if present, may be selected from the following group: E, EI, EID, EIDK (SEQ ID NO: 384), EIE and EIEK (SEQ ID NO: 635); X2 is selected from the hinge region sequences SEQ ID NO: 317-321; given Fc- GVSDVPRDLEVVAATPTSLLISWEPFSR LPGGGEYYRITYGETGGNSPLQQFTVPGSKGTA TISGLKPGVDYTITVYAVEY PYAH SGY FHRPIS INYRT-Xx-Xg- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYT QKSLSLSPG

- 25 044608

fragment may include C-terminal residue K 81 2382 AOl-Fc fusion protein XI is optional and, if present, may be selected from the following group: E, EI, EID, EIDK (SEQ ID NO: 384), EIE and EIEK (SEQ ID NO: 635); X2 is selected from the hinge region sequences SEQ ID NO: 317-321; this Fc fragment may include a C-terminal K residue GVSDVPRDLEVVAATPT SLLISWAAPAG GGYGYYRITYGETGGNSPVQEFTVPVSKGTATI SGLKPGVDYTITVYAVEYDFPGAGYYHRPISIN YRT-Хх-Хг- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYT QKSLSLSPG 82 2382V 10-Fc fusion protein XI is optional and, if present, may be selected from the following group: E, EI, EID, EIDK (SEQ ID NO: 384), EIE and EIEK (SEQ ID NO: 635); X2 is selected from the hinge region sequences SEQ ID NO: 317-321; this Fc fragment may include a C-terminal K residue GVSDVPRDLEVVAAT PT SLLISWDAPAD AYGYYRITYGETGGNSPVQEFTVPSSKGTATIS GLKPGVDYTITVYAVEYDFPGSGYYHRPISINY RT-Хх-Хг- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYT QKSLSLSPG 83 2382V 09-Fc fusion protein XI is optional and, if present, may be selected from the following group: E, EI, EID, EIDK (SEQ ID NO: 384), EIE and EIEK (SEQ ID NO: 635); X2 GVSDVPRDLEVVAAT PT SLLISWDAPAD AYGYYRITYGETGGNSPVQEFTVPVSKGTATIS GLKPGVDYTITVYAVEFDYPGSGYYHRPISINY RT-Xx-Xz- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK

- 26 044608

selected from the hinge region sequences SEQ ID NO: 317-321; this Fc fragment may include a C-terminal K residue AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPG 84 2382C 05-Fc fusion protein XI is optional and, if present, may be selected from the following group: E, EI, EID, EIDK (SEQ ID NO: 384), EIE and EIEK (SEQ ID NO: 635); X2 is selected from the hinge region sequences SEQ ID NO: 317-321; this Fc fragment may include a C-terminal K residue GVSDVPRDLEVVAAT PT SLLISWDAPAD GAYGYYRITYGETGGNSPVQEFTVPVSKGTATI SGLKPGVDYTITVYAVEYS FPGAGYYHRPISIN YRT-Xi-Хг- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYT QKSLSLSPG 85 2382С 09-Fc fusion protein XI is optional and, if present, may be selected from the following group: E, EI, EID, EIDK (SEQ ID NO: 384), EIE and EIEK (SEQ ID NO: 635); X2 is selected from the hinge region sequences SEQ ID NO: 317-321; this Fc fragment may include a C-terminal K residue GVSDVPRDLEVVAAT PT SLLISWDAPAE GYGYYRITYGETGGNSPVQEFTVPVSKGTATIS GLKPGVDYTITVYAVEFDFPGSGYYHRPISINY RT-Xx-Xz- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYT QKSLSLSPG 86 2382 D03-Fc fusion protein XI is optional and, if present, may be GVSDVPRDLEVVAAT PT SLLISWDAPAD EAYGYYRITYGETGGNSPVQEFTVPVSKGTATI SGLKPGVDYTITVYAVE FDFPGAGYYHRPISIN

- 27 044608

selected from the following group: E, EI, EID, EIDK (SEQ ID NO: 384), EIE and EIEK (SEQ ID NO: 635); X2 is selected from the hinge region sequences SEQ ID NO: 317-321; this Fc fragment may include a C-terminal K residue YRT-Xi-Xr- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYT QKSLSLSPG 87 2382 D05-Fc fusion protein XI is optional and, if present, may be selected from the following group: E, EI, EID, EIDK (SEQ ID NO: 384), EIE and EIEK (SEQ ID NO: 635); X2 is selected from the hinge region sequences SEQ ID NO: 317-321; this Fc fragment may include a C-terminal K residue GVSDVPRDLEVVAAT PT SLLISWDAPAD GGYGYYRITYGETGGNSPVQEFTVPVSKGTATI SGLKPGVDYTITVYAVE FDFPGAGYYHRPISIN YRT-Xi-Xz- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPG 88 2382 D08-Fc fusion protein XI is optional and, if present, may be selected from the following group: E, EI, EID, EIDK (SEQ ID NO: 384), EIE and EIEK (SEQ ID NO: 635); X2 is selected from the hinge region sequences SEQ ID NO: 317-321; this F fragment may include GVSDVPRDLEVVAAT PT SLLISWDAPAD GYGYYRITYGETGGNSPVQEFTVPVSKGTATIS GLKPGVDYTITVYAVEFPFPGSGYYHRPISINY RT-X1-X2- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYT QKSLSLSPG

- 28 044608

C-terminal residue K 89 2382 D09-Fc fusion protein XI is optional and, if present, may be selected from the following group: E, EI, EID, EIDK (SEQ ID NO: 384), EIE and EIEK (SEQ ID NO: 635); X2 is selected from the hinge region sequences SEQ ID NO: 317-321; this Fc fragment may include a C-terminal K residue GVSDVPRDLEVVAATPT SLLISWDAPAE GYGYYRITYGETGGNSPVQEFTVPVSKGTATIS GLKPGVDYTITVYAVEFDFPGAGYYHRPISINY RT-X1-X2- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYT QKSLSLSPG 90 2382F 02-Fc fusion protein XI is optional and, if present, may be selected from the following group: E, EI, EID, EIDK (SEQ ID NO: 384), EIE and EIEK (SEQ ID NO: 635); X2 is selected from the hinge region sequences SEQ ID NO: 317-321; this Fc fragment may include a C-terminal K residue GVSDVPRDLEVVAAT PT SLLISWDAPAG GGYGYYRITYGETGGNSPVQEFTVPVSKGTATI SGLKPGVDYTITVYAVE FDFPGSGYYHRPISIN YRT-X1-X2- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPG 91 2382F OZ-Fc fusion protein XI is optional and, if present, may be selected from the following group: E, EI, EID, EIDK (SEQ ID NO: 384), EIE and EIEK (SEQ ID NO: 635); X2 is selected from GVSDVPRDLEVVAAT PT SLLISWDAPAA DAYGYYRITYGETGGNSPVQEFTVPVSKGTATI SGLKPGVDYTITVYAVE FNFPGAGYYHRPISIN YRT-Xx-Xz- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG

- 29 044608

hinge region sequences SEQ ID NO: 317-321; this Fc fragment may include a C-terminal K residue FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPG 92 2382F 05-Fc fusion protein XI is optional and, if present, may be selected from the following group: E, EI, EID, EIDK (SEQ ID NO: 384), EIE and EIEK (SEQ ID NO: 635); X2 is selected from the hinge region sequences SEQ ID NO: 317-321; this Fc fragment may include a C-terminal K residue GVSDVPRDLEVVAAT PT SLLISWDAPAE AGKHYGYYRITYGETGGNSPVQEFTVPVSKGTA TISGLKPGVDYTITVYAVEFDFPGAGYYHRPIS INYRT-Xi-Хг- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYT QKSLSLSPG 93 2382F 08-Fc fusion protein XI is optional and, if present, may be selected from the following group: E, EI, EID, EIDK (SEQ ID NO: 384), EIE and EIEK (SEQ ID NO: 635); X2 is selected from the hinge region sequences SEQ ID NO: 317-321; this Fc fragment may include a C-terminal K residue GVSDVPRDLEVVAAT PT SLLISWDAPAE AYGYYRITYGETGGNSPVQEFTVPVSKGTATIS GLKPGVDYTITVYAVEFTYPGSGYYHRPISINY RT-Хх-Хг- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYT QKSLSLSPG 94 2382F 09-Fc fusion protein XI is optional and, if present, can be selected from the following GVSDVPRDLEVVAAT PT SLLISWDAPAA AYGYYRITYGETGGNSPVQEFTVPVSKGTATIS GLKPGVDYTITVYAVEYDFPGSGYYHRPISINY RT-Xx-Xz-

- 30 044608

groups: E, EI, EID, EIDK (SEQ ID NO: 384), EIE and EIEK (SEQ ID NO: 635); X2 is selected from the hinge region sequences SEQ ID NO: 317-321; this Fc fragment may include a C-terminal K residue VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYT QKSLSLSPG 95 2382 G04-Fc fusion protein XI is optional and, if present, may be selected from the following group: E, EI, EID, EIDK (SEQ ID NO: 384), EIE and EIEK (SEQ ID NO: 635); X2 is selected from the hinge region sequences SEQ ID NO: 317-321; this Fc fragment may include a C-terminal K residue GVSDVPRDLEVVAAT PT SLLISWDAPAG GGYGYYRITYGETGGNSPVQEFTVPSSKGTATI SGLKPGVDYTITVYAVE FDFPGAGYYHRPISIN YRT-Xx-Xz- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYT QKSLSLSPG 96 2382 H10-Fc fusion protein XI is optional and, if present, may be selected from the following group: E, EI, EID, EIDK (SEQ ID NO: 384), EIE and EIEK (SEQ ID NO: 635); X2 is selected from the hinge region sequences SEQ ID NO: 317-321; this Fc fragment may include a C-terminal K residue GVSDVPRDLEVVAAT PT SLLISWDAPAG GYGYYRITYGETGGNSPVQEFTVPVSKGTATIS GLKPGVDYTITVYAVEFDFPGSGYYHRPISINY RT-Xx-Xz- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYT QKSLSLSPG

- 31 044608

97 2382 Hll-Fc fusion protein XI is optional and, if present, may be selected from the following group: E, EI, EID, EIDK (SEQ ID NO: 384), EIE and EIEK (SEQ ID NO: 635); X2 is selected from the hinge region sequences SEQ ID NO: 317-321; this Fc fragment may include a C-terminal K residue GVSDVPRDLEVVAAT PT SLLISWDAPAD GYGYYRITYGETGGNSPVQEFTVPVFKGTATIS GLKPGVDYTITVYAVEFDYPGSGYYHRPISINY RT-Xx-Xz- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYT QKSLSLSPG 98 2382 H04-Fc fusion protein XI is optional and, if present, may be selected from the following group: E, EI, EID, EIDK (SEQ ID NO: 384), EIE and EIEK (SEQ ID NO: 635); X2 is selected from the hinge region sequences SEQ ID NO: 317-321; this Fc fragment may include a C-terminal K residue GVSDVPRDLEVVAAT PT SLLISWDAPAA GGYGYYRITYGETGGNSPVQEFTVPSSKGTATI SGLKPGVDYTITVYAVEYDFPGAGYYHRPISIN YRT-Xx-Xz- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYT QKSLSLSPG 99 2382 H07-Fc fusion protein XI is optional and, if present, may be selected from the following group: E, EI, EID, EIDK (SEQ ID NO: 384), EIE and EIEK (SEQ ID NO: 635); X2 is selected from sequences GVSDVPRDLEVVAAT PT SLLISWDAPAD AYGYYRITYGETGGNSPVQEFTVPGSKGTATIS GLKPGVDYTITVYAVEFDFPGSGYYHRPISINY RT-Xx-Xz- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF

- 32 044608

hinge region SEQ ID NO: 317-321; this Fc fragment may include a C-terminal K residue FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPG 00 2382 H09-Fc fusion protein XI is optional and, if present, may be selected from the following group: E, EI, EID, EIDK (SEQ ID NO: 384), EIE and EIEK (SEQ ID NO: 635); X2 is selected from the hinge region sequences SEQ ID NO: 317-321; this Fc fragment may include a C-terminal K residue GVSDVPRDLEVVAAT PT SLLISWDAPAA AYGYYRITYGETGGNSPVQEFTVPSSKGTATIS GLKPGVDYTITVYAVEFDFPGSGYYHRPISINY RT-Xx-Xz- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYT QKSLSLSPG 01 2451 A02-Fc fusion protein XI is optional and, if present, may be selected from the following group: E, EI, EID, EIDK (SEQ ID NO: 384), EIE and EIEK (SEQ ID NO: 635); X2 is selected from the hinge region sequences SEQ ID NO: 317-321; this Fc fragment may include a C-terminal K residue GVSDVPRDLEVVAAT PT SLLISWDAPAA GYGYYRITYGETGGNSPVQEFTVPVSKGTATIS GLKPGVDYTITVYAVEFPFPGSGYYHRPISINY RT-Xi-Xz- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYT QKSLSLSPG 02 2451B 05-Fc fusion protein XI is optional and, if present, can be selected from the following group: E, EI, EID, EIDK GVSDVPRDLEVVAAT PT SLLISWDAPAG GYGYYRITYGETGGNSPVQEFTVPSSKGTATIS GLKPGVDYTITVYAVEFDYPGSGYYHRPISINY RT-Xx-Xz- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE

- 33 044608

(SEQ ID NO: 384), EIE and EIEK (SEQ ID NO: 635); X2 is selected from the hinge region sequences SEQ ID NO: 317-321; this Fc fragment may include a C-terminal K residue VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPG 03 2451B 06-Fc fusion protein (equivalent to 2382D05) XI is optional and, if present, may be selected from the following group: E, EI, EID, EIDK (SEQ ID NO: 384), EIE and EIEK (SEQ ID NO: 635); X2 is selected from the hinge region sequences SEQ ID NO: 317-321; this Fc fragment may include a C-terminal K residue GVSDVPRDLEVVAAT PT SLLISWDAPAD GGYGYYRITYGETGGNSPVQEFTVPVSKGTATI SGLKPGVDYTITVYAVE FDFPGAGYYHRPISIN YRT-Xx-Xz- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYT QKSLSLSPG 04 2451C 06-Fc fusion protein XI is optional and, if present, may be selected from the following group: E, EI, EID, EIDK (SEQ ID NO: 384), EIE and EIEK (SEQ ID NO: 635); X2 is selected from the hinge region sequences SEQ ID NO: 317-321; this Fc fragment may include a C-terminal K residue GVSDVPRDLEVVAAT PT SLLISWDAPAG AASYGYYRITYGETGGNSPVQEFTVPVSKGTAT ISGLKPGVDYTITVYAVEFPFPGAGYYHRPISI NYRT-Xi-X ₂ - VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDW LNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPG 2451 XI is GVSDVPRDLEVVAAT PT SLLISWDAPAG

- 34 044608

05 D05-Fc fusion protein optional and, if present, may be selected from the following group: E, EI, EID, EIDK (SEQ ID NO: 384), EIE and EIEK (SEQ ID NO: 635); X2 is selected from the hinge region sequences SEQ ID NO: 317-321; this Fc fragment may include a C-terminal K residue AYGYYRITYGETGGNSPVQEFTVPVSKGTATIS GLKPGVDYTITVYAVEFDFPGSGYYHRPISINY RT-Xx-Xz- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYT QKSLSLSPG 06 245IF OZ-Fc fusion protein XI is optional and, if present, may be selected from the following group: E, EI, EID, EIDK (SEQ ID NO: 384), EIE and EIEK (SEQ ID NO: 635); X2 is selected from the hinge region sequences SEQ ID NO: 317-321; this Fc fragment may include a C-terminal K residue GVSDVPRDLEVVAATPTSLLISWDPPAE GYGYYRITYGETGGNSPVQEFTVPVSKGTATIS GLKPGVDYTITVYAVEFNFPGSGYYHRPISINY RT-Xx-Xz- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYT QKSLSLSPG 07 2451 G01-Fc fusion protein XI is optional and, if present, may be selected from the following group: E, EI, EID, EIDK (SEQ ID NO: 384), EIE and EIEK (SEQ ID NO: 635); X2 is selected from the hinge region sequences SEQ ID GVSDVPRDLEVVAAT PT SLLISWDAPAG GYGYYRITYGETGGNSPVQEFTVPSSKGTATIS GLKPGVDYTITVYAVEFDFPGSGYYHRPISINY RT-Xx-Xz- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT

- 35 044608

NO: 317-321; this Fc fragment may include a C-terminal K residue QKSLSLSPG 08 2451 H07-Fc fusion protein XI is optional and, if present, may be selected from the following group: E, EI, EID, EIDK (SEQ ID NO: 384), EIE and EIEK (SEQ ID NO: 635); X2 is selected from the hinge region sequences SEQ ID NO: 317-321; this Fc fragment may include a C-terminal K residue GITDVPRDLEVVAATPTSLLISWNPPDV NYGYYRITYGETGGNSPLQEFTVPVSKGTATIS GLKPGVDYTITVYAVEYPYAHAGYYHRPISINY RT-Xi-Хг- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPG 09 2382E OZ-Fc fusion protein XI is optional and, if present, may be selected from the following group: E, EI, EID, EIDK (SEQ ID NO: 384), EIE and EIEK (SEQ ID NO: 635); X2 is selected from the hinge region sequences SEQ ID NO: 317-321; this Fc fragment may include a C-terminal K residue GVSDVPRDLEVVAAT PT SLLISWDAPAG DGYGYYRITYGETGGNSPVQEFTVPVSKGTATI SGLKPGVDYTITVYAVE FDFPGAGYYHRPISIN YRT-Хх-Хг- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYT QKSLSLSPG 10 2382E 04-Fc fusion protein XI is optional and, if present, can be selected from the following group: E, EI, EID, EIDK (SEQ ID NO: 384), EIE and GVSDVPRDLEVVAAT PT SLLISWDAPAG GGYGYYRITYGETGGNSPVQEFTVPVSKGTATI SGLKPGVDYTITVYAVE FT FPGAGYYHRPISIN YRT-Xx-Xg- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV

- 36 044608

EIEK (SEQ ID NO: 635); X2 is selected from the hinge region sequences SEQ ID NO: 317-321; this Fc fragment may include a C-terminal K residue LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPG eleven 2382E 05-Fc fusion protein XI is optional and, if present, may be selected from the following group: E, EI, EID, EIDK (SEQ ID NO: 384), EIE and EIEK (SEQ ID NO: 635); X2 is selected from the hinge region sequences SEQ ID NO: 317-321; this Fc fragment may include a C-terminal K residue GVSDVPRDLEVVAAT PT SLLISWDAPAE GGYGYYRITYGETGGNSPVQEFTVPVSKGTATI SGLKPGVDYTITVYAVE FDFPGAGYYHRPISIN YRT-Xx-Xz- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYT QKSLSLSPG 12 2382E 09-Fc fusion protein XI is optional and, if present, may be selected from the following group: E, EI, EID, EIDK (SEQ ID NO: 384), EIE and EIEK (SEQ ID NO: 635); X2 is selected from the hinge region sequences SEQ ID NO: 317-321; this Fc fragment may include a C-terminal K residue GVSDVPRDLEVVAAT PT SLLISWDAPAE AYGYYRITYGETGGNSPVQEFTVPVSKGTATIS GLKPGVDYTITVYAVEYDFPGSGYYHRPISINY RT-Xi-Xz- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPG 13 2381 A04-Fc XI is optional and if GVSDVPRDLEVVAATPTSLLISWEPFSR LPGGGEYYRITYGETGGNSPLQQFTVPGSKGTA

- 37 044608

fusion protein present, can be selected from the following group: E, EI, EID, EIDK (SEQ IDNO: 384), EIE and EIEK (SEQ ID NO: 635); X2 is selected from the hinge region sequences SEQ ID NO: 317-321; this Fc fragment may include a C-terminal K residue TISGLKPGVDYTITVYAVEYPYPFSGYYHRPIS INYRT-X1-X2- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYT QKSLSLSPG 14 2381 A08-Fc fusion protein XI is optional and, if present, may be selected from the following group: E, EI, EID, EIDK (SEQ IDNO: 384), EIE and EIEK (SEQ ID NO: 635); X2 is selected from the hinge region sequences SEQ ID NO: 317-321; this Fc fragment may include a C-terminal K residue GVSDVPRDLEVVAAT PT SLLISWDAPAD GGYGYYRITYGETGGNSPVQEFTVPGSKGTATI SGLKPGVDYTITVYAVEYDFPGAGYYHRPISIN YRT-Хх-Хг- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYT QKSLSLSPG 15 2381B ΊΟ-Fc fusion protein XI is optional and, if present, may be selected from the following group: E, EI, EID, EIDK (SEQ IDNO: 384), EIE and EIEK (SEQ ID NO: 635); X2 is selected from the hinge region sequences SEQ ID NO: 317-321; given Fc- GVSDVPRDLEVVAAT PT SLLISWDAPAG GGYGYYRITYGETGGNSPVQEFTVPVSKGTATI SGLKPGVDYTITVYAVEYNFIGAGYYHRPISIN YRT-Хх-Хг- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYT QKSLSLSPG

- 38 044608

fragment may include C-terminal residue K 16 2381C 08-Fc fusion protein XI is optional and, if present, may be selected from the following group: E, EI, EID, EIDK (SEQ ID NO: 384), EIE and EIEK (SEQ ID NO: 635); X2 is selected from the hinge region sequences SEQ ID NO: 317-321; this Fc fragment may include a C-terminal K residue GVSDVPRDLEVVAAT PT SLLISWDAPAD GAYGYYRITYGETGGNSPVQEFTVPVSKGTATI SGLKPGVDYTITVYAVEFPYPFAGYYHRPISIN YRT-Xx-Xz- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYT QKSLSLSPG 17 2381 G06-Fc fusion protein XI is optional and, if present, may be selected from the following group: E, EI, EID, EIDK (SEQ ID NO: 384), EIE and EIEK (SEQ ID NO: 635); X2 is selected from the hinge region sequences SEQ ID NO: 317-321; this Fc fragment may include a C-terminal K residue GVSDVPRDLEVVAATPTSLLISWSEKLD GKARRGYYRITYGETGGNSPVQQFTVPGSKGTA TISGLKPGVDYTITVYAVEFPYDHSGYYHRPIS INYRT-Xi-X ₂ - VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDW LNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPG 18 2381 H01-Fc fusion protein XI is optional and, if present, may be selected from the following group: E, EI, EID, EIDK (SEQ ID NO: 384), EIE and EIEK (SEQ ID NO: 635); X2 GVSDVPRDLEVVAATPTSLLISWSPRDS TGLVRRGYYRITYGETGGNSPVQQFTVPGSKGT ATISGLKPGVDYTITVYAVEYPYDHSGYYHRPI SINYRT-Xi-X ₂ - VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQD WLNGKEYKCKVSNKALPAPIEKTISK

- 39 044608

selected from the hinge region sequences SEQ ID NO: 317-321; this Fc fragment may include a C-terminal K residue AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPG 19 2381 NOB-Fc fusion protein XI is optional and, if present, may be selected from the following group: E, EI, EID, EIDK (SEQ ID NO: 384), EIE and EIEK (SEQ ID NO: 635); X2 is selected from the hinge region sequences SEQ ID NO: 317-321; this Fc fragment may include a C-terminal K residue GVSDVPRDLEVVAAT PT SLLISWGDVRT NEARQGYYRITYGETGGNSPLQGFTVPGSKGTA TISGLKPGVDYTITVYAVEYTYEHSGYYHRPIS INYRT-Xi-X ₂ - VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLH QDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPG 20 2381 H09-Fc fusion protein XI is optional and, if present, may be selected from the following group: E, EI, EID, EIDK (SEQ ID NO: 384), EIE and EIEK (SEQ ID NO: 635); X2 is selected from the hinge region sequences SEQ ID NO: 317-321; this Fc fragment may include a C-terminal K residue GVSDVPRDLEVVAAT PT SLLISWDAPAG GGYGYYRITYGETGGNSPVQEFTVPVSKGTATI SGLKPGVDYTITVYAVE FDFVGAGYYHRPISIN YRT-Xx-Xz- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYT QKSLSLSPG 21 2382V 1 Ι-Fc fusion protein XI is optional and, if present, may be GVSDVPRDLEVVAAT PT SLLISWDAPAA AYGYYRITYGETGGNSPVQEFTVPVSKGTATIS GLKPGVDYTITVYAVEYDFAGSGYYHRPISINY

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selected from the following group: E, EI, EID, EIDK (SEQ ID NO: 384), EIE and EIEK (SEQ ID NO: 635); X2 is selected from the hinge region sequences SEQ ID NO: 317-321; this Fc fragment may include a C-terminal K residue RT-Xx-Xz- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYT QKSLSLSPG 22 2382V 08-Fc fusion protein XI is optional and, if present, may be selected from the following group: E, EI, EID, EIDK (SEQ ID NO: 384), EIE and EIEK (SEQ ID NO: 635); X2 is selected from the hinge region sequences SEQ ID NO: 317-321; this Fc fragment may include a C-terminal K residue GVSDVPRDLEVVAAT PT SLLISWDAPAD AYGYYRITYGETGGNSPVQEFTVPSSKGTATIS GLKPGVDYTITVYAVEFAFPGAGYYHRPISINY RT-Xx-Xz- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPG 23 2382С 11-Fc fusion protein XI is optional and, if present, may be selected from the following group: E, EI, EID, EIDK (SEQ ID NO: 384), EIE and EIEK (SEQ ID NO: 635); X2 is selected from the hinge region sequences SEQ ID NO: 317-321; this F fragment may include GVSDVPRDLEVVAAT PT SLLISWDAPAG GYGYYRITYGETGGNSPVQEFTVPVSKGTATIS GLKPGVDYTITVYAVEYDFAGSGYYHRPISINY RT-Xi-Xz- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYT QKSLSLSPG

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C-terminal residue K 24 2382 G03-Fc fusion protein XI is optional and, if present, may be selected from the following group: E, EI, EID, EIDK (SEQ ID NO: 384), EIE and EIEK (SEQ ID NO: 635); X2 is selected from the hinge region sequences SEQ ID NO: 317-321; this Fc fragment may include a C-terminal K residue GVSDVPRDLEVVAAT PT SLLISWDAPAE AEAYGYYRITYGETGGNSPVQEFTVPVSKGTAT ISGLKPGVDYTITVYAVEYVFPGAGYYHRPISI NYRT-X1-X2- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYT QKSLSLSPG 25 2382 NOZ-Fc fusion protein XI is optional and, if present, may be selected from the following group: E, EI, EID, EIDK (SEQ ID NO: 384), EIE and EIEK (SEQ ID NO: 635); X2 is selected from the hinge region sequences SEQ ID NO: 317-321; this Fc fragment may include a C-terminal K residue GVSDVPRDLEVVAAT PT SLLISWDAPAE GAYGYYRITYGETGGNSPVQEFTVPVSKGTATI SGLKPGVDYTITVYAVEYPYPFAGYYHRPISIN YRT-X1-X2- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYT QKSLSLSPG 26 2451 AlO-Fc fusion protein XI is optional and, if present, may be selected from the following group: E, EI, EID, EIDK (SEQ ID NO: 384), EIE and EIEK (SEQ ID NO: 635); X2 is selected from GVTDVPRDMEVVAATPTSLLISWQPPAV TYGYYRITYGETGGNSTLQQFTVPVYKGTATIS GLKPGVDYTITVYAVEYPYDHSGYYHRPISINY RT-Xx-Xz- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG

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hinge region sequences SEQ ID NO: 317-321; this Fc fragment may include a C-terminal K residue FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPG 27 2451B 02-Fc fusion protein XI is optional and, if present, may be selected from the following group: E, EI, EID, EIDK (SEQ ID NO: 384), EIE and EIEK (SEQ ID NO: 635); X2 is selected from the hinge region sequences SEQ ID NO: 317-321; this Fc fragment may include a C-terminal K residue GVSDVPRDLEVVAAT PT SLLISWDAPAA AYGYYRITYGETGGNSPVQEFTVPVSKGTATIS GLKPGVDYTITVYAVEFDYPGSGYYHRPISINY RT-Xx-Xz- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYT QKSLSLSPG 28 2451C 1 Ι-Fc fusion protein XI is optional and, if present, may be selected from the following group: E, EI, EID, EIDK (SEQ ID NO: 384), EIE and EIEK (SEQ ID NO: 635); X2 is selected from the hinge region sequences SEQ ID NO: 317-321; this Fc fragment may include a C-terminal K residue GIVDVPRDLEVVAATPTSLLISWDPPAG AYGYYRITYGETGGNSPKQQFTVPGYKGTATIS GLKPGVDYTITVYAVEYPYDHSGYYHRPISINY RT-Xx-Xz- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYT QKSLSLSPGK 29 2451 HOl-Fc fusion protein XI is optional and, if present, can be selected from the following GVSDVPRDLEVVAAT PT SLLISWDAPAA GYGYYRITYGETGGNSPVQEFTVPVSKGTATIS GLKPGVDYTITVYAVEYDFPGSGYYHRPISINY RT-Xx-Xz-

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groups: E, EI, EID, EIDK (SEQ ID NO: 384), EIE and EIEK (SEQ ID NO: 635); X2 is selected from the hinge region sequences SEQ ID NO: 317-321; this Fc fragment may include a C-terminal K residue VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYT QKSLSLSPG thirty 2011В 11-Fc fusion protein XI is optional and, if present, may be selected from the following group: E, EI, EID, EIDK (SEQ ID NO: 384), EIE and EIEK (SEQ ID NO: 635); X2 is selected from the hinge region sequences SEQ ID NO: 317-321; this Fc fragment may include a C-terminal K residue GVSDVPRDLEVVAATPTSLLISWAPPSD AYGYYRITYGETGGNSPVQEFTVPIGKGTATIS GLKPGVDYTITVYAVEYPYSHAGYYHRPISINY RT-Xx-Xz- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYT QKSLSLSPG 31 2971 АОЗ-Fc fusion protein XI is optional and, if present, may be selected from the following group: E, EI, EID, EIDK (SEQ ID NO: 384), EIE and EIEK (SEQ ID NO: 635); X2 is selected from the hinge region sequences SEQ ID NO: 317-321; this Fc fragment may include a C-terminal K residue GVSDVPRDLEVVAATPTSLLISWDPPSD DYGYYRITYGETGGNSPVQEFTVPIGKGTATIS GLKPGVDYTITVYAVEFPWPHAGYYHRPISINY RT-Xx-Xz- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYT QKSLSLSPG

- 44 044608

32 2971 A09-Fc fusion protein XI is optional and, if present, may be selected from the following group: E, EI, EID, EIDK (SEQ ID NO: 384), EIE and EIEK (SEQ ID NO: 635); X2 is selected from the hinge region sequences SEQ ID NO: 317-321; this Fc fragment may include a C-terminal K residue GVSDVPRDLEVVAAT PT SLLISWDAPAD DYGYYRITYGETGGNSPVQEFTVPIGKGTATIS GLKPGVDYTITVYAVEFPWPHAGYYHRPISINY RT-Хх-Хг- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYT QKSLSLSPG 33 2971E 02-Fc fusion protein XI is optional and, if present, may be selected from the following group: E, EI, EID, EGK (SEQ ID NO: 384), EIE and EIEK (SEQ ID NO: 635); X2 is selected from the hinge region sequences SEQ GO NO: 317-321; this Fc fragment may include a C-terminal K residue GVSDVPRDLEVVAATPTSLLISWDAPSD DYGYYRITYGETGGNSPVQEFTVPIGKGTATIS GLKPGVDYTITVYAVEFPWPHAGYYHRPISINY RT-Xi-Хг- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYT QKSLSLSPG Xi-Fc-X2-PCSK9 Adnectin N-terminal variants 34 Fc- 1459D05 fusion protein XI is selected from the hinge region sequences SEQ ID NO: 317-321; X2 is selected from the linker sequences of SEQ ID NOs: 310-314 and 620-634; HZ is Xi- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPG-X ₂ -

- 45 044608

optional and, if present, may be a C-terminal tail sequence selected from SEQ ID NO: 380-395 and EIEK (SEQ ID NO: 635) VSDVPRDLEVVAATPTSLLISWPPPSHGYGYYR ITYGETGGNSPVQEFTVPPGKGTATISGLKPGV DYTITVYAVEYPYKHSGYYHRPISINYRT-Хз 35 Fc- 1784F03 fusion protein XI is selected from the hinge region sequences SEQ ID NO: 317-321; X2 is selected from the linker sequences of SEQ ID NOs: 310-314 and 620-634; X3 is optional and, if present, may be a C-terminal tail sequence selected from SEQ ID NO: 380-395 and EIEK (SEQ ID NO: 635) Х1- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYT qkslslspg-x ₂ - VSDVPRDLEVVAATPTSLLISWRPPIHAYGYYR ITYGETGGNSPVQEFTVPIVEGTATISGLKPGV DYTITVYAVEYTFKHSGYYHRPISINYRT-Хз 36 Fc1784F03-ml fusion protein XI is selected from the hinge region sequences SEQ ID NO: 317-321; X2 is selected from the linker sequences of SEQ ID NOs: 310-314 and 620-634; CH is optional and, if present, may be C-terminal "tail" Х1- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYT qkslslspg-x ₂ - VSDVPRDLEVVAATPTSLLISWDAPIHAYGYYR ITYGETGGNSPVQEFTVPGSEGTATISGLKPGV DYTITVYAVEYTFKHSGYYHRPISINYRT-Хз

- 46 044608

a sequence selected from SEQ ID NO: 380-395 and EIEK (SEQ ID NO: 635) 37 Fc1784F03-m2 fusion protein XI is selected from the hinge region sequences SEQ ID NO: 317-321; X2 is selected from the linker sequences of SEQ ID NOs: 310-314 and 620-634; X3 is optional and, if present, may be a C-terminal tail sequence selected from SEQ ID NO: 380-395 and EIEK (SEQ ID NO: 635) Х1- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYT qkslslspg-x ₂ - VSDVPRDLEVVAATPTSLLISWDAPAHAYGYYR ITYGETGGNSPVQEFTVPGSKGTATISGLKPGV DYTITVYAVEYTFKHSGYYHRPISINYRT-Xs 38 Fc- 1784F03-m3 fusion protein XI is selected from the hinge region sequences SEQ ID NO: 317-321; X2 is selected from the linker sequences of SEQ ID NOs: 310-314 and 620-634; X3 is optional and, if present, may be a C-terminal tail sequence selected from SEQ Х1- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYT qkslslspg-x ₂ - VSDVPRDLEVVAATPTSLISWDAPAVTYGYYR ITYGETGGNSPVQEFTVPGSKSTATISGLKPGV DYTITVYAVEYTFKHSGYYHRPISINYRT-Хз

- 47 044608

ID NO: 380-395 and EIEK (SEQ ID NO: 635) 39 Fc- 1813E02 fusion protein XI is selected from the hinge region sequences SEQ ID NO: 317-321; X2 is selected from the linker sequences SEQ ID NO: 310-314 and 620-634; X3 is optional and, if present, may be a C-terminal tail sequence selected from SEQ ID NO: 380-395 and EIEK (SEQ ID NO: 635) Xi- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYT qkslslspg-x ₂ - VSDVPRDLEVVAATPTSLLISWSPPANGYGYYR ITYGETGGNSPVQEFTVPVGRGTATISGLKPGV DYTITVYAVEYTYKGSGYYHRPISINYRT-Хз 40 Fc- 1923B02 fusion protein XI is selected from the hinge region sequences SEQ ID NO: 317-321; X2 is selected from the linker sequences of SEQ ID NOs: 310-314 and 620-634; X3 is optional and, if present, may be a C-terminal tail sequence selected from SEQ ID NO: 380-395 and EIEK (SEQ ID NO: 635) Xi- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYT qkslslspg-x ₂ - VSDVPRDLEVVAATPTSLLISWTPPPKGYGYYR ITYGETGGNSPVQEFTVPVGEGTATISGLKPGV DYTITVYAVEYTYNGAGYYHRPISINYRT-Хз Fc- XI is selected from Xi-

- 48 044608

41 1923B02(N82 I) fusion protein hinge region sequences SEQ ID NO: 317-321; X2 is selected from the linker sequences SEQ ID NO: 310-314 and 620-634; X3 is optional and, if present, may be a C-terminal tail sequence selected from SEQ ID NO: 380-395 and EIEK (SEQ ID NO: 635) VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYT qkslslspg-x ₂ - VSDVPRDLEVVAATPTSLLISWTPPPKGYGYYR ITYGETGGNSPVQEFTVPVGEGTATISGLKPGV DYTITVYAVEYTYIGAGYYHRPISINYRT-Хз 42 Fc1923B02(N82 E) fusion protein XI is selected from the hinge region sequences SEQ ID NO: 317-321; X2 is selected from the linker sequences of SEQ ID NOs: 310-314 and 620-634; X3 is optional and, if present, may be a C-terminal tail sequence selected from SEQ ID NO: 380-395 and EIEK (SEQ ID NO: 635) Xi- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYT qkslslspg-x ₂ - VSDVPRDLEVVAATPTSLLISWTPPPKGYGYYR ITYGETGGNSPVQEFTVPVGEGTATISGLKPGV DYTITVYAVEYTYEGAGYYHRPISINYRT-Хз 43 Fc1923B02(T80 A) fusion protein XI is selected from the hinge region sequences SEQ ID NO: 317-321; X2 Xi- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK

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selected from linker sequences SEQ ID NO: 310-314 and 620-634; X3 is optional and, if present, may be a C-terminal tail sequence selected from SEQ ID NO: 380-395 and EIEK (SEQ ID NO: 635) AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPG-X ₂ - VSDVPRDLEVVAATPTSLLISWTPPPKGYGYYR ITYGETGGNSPVQEFTVPVGEGTATISGLKPGV DYTITV YAVEYAYNGAGYYHRPISINYRT-Xs 44 Fc- 1922G04 fusion protein XI is selected from the hinge region sequences SEQ ID NO: 317-321; X2 is selected from the linker sequences of SEQ ID NOs: 310-314 and 620-634; X3 is optional and, if present, may be a C-terminal tail sequence selected from SEQ ID NO: 380-395 and EIEK (SEQ ID NO: 635) Х1- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPG-X ₂ - VSDVPRDLEVVAATPTSLLISWRPPSHAYGYYR IT YGE T GGN S РVQ E FTVPIGKGTATIS GL KPGV DYTITVYAVEYPWKGSGYYHRPISINYRT-Хз 45 Fc1922G04(R25 D) fusion protein XI is selected from the hinge region sequences SEQ ID NO: 317-321; X2 is selected from the linker sequences of SEQ ID NOs: 310-314 and 620-634; X1- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYT

- 50 044608

X3 is optional and, if present, may be a C-terminal tail sequence selected from SEQ ID NO: 380-395 and EIEK (SEQ ID NO: 635) qkslslspg-x ₂ - VSDVPRDLEVVAATPTSLLISWDPPSHAYGYYR IT YGE T GGN S РVQ E FTVPIGKGTATIS GL KPGV DYTITVYAVEYPWKGSGYYHRPISINYRT-Хз 46 Fc1922G04(R25 E) fusion protein XI is selected from the hinge region sequences SEQ ID NO: 317-321; X2 is selected from the linker sequences of SEQ ID NOs: 310-314 and 620-634; X3 is optional and, if present, may be a C-terminal tail sequence selected from SEQ ID NO: 380-395 and EIEK (SEQ ID NO: 635) Х1- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYT qkslslspg-x ₂ - VSDVPRDLEVVAATPTSLLISWEPPSHAYGYYR IT YGE T GGN S РVQ E FTVPIGKGTATIS GL KPGV DYTITVYAVEYPWKGSGYYHRPISINYRT-Хз 47 Fc1922G04(R25 S) fusion protein XI is selected from the hinge region sequences SEQ ID NO: 317-321; X2 is selected from the linker sequences of SEQ ID NOs: 310-314 and 620-634; HZ is optional and, if present, may be Xi- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYT qkslslspg-x ₂ - VSDVPRDLEVVAATPTSLLISWSPPSHAYGYYR IT YGE T GGN S PVQ E FTVPIGKGTATIS GL KPGV

- 51 044608

C-terminal tail sequence selected from SEQ ID NO: 380-395 and EIEK (SEQ ID NO: 635) DYTITVYAVEYPWKGSGYYHRPISINYRT-X ₃ 48 Fc- 2012A04 fusion protein XI is selected from the hinge region sequences SEQ ID NO: 317-321; X2 is selected from the linker sequences of SEQ ID NOs: 310-314 and 620-634; X3 is optional and, if present, may be a C-terminal tail sequence selected from SEQ ID NO: 380-395 and EIEK (SEQ ID NO: 635) Х1- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYT qkslslspg-x ₂ - VSDVPRDLEVVAATPTSLLISWRPPSNGHGYYR IT YGE T GGN S РVQ E FTVPVNE GTATIS GL KPGV DYTITVYAVEFPFKWSGYYHRPISINYRT-Хз 49 Fc- 2013E01 fusion protein XI is selected from the hinge region sequences SEQ ID NO: 317-321; X2 is selected from the linker sequences of SEQ ID NOs: 310-314 and 620-634; X3 is optional and, if present, may be a C-terminal tail sequence selected from X1- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYT QKSLSLSPG-Хг- VSDVPRDLEVVAATPTSLLISWVPPSDDYGYYR IT YGE T GGN S РVQ E FTVPIGKGTATIS GL KPGV DYTITVYAVEFPWPHAGYYHRPISINYRT-Хз

- 52 044608

sequences SEQ ID NO: 380-395 and EIEK (SEQ ID NO: 635) 50 Fc- 2011H05 fusion protein XI is selected from the hinge region sequences SEQ ID NO: 317-321; X2 is selected from the linker sequences SEQ ID NO: 310-314 and 620-634; X3 is optional and, if present, may be a C-terminal tail sequence selected from SEQ ID NO: 380-395 and EIEK (SEQ ID NO: 635) Xi- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYT qkslslspg-x ₂ - VSDVPRDLEVVAATPTSLLISWVPSSHAYGYYR IT YGE T GGN S PVQ E FTVPVGVGTATIS GL KPGV DYTITVYAVEYAFEGAGYYHRPISINYRT-Хз 51 Fc2011H05(V23 D) fusion protein XI is selected from the hinge region sequences SEQ ID NO: 317-321; X2 is selected from the linker sequences of SEQ ID NOs: 310-314 and 620-634; X3 is optional and, if present, may be a C-terminal tail sequence selected from SEQ ID NO: 380-395 and EIEK (SEQ ID NO: 635) Xi- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYT qkslslspg-x ₂ - VSDVPRDLEVVAATPTSLLISWDPSSHAYGYYR IT YGE T GGN S PVQ E FTVPVGVGTATIS GL KPGV DYTITVYAVEYAFEGAGYYHRPISINYRT-Хз

- 53 044608

52 Fc2011H05(V23 E) fusion protein XI is selected from the hinge region sequences SEQ ID NO: 317-321; X2 is selected from the linker sequences of SEQ ID NOs: 310-314 and 620-634; X3 is optional and, if present, may be a C-terminal tail sequence selected from SEQ ID NO: 380-395 and EIEK (SEQ ID NO: 635) Х1- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYT qkslslspg-x ₂ - VSDVPRDLEVVAATPTSLLISWEPSSHAYGYYR IT YGE T GGN S РVQ E FTVPVGVGTATIS GL KPGV DYTITVYAVEYAFEGAGYYHRPISINYRT-Хз 53 Fc- 2381B02 fusion protein XI is selected from the hinge region sequences SEQ ID NO: 317-321; X2 is selected from the linker sequences of SEQ ID NOs: 310-314 and 620-634; X3 is optional and, if present, may be a C-terminal tail sequence selected from SEQ ID NO: 380-395 and EIEK (SEQ ID NO: 635) Х1- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYT qkslslspg-x ₂ - VSDVPRDLEVVAATPTSLLISWEPFSRLPGGGE YYRITYGETGGNSPLQQFTVPGSKGTATISGLK PGVDYTITVYAVEYPYDYSGYYHRPISINYRTХз 54 Fc- 2381B04 fusion protein XI is selected from the hinge region sequences SEQ ID X1- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV

- 54 044608

NO: 317-321; X2 is selected from the linker sequences of SEQ ID NOs: 310-314 and 620-634; X3 is optional and, if present, may be a C-terminal tail sequence selected from SEQ ID NO: 380-395 and EIEK (SEQ ID NO: 635) LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT qkslslspg-x ₂ - VSDVPRDLEVVAATPTSLLISWEPFSRLPGGGE YY RITYGETGGNSPLQQFTVPGSKGTATISGLK PGVDYTITVYAVEYPYEHSGYYHRPISINYRTХз 55 Fc- 2381B06 fusion protein XI is selected from the hinge region sequences SEQ ID NO: 317-321; X2 is selected from the linker sequences of SEQ ID NOs: 310-314 and 620-634; X3 is optional and, if present, may be a C-terminal tail sequence selected from SEQ ID NO: 380-395 and EIEK (SEQ ID NO: 635) Х1- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYT qkslslspg-x ₂ - VSDVPRDLEVVAATPTSLLISWEPFSRLPGGGE YYRITYGETGGNSPLQQFTVPGSKGTATISGLK PGVDYTITVYAVEYPYPHSGYYHRPISINYRTХз 56 Fc- 2381B08 fusion protein XI is selected from the hinge region sequences SEQ ID NO: 317-321; X2 is selected from linker sequences SEQ X1- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF

- 55 044608

IDNO: 310-314 and 620-634; X3 is optional and, if present, may be a C-terminal tail sequence selected from SEQ IDNO: 380-395 and EIEK (SEQ IDNO: 635) FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPG-X ₂ - VSDVPRDLEVVAATPTSLLISWDAPADGGYGYY RITYGETGGNSPVQEFTVPSSKGTATISGLKPG VDYTITVYAVEYTFPGAGYYHRPISINYRT-Хз 57 Fc- 2381D02 fusion protein XI is selected from the hinge region sequences SEQ ID NO: 317-321; X2 is selected from the linker sequences of SEQ IDNO: 310-314 and 620-634; X3 is optional and, if present, may be a C-terminal tail sequence selected from SEQ IDNO: 380-395 and EIEK (SEQ IDNO: 635) Х1- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYT qkslslspg-x ₂ - VSDVPRDLEVVAATPTSLLISWEPFSRLPGGGE YYRITYGETGGNSPLQQFTVPGSKGTATISGLK PGVDYTITVYAVEYPYDHSGYYHRPISINYRTХз 58 Fc- 2381D04 fusion protein XI is selected from the hinge region sequences SEQ ID NO: 317-321; X2 is selected from the linker sequences of SEQ IDNO: 310-314 and 620-634; HZ is optional and, if Х1- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYT qkslslspg-x ₂ - VSDVPRDLEVVAATPTSLLISWEPFSRLPGGGE

- 56 044608

present, may be a C-terminal tail sequence selected from SEQ ID NO: 380-395 and EIEK (SEQ ID NO: 635) YYRITYGETGGNSPLQQFTVPGSKGTATISGLK PGVDYTITVYAVEFPYDHSGYYHRPISINYRT- Idk 59 Fc- 2381F11 fusion protein XI is selected from the hinge region sequences SEQ ID NO: 317-321; X2 is selected from the linker sequences of SEQ ID NOs: 310-314 and 620-634; X3 is optional and, if present, may be a C-terminal tail sequence selected from SEQ ID NO: 380-395 and EIEK (SEQ ID NO: 635) Х1- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYT qkslslspg-x ₂ - VSDVPRDLEVVAATPTSLLISWDAPADGGYGYY RITYGETGGNSPVQEFTVPVSKSTATISGLKPG VDYTITVYAVEYTFPGAGYYHRPISINYRT-Хз 60 Fc- 2381G03 fusion protein XI is selected from the hinge region sequences SEQ ID NO: 317-321; X2 is selected from the linker sequences of SEQ ID NOs: 310-314 and 620-634; X3 is optional and, if present, may be the C-terminal tail sequence, Х1- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYT qkslslspg-x ₂ - VSDVPRDLEVVAATPTSLLISWEPFSRLPGGGE YYRITYGETGGNSPLQQFTVPGSKGTATISGLK PGVDYTITVYAVE FPYAHSGYYHRPISINYRTХз

- 57 044608

selected from the sequences SEQ ID NO: 380-395 and EIEK (SEQ ID NO: 635) 61 Fc- 2381G09 fusion protein XI is selected from the hinge region sequences SEQ ID NO: 317-321; X2 is selected from the linker sequences of SEQ ID NOs: 310-314 and 620-634; X3 is optional and, if present, may be a C-terminal tail sequence selected from SEQ ID NO: 380-395 and EIEK (SEQ ID NO: 635) Х1- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYT qkslslspg-x ₂ - VSDVPRDLEVVAATPTSLLISWDAPAGDGYGYY RITYGETGGNSPVQEFTVPVSKGTATISGLKPG VDYTITVYAVEFTFPGAGYYHRPISINYRT-Xs 62 Fc- 2381H03 fusion protein XI is selected from the hinge region sequences SEQ ID NO: 317-321; X2 is selected from the linker sequences of SEQ ID NOs: 310-314 and 620-634; X3 is optional and, if present, may be a C-terminal tail sequence selected from SEQ ID NOs: 380-395 and EIEK Х1- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYT qkslslspg-x ₂ - VSDVPRDLEVVAATPTSLLISWEPFSRLPGGGE YYRITYGETGGNSPLQQFTVPGSKGTATISGLK PGVDYTITVYAVEYPYAHSGYFHRPISINYRTХз

- 58 044608

(SEQ ID NO: 635) 63 Fc- 2382A01 fusion protein XI is selected from the hinge region sequences SEQ ID NO: 317-321; X2 is selected from the linker sequences of SEQ ID NOs: 310-314 and 620-634; X3 is optional and, if present, may be a C-terminal tail sequence selected from SEQ ID NO: 380-395 and EIEK (SEQ ID NO: 635) Х1- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYT qkslslspg-x ₂ - VSDVPRDLEVVAATPTSLLISWAAPAGGGYGYY RITYGETGGNSPVQEFTVPVSKGTATISGLKPG VDYTITVYAVEYDFPGAGYYHRPISINYRT-Хз 64 Fc- 2382B10 fusion protein XI is selected from the hinge region sequences SEQ ID NO: 317-321; X2 is selected from the linker sequences of SEQ ID NOs: 310-314 and 620-634; X3 is optional and, if present, may be a C-terminal tail sequence selected from SEQ ID NO: 380-395 and EIEK (SEQ ID NO: 635) Х1- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYT qkslslspg-x ₂ - VSDVPRDLEVVAATPTSLLISWDAPADAYGYYR ITYGETGGNSPVQEFTVPSSKGTATISGLKPGV DYTITVYAVEYDFPGSGYYHRPISINYRT-Хз 65 Fc- 2382В09 XI is selected from sequences X1- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE

- 59 044608

fusion protein hinge region SEQ ID NO: 317-321; X2 is selected from the linker sequences of SEQ ID NOs: 310-314 and 620-634; X3 is optional and, if present, may be a C-terminal tail sequence selected from SEQ ID NO: 380-395 and EIEK (SEQ ID NO: 635) VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT qkslslsp g-x ₂ - VSDVPRDLEVVAATPTSLLISWDAPADAYGYYR ITYGETGGNSPVQEFTVPVSKGTATISGLKPGV DYTITVYAVEFDYPGSGYYHRPISINYRT-Хз 66 Fc- 2382C05 fusion protein XI is selected from the hinge region sequences SEQ ID NO: 317-321; X2 is selected from the linker sequences of SEQ ID NOs: 310-314 and 620-634; X3 is optional and, if present, may be a C-terminal tail sequence selected from SEQ ID NO: 380-395 and EIEK (SEQ ID NO: 635) Х1- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYT qkslslspg-x ₂ - VSDVPRDLEVVAATPTSLLISWDAPADGAYGYY RITYGETGGNSPVQEFTVPVSKGTATISGLKPG VDYTITVYAVEYSFPGAGYYHRPISINYRT-Xs 67 Fc- 2382С09 fusion protein XI is selected from the hinge region sequences SEQ ID NO: 317-321; X2 is selected from the linker X1- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG

- 60 044608

sequences SEQ ID NO: 310-314 and 620-634; X3 is optional and, if present, may be a C-terminal tail sequence selected from SEQ ID NO: 380-395 and EIEK (SEQ ID NO: 635) FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT qkslslspg-x ₂ - VSDVPRDLEVVAATPTSLLISWDAPAEGYGYYR ITYGETGGNSPVQEFTVPVSKGTATISGLKPGV DYTITVYAVEFDFPGSGYYHRPISINYRT-Xs 68 Fc- 2382D03 fusion protein XI is selected from the hinge region sequences SEQ ID NO: 317-321; X2 is selected from the linker sequences of SEQ ID NOs: 310-314 and 620-634; X3 is optional and, if present, may be a C-terminal tail sequence selected from SEQ ID NO: 380-395 and EIEK (SEQ ID NO: 635) Х1- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYT qkslslspg-x ₂ - VSDVPRDLEVVAATPTSLLISWDAPADEAYGYY RITYGETGGNSPVQEFTVPVSKGTATISGLKPG VDYTITVYAVEFDFPGAGYYHRPISINYRT-Хз 69 Fc- 2382D05 fusion protein XI is selected from the hinge region sequences SEQ ID NO: 317-321; X2 is selected from the linker sequences of SEQ ID NOs: 310-314 and 620-634; HZ is Х1- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYT qkslslspg-x ₂ -

- 61 044608

optional and, if present, may be a C-terminal tail sequence selected from SEQ ID NO: 380-395 and EIEK (SEQ ID NO: 635) VSDVPRDLEVVAATPTSLLISWDAPADGGYGYY RITYGETGGNSPVQEFTVPVSKGTATISGLKPG VDYTITVYAVEFDFPGAGYYHRPISINYRT-Хз 70 Fc- 2382D08 fusion protein XI is selected from the hinge region sequences SEQ ID NO: 317-321; X2 is selected from the linker sequences of SEQ ID NOs: 310-314 and 620-634; X3 is optional and, if present, may be a C-terminal tail sequence selected from SEQ ID NO: 380-395 and EIEK (SEQ ID NO: 635) Х1- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYT qkslslspg-x ₂ - VSDVPRDLEVVAATPTSLLISWDAPADGYGYYR ITYGETGGNSPVQEFTVPVSKGTATISGLKPGV DYTITVYAVEFPFPGSGYYHRPISINYRT-Xs 71 Fc- 2382D09 fusion protein XI is selected from the hinge region sequences SEQ ID NO: 317-321; X2 is selected from the linker sequences of SEQ ID NOs: 310-314 and 620-634; CH is optional and, if present, may be C-terminal "tail" Х1- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYT qkslslspg-x ₂ - VSDVPRDLEVVAATPTSLLISWDAPAEGYGYYR ITYGETGGNSPVQEFTVPVSKGTATISGLKPGV DYTITVYAVEFDFPGAGYYHRPISINYRT-Хз

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a sequence selected from SEQ ID NO: 380-395 and EIEK (SEQ ID NO: 635) 72 Fc- 2382F02 fusion protein XI is selected from the hinge region sequences SEQ ID NO: 317-321; X2 is selected from the linker sequences of SEQ ID NOs: 310-314 and 620-634; X3 is optional and, if present, may be a C-terminal tail sequence selected from SEQ ID NO: 380-395 and EIEK (SEQ ID NO: 635) Х1- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYT qkslslspg-x ₂ - VSDVPRDLEVVAATPTSLLISWDAPAGGYGYY RITYGETGGNSPVQEFTVPVSKGTATISGLKPG VDYTITVYAVEFDFPGSGYYHRPISINYRT-Хз 73 Fc- 2382F03 fusion protein XI is selected from the hinge region sequences SEQ ID NO: 317-321; X2 is selected from the linker sequences of SEQ ID NOs: 310-314 and 620-634; X3 is optional and, if present, may be a C-terminal tail sequence selected from SEQ Х1- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYT qkslslspg-x ₂ - VSDVPRDLEVVAATPTSLLISWDAPAADAYGYY RITYGETGGNSPVQEFTVPVSKGTATISGLKPG VDYTITVYAVEFNFPGAGYYHRPISINYRT-Xs

- 63 044608

ID NO: 380-395 and EIEK (SEQ ID NO: 635) 74 Fc- 2382F05 fusion protein XI is selected from the hinge region sequences SEQ ID NO: 317-321; X2 is selected from the linker sequences SEQ ID NO: 310-314 and 620-634; X3 is optional and, if present, may be a C-terminal tail sequence selected from SEQ ID NO: 380-395 and EIEK (SEQ ID NO: 635) Xi- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYT qkslslspg-x ₂ - VSDVPRDLEVVAATPTSLLISWDAPAEAGKHYG YYRITYGETGGNSPVQEFTVPVSKGTATISGLK PGVDYTITVYAVEFDFPGAGYYHRPISINYRTХз 75 Fc- 2382F08 fusion protein XI is selected from the hinge region sequences SEQ ID NO: 317-321; X2 is selected from the linker sequences of SEQ ID NOs: 310-314 and 620-634; X3 is optional and, if present, may be a C-terminal tail sequence selected from SEQ ID NO: 380-395 and EIEK (SEQ ID NO: 635) Xi- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYT qkslslspg-x ₂ - VSDVPRDLEVVAATPTSLLISWDAPAEAYGYYR ITYGETGGNSPVQEFTVPVSKGTATISGLKPGV DYTITVYAVEFTYPGSGYYHRPISINYRT-Хз Fc- XI is selected from Xi-

- 64 044608

76 2382F09 fusion protein hinge region sequences SEQ ID NO: 317-321; X2 is selected from the linker sequences of SEQ ID NOs: 310-314 and 620-634; X3 is optional and, if present, may be a C-terminal tail sequence selected from SEQ ID NO: 380-395 and EIEK (SEQ ID NO: 635) VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYT qkslslspg-x ₂ - VSDVPRDLEVVAATPTSLLISWDAPAAAYGYYR ITYGETGGNSPVQEFTVPVSKGTATISGLKPGV DYTITVYAVEYDFPGSGYYHRPISINYRT-Хз 77 Fc- 2382G04 fusion protein XI is selected from the hinge region sequences SEQ ID NO: 317-321; X2 is selected from the linker sequences of SEQ ID NOs: 310-314 and 620-634; X3 is optional and, if present, may be a C-terminal tail sequence selected from SEQ ID NO: 380-395 and EIEK (SEQ ID NO: 635) Xi- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYT qkslslspg-x ₂ - VSDVPRDLEVVAATPTSLLISWDAPAGGYGYY RITYGETGGNSPVQEFTVPSSKGTATISGLKPG VDYTITVYAVEFDFPGAGYYHRPISINYRT-Xs 78 Fc- 2382H10 fusion protein XI is selected from the hinge region sequences SEQ ID NO: 317-321; X2 Xi- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK

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selected from linker sequences SEQ ID NO: 310-314 and 620-634; X3 is optional and, if present, may be a C-terminal tail sequence selected from SEQ ID NO: 380-395 and EIEK (SEQ ID NO: 635) AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT qkslslspg-x ₂ - VSDVPRDLEVVAATPTSLLISWDAPAGGYGYYR ITYGETGGNSPVQEFTVPVSKGTATISGLKPGV DY TITVYAVEFDFPGSGYYHRPISINYRT-Хз 79 Fc- 2382H11 fusion protein XI is selected from the hinge region sequences SEQ ID NO: 317-321; X2 is selected from the linker sequences of SEQ ID NOs: 310-314 and 620-634; X3 is optional and, if present, may be a C-terminal tail sequence selected from SEQ ID NO: 380-395 and EIEK (SEQ ID NO: 635) Х1- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYT qkslslspg-x ₂ - VSDVPRDLEVVAATPTSLLISWDAPADGYGYYR IT YGE T GGN S РVQ E FTVPV FKGTATIS GL KPGV DYTITVYAVEFDYPGSGYYHRPISINYRT-Хз 80 Fc- 2382H04 fusion protein XI is selected from the hinge region sequences SEQ ID NO: 317-321; X2 is selected from the linker sequences of SEQ ID NOs: 310-314 and 620-634; X1- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYT

- 66 044608

X3 is optional and, if present, may be a C-terminal tail sequence selected from SEQ ID NO: 380-395 and EIEK (SEQ ID NO: 635) QKSLSLSPG-X ₂ - VSDVPRDLEVVAATPTSLLISWDAPAAGGYGYY RITYGETGGNSPVQEFTVPSSKGTATISGLKPG VDYTITVYAVEYDFPGAGYYHRPISINYRT-Xs 81 Fc- 2382H07 fusion protein XI is selected from the hinge region sequences SEQ ID NO: 317-321; X2 is selected from the linker sequences of SEQ ID NOs: 310-314 and 620-634; X3 is optional and, if present, may be a C-terminal tail sequence selected from SEQ ID NO: 380-395 and EIEK (SEQ ID NO: 635) Х1- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYT qkslslspg-x ₂ - VSDVPRDLEVVAATPTSLLISWDAPADAYGYYR ITYGETGGNSPVQEFTVPGSKGTATISGLKPGV DYTITVYAVEFDFPGSGYYHRPISINYRT-Хз 82 Fc- 2382H09 fusion protein XI is selected from the hinge region sequences SEQ ID NO: 317-321; X2 is selected from the linker sequences of SEQ ID NOs: 310-314 and 620-634; HZ is optional and, if present, may be Х1- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYT qkslslspg-x ₂ - VSDVPRDLEVVAATPTSLLISWDAPAAAYGYYR ITYGETGGNSPVQEFTVPSSKGTATISGLKPGV

- 67 044608

C-terminal tail sequence selected from SEQ ID NO: 380-395 and EIEK (SEQ ID NO: 635) DYTITVYAVEFDFPGSGYYHRPISINYRT-X ₃ 83 Fc- 2451A02 fusion protein XI is selected from the hinge region sequences SEQ ID NO: 317-321; X2 is selected from the linker sequences of SEQ ID NOs: 310-314 and 620-634; X3 is optional and, if present, may be a C-terminal tail sequence selected from SEQ ID NO: 380-395 and EIEK (SEQ ID NO: 635) Х1- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYT qkslslspg-x ₂ - VSDVPRDLEVVAATPTSLLISWDAPAAGYGYYR ITYGETGGNSPVQEFTVPVSKGTATISGLKPGV DYTITVYAVEFPFPGSGYYHRPISINYRT-Хз 84 Fc- 2451B05 fusion protein XI is selected from the hinge region sequences SEQ ID NO: 317-321; X2 is selected from the linker sequences of SEQ ID NOs: 310-314 and 620-634; X3 is optional and, if present, may be a C-terminal tail sequence selected from Х1- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYT qkslslspg-x ₂ - VSDVPRDLEVVAATPTSLLISWDAPAGGYGYYR ITYGETGGNSPVQEFTVPSSKGTATISGLKPGV DYTITVYAVEFDYPGSGYYHRPISINYRT-Хз

- 68 044608

sequences SEQ ID NO: 380-395 and EIEK (SEQ ID NO: 635) 85 Fc- 2451B06 fusion protein (equivalent to 2382D05) XI is selected from the hinge region sequences SEQ ID NO: 317-321; X2 is selected from the linker sequences of SEQ ID NOs: 310-314 and 620-634; X3 is optional and, if present, may be a C-terminal tail sequence selected from SEQ ID NO: 380-395 and EIEK (SEQ ID NO: 635) Xi- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYT qkslslspg-x ₂ - VSDVPRDLEVVAATPTSLLISWDAPADGGYGYY RITYGETGGNSPVQEFTVPVSKGTATISGLKPG VDYTITVYAVEFDFPGAGYYHRPISINYRT-Xs 86 Fc- 2451C06 fusion protein XI is selected from the hinge region sequences SEQ ID NO: 317-321; X2 is selected from the linker sequences of SEQ ID NOs: 310-314 and 620-634; X3 is optional and, if present, may be a C-terminal tail sequence selected from SEQ ID NO: 380-395 and EIEK (SEQ ID NO: 635) Xi- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYT qkslslspg-x ₂ - VSDVPRDLEVVAATPTSLLISWDAPAGAASYGY YRITYGETGGNSPVQEFTVPVSKGTATISGLKP GVDYTITVYAVEFPFPGAGYYHRPISINYRTХз

- 69 044608

87 Fc- 2451D05 fusion protein XI is selected from the hinge region sequences SEQ ID NO: 317-321; X2 is selected from the linker sequences of SEQ ID NOs: 310-314 and 620-634; X3 is optional and, if present, may be a C-terminal tail sequence selected from SEQ ID NO: 380-395 and EIEK (SEQ ID NO: 635) Х1- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYT qkslslspg-x ₂ - VSDVPRDLEVVAATPTSLLISWDAPAGAYGYYR ITYGETGGNSPVQEFTVPVSKGTATISGLKPGV DYTITVYAVEFDFPGSGYYHRPISINYRT-Хз 88 Fc- 2451F03 fusion protein XI is selected from the hinge region sequences SEQ ID NO: 317-321; X2 is selected from the linker sequences of SEQ ID NOs: 310-314 and 620-634; X3 is optional and, if present, may be a C-terminal tail sequence selected from SEQ ID NO: 380-395 and EIEK (SEQ ID NO: 635) Х1- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYT qkslslspg-x ₂ - VSDVPRDLEVVAATPTSLLISWDPPAEGYGYYR ITYGETGGNSPVQEFTVPVSKGTATISGLKPGV DYTITVYAVEFNFPGSGYYHRPISINYRT-Хз 89 Fc- 2451 GO 1 fusion protein XI is selected from the hinge region sequences SEQ ID X1- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV

- 70 044608

NO: 317-321; X2 is selected from the linker sequences of SEQ ID NOs: 310-314 and 620-634; X3 is optional and, if present, may be a C-terminal tail sequence selected from SEQ ID NO: 380-395 and EIEK (SEQ ID NO: 635) LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT qkslslspg-x ₂ - VSDVPRDLEVVAATPTSLLISWDAPAGGYGYYR ITYGETGGNSPVQEFTVPSSKGTATISGLKPGV DYTITVYAVEFDFPGSGYYHRPISINYRT-Хз 90 Fc- 2451H07 fusion protein XI is selected from the hinge region sequences SEQ ID NO: 317-321; X2 is selected from the linker sequences of SEQ ID NOs: 310-314 and 620-634; X3 is optional and, if present, may be a C-terminal tail sequence selected from SEQ ID NO: 380-395 and EIEK (SEQ ID NO: 635) Х1- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYT qkslslspg-x ₂ - ITDVPRDLEVVAATPTSLLISWNPPDVNYGYYR ITYGETGGNSPLQEFTVPVSKGTATISGLKPGV DYTITVYAVEYPYAHAGYYHRPISINYRT-Хз 91 Fc- 2382E03 fusion protein XI is selected from the hinge region sequences SEQ ID NO: 317-321; X2 is selected from linker sequences SEQ X1- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF

- 71 044608

ID NO: 310-314 and 620-634; X3 is optional and, if present, may be a C-terminal tail sequence selected from SEQ ID NO: 380-395 and EIEK (SEQ ID NO: 635) FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPG-X ₂ - VSDVPRDLEVVAATPTSLLISWDAPAGDGYGYY RITYGETGGNSPVQEFTVPVSKGTATISGLKPG VDYTITVYAVEFDFPGAGYYHRPISINYRT-Хз 92 Fc- 2382E04 fusion protein XI is selected from the hinge region sequences SEQ ID NO: 317-321; X2 is selected from the linker sequences of SEQ ID NOs: 310-314 and 620-634; X3 is optional and, if present, may be a C-terminal tail sequence selected from SEQ ID NO: 380-395 and EIEK (SEQ ID NO: 635) X1- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYT QKSLSLSPG-Xz- VSDVPRDLEVVAATPTSLLISWDAPAGGGYGYY RITYGETGGNSPVQEFTVPVSKGTATISGLKPG VDYTITVYAVEFTFPGAGYYHRPISINYRT-Xs 93 Fc- 2382E05 fusion protein XI is selected from the hinge region sequences SEQ ID NO: 317-321; X2 is selected from the linker sequences of SEQ ID NOs: 310-314 and 620-634; HZ is optional and, if Х1- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPG-X ₂ - VSDVPRDLEVVAATPTSLLISWDAPAEGGYGYY

- 72 044608

present, may be a C-terminal tail sequence selected from SEQ ID NO: 380-395 and EIEK (SEQ ID NO: 635) RITYGETGGNSPVQEFTVPVSKGTATISGLKPG VDYTITVYAVEFDFPGAGYYHRPISINYRT-Хз 94 Fc- 2382E09 fusion protein XI is selected from the hinge region sequences SEQ ID NO: 317-321; X2 is selected from the linker sequences of SEQ ID NOs: 310-314 and 620-634; X3 is optional and, if present, may be a C-terminal tail sequence selected from SEQ ID NO: 380-395 and EIEK (SEQ ID NO: 635) Х1- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYT qkslslspg-x ₂ - VSDVPRDLEVVAATPTSLLISWDAPAEAYGYYR ITYGETGGNSPVQEFTVPVSKGTATISGLKPGV DYTITVYAVEYDFPGSGYYHRPISINYRT-Хз 95 Fc- 2381A04 fusion protein XI is selected from the hinge region sequences SEQ ID NO: 317-321; X2 is selected from the linker sequences of SEQ ID NOs: 310-314 and 620-634; X3 is optional and, if present, may be the C-terminal tail sequence, Х1- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYT qkslslspg-x ₂ - VSDVPRDLEVVAATPTSLLISWEPFSRLPGGGE YYRITYGETGGNSPLQQFTVPGSKGTATISGLK PGVDYTITVYAVEYPYPFSGYYHRPISINYRTХз

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selected from the sequences SEQ ID NO: 380-395 and EIEK (SEQ ID NO: 635) 96 Fc- 2381A08 fusion protein XI is selected from the hinge region sequences SEQ ID NO: 317-321; X2 is selected from the linker sequences of SEQ ID NOs: 310-314 and 620-634; X3 is optional and, if present, may be a C-terminal tail sequence selected from SEQ ID NO: 380-395 and EIEK (SEQ ID NO: 635) Х1- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYT qkslslspg-x ₂ - VSDVPRDLEVVAATPTSLLISWDAPADGGYGYY RITYGETGGNSPVQEFTVPGSKGTATISGLKPG VDYTITVYAVEYDFPGAGYYHRPISINYRT-Xs 97 Fc- 2381B10 fusion protein XI is selected from the hinge region sequences SEQ ID NO: 317-321; X2 is selected from the linker sequences of SEQ ID NOs: 310-314 and 620-634; X3 is optional and, if present, may be a C-terminal tail sequence selected from SEQ ID NOs: 380-395 and EIEK Х1- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYT qkslslspg-x ₂ - VSDVPRDLEVVAATPTSLLISWDAPAGGYGYY RITYGETGGNSPVQEFTVPVSKGTATISGLKPG VDYTITVYAVEYNFIGAGYYHRPISINYRT-Xs

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(SEQ ID NO: 635) 98 Fc2381C08 fusion protein XI is selected from the hinge region sequences SEQ ID NO: 317-321; X2 is selected from the linker sequences of SEQ ID NOs: 310-314 and 620-634; X3 is optional and, if present, may be a C-terminal tail sequence selected from SEQ ID NO: 380-395 and EIEK (SEQ ID NO: 635) Х1- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYT qkslslspg-x ₂ - VSDVPRDLEVVAATPTSLLISWDAPADGAYGYY RITYGETGGNSPVQEFTVPVSKGTATISGLKPG VDYTITVYAVEFPYPFAGYYHRPISINYRT-Хз 99 Fc- 2381G06 fusion protein XI is selected from the hinge region sequences SEQ ID NO: 317-321; X2 is selected from the linker sequences of SEQ ID NOs: 310-314 and 620-634; X3 is optional and, if present, may be a C-terminal tail sequence selected from SEQ ID NO: 380-395 and EIEK (SEQ ID NO: 635) Х1- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYT qkslslspg-x ₂ - VSDVPRDLEVVAATPTSLLISWSEKLDGKARRG YYRITYGETGGNSPVQQFTVPGSKGTATISGLK PGVDYTITVYAVEFPYDHSGYYHRPISINYRTХз 00 Fc- 2381N01 XI is selected from sequences X1- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE

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fusion protein hinge region SEQ ID NO: 317-321; X2 is selected from the linker sequences of SEQ ID NOs: 310-314 and 620-634; X3 is optional and, if present, may be a C-terminal tail sequence selected from SEQ ID NO: 380-395 and EIEK (SEQ ID NO: 635) VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT qkslslsp g-x ₂ - VSDVPRDLEVVAATPTSLLISWSPRDSTGLVRR GYYRITYGETGGNSPVQQFTVPGSKGTATISGL KPGVDYTITVYAVEYPYDHSGYYHRPISINYRT -Хз 01 Fc- 2381H06 fusion protein XI is selected from the hinge region sequences SEQ ID NO: 317-321; X2 is selected from the linker sequences of SEQ ID NOs: 310-314 and 620-634; X3 is optional and, if present, may be a C-terminal tail sequence selected from SEQ ID NO: 380-395 and EIEK (SEQ ID NO: 635) Х1- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYT qkslslspg-x ₂ - VSDVPRDLEVVAATPTSLLISWGDVRTNEARQG YYRITYGETGGNSPLQGFTVPGSKGTATISGLK PGVDYTITVYAVEYTYEHSGYYHRPISINYRTХз 02 Fc- 2381H09 fusion protein XI is selected from the hinge region sequences SEQ ID NO: 317-321; X2 is selected from the linker X1- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG

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sequences SEQ ID NO: 310-314 and 620-634; X3 is optional and, if present, may be a C-terminal tail sequence selected from SEQ ID NO: 380-395 and EIEK (SEQ ID NO: 635) FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT qkslslspg-x ₂ - VSDVPRDLEVVAATPTSLLISWDAPAGGGYGYY RITYGETGGNSPVQEFTVPVSKGTATISGLKPG VDYTITVYAVEFDFVGAGYYHRPISINYRT-Xs 03 Fc- 2382B11 merged th protein XI is selected from the hinge region sequences SEQ ID NO: 317-321; X2 is selected from the linker sequences of SEQ ID NOs: 310-314 and 620-634; X3 is optional and, if present, may be a C-terminal tail sequence selected from SEQ ID NO: 380-395 and EIEK (SEQ ID NO: 635) Х1- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYT qkslslspg-x ₂ - VSDVPRDLEVVAATPTSLISWDAPAAAYGYYR ITYGETGGNSPVQEFTVPVSKGTATISGLKPGV DYTITVYAVEYDFAGSGYYHRPISINYRT-Хз 04 Fc- 2382B08 fusion protein XI is selected from the hinge region sequences SEQ ID NO: 317-321; X2 is selected from the linker sequences of SEQ ID NOs: 310-314 and 620-634; HZ is Х1- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYT qkslslspg-x ₂ -

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optional and, if present, may be a C-terminal tail sequence selected from SEQ ID NO: 380-395 and EIEK (SEQ ID NO: 635) VSDVPRDLEVVAATPTSLLISWDAPADAYGYYR ITYGETGGNSPVQEFTVPSSKGTATISGLKPGV DYTITVYAVEFAFPGAGYYHRPISINYRT-Xs 05 Fc- 2382C11 merged th protein XI is selected from the hinge region sequences SEQ ID NO: 317-321; X2 is selected from the linker sequences of SEQ ID NOs: 310-314 and 620-634; X3 is optional and, if present, may be a C-terminal tail sequence selected from SEQ ID NO: 380-395 and EIEK (SEQ ID NO: 635) Х1- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYT qkslslspg-x ₂ - VSDVPRDLEVVAATPTSLLISWDAPAGGYGYYR ITYGETGGNSPVQEFTVPVSKGTATISGLKPGV DYTITVYAVEYDFAGSGYYHRPISINYRT-Хз 06 Fc- 2382G03 fusion protein XI is selected from the hinge region sequences SEQ ID NO: 317-321; X2 is selected from the linker sequences of SEQ ID NOs: 310-314 and 620-634; CH is optional and, if present, may be C-terminal "tail" Х1- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYT qkslslspg-x ₂ - VSDVPRDLEVVAATPTSLLISWDAPAEAEAYGY YRITYGETGGNSPVQEFTVPVSKGTATISGLKP GVDYTITVYAVEYVFPGAGYYHRPISINYRT-

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sequence selected from SEQ IDNO: 380-395 and EIEK (SEQ IDNO: 635) Idk 07 Fc- 2382H03 fusion protein XI is selected from the hinge region sequences SEQ ID NO: 317-321; X2 is selected from the linker sequences of SEQ IDNO: 310-314 and 620-634; X3 is optional and, if present, may be a C-terminal tail sequence selected from SEQ IDNO: 380-395 and EIEK (SEQ IDNO: 635) Х1- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYT qkslslspg-x ₂ - VSDVPRDLEVVAATPTSLLISWDAPAEGAYGYY RITYGETGGNSPVQEFTVPVSKGTATISGLKPG VDYTITVYAVEYPYPFAGYYHRPISINYRT-Хз 08 Fc- 2451A10 fusion protein XI is selected from the hinge region sequences SEQ ID NO: 317-321; X2 is selected from the linker sequences of SEQ IDNO: 310-314 and 620-634; X3 is optional and, if present, may be a C-terminal tail sequence selected from SEQ Х1- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYT qkslslspg-x ₂ - VTDVPRDMEVVAATPTSLLISWQPPAVTYGYYR ITYGETGGNSTLQQFTVPVYKGTATISGLKPGV DYTITVYAVEYPYDHSGYYHRPISINYRT-Хз

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ID NO: 380-395 and EIEK (SEQ ID NO: 635) 09 Fc- 2451B02 fusion protein XI is selected from the hinge region sequences SEQ ID NO: 317-321; X2 is selected from the linker sequences SEQ ID NO: 310-314 and 620-634; X3 is optional and, if present, may be a C-terminal tail sequence selected from SEQ ID NO: 380-395 and EIEK (SEQ ID NO: 635) Xi- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYT qkslslspg-x ₂ - VSDVPRDLEVVAATPTSLISWDAPAAAYGYYR ITYGETGGNSPVQEFTVPVSKGTATISGLKPGV DYTITVYAVEFDYPGSGYYHRPISINYRT-Хз 10 Fc- 2451C11 fusion protein XI is selected from the hinge region sequences SEQ ID NO: 317-321; X2 is selected from the linker sequences of SEQ ID NOs: 310-314 and 620-634; X3 is optional and, if present, may be a C-terminal tail sequence selected from SEQ ID NO: 380-395 and EIEK (SEQ ID NO: 635) Xi- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYT qkslslspg-x ₂ - IVDVPRDLEVVAATPTSLLISWDPPAGAYGYYR ITYGETGGNSPKQQFTVPGYKGTATISGLKPGV DYTITVYAVEYPYDHSGYYHRPISINYRT-Xs Fc- XI is selected from Xi-

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eleven 2451H01 fusion protein hinge region sequences SEQ ID NO: 317-321; X2 is selected from the linker sequences of SEQ ID NOs: 310-314 and 620-634; X3 is optional and, if present, may be a C-terminal tail sequence selected from SEQ ID NO: 380-395 and EIEK (SEQ ID NO: 635) VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYT qkslslspg-x ₂ - VSDVPRDLEVVAATPTSLLISWDAPAAGYGYYR ITYGETGGNSPVQEFTVPVSKGTATISGLKPGV DYTITVYAVEYDFPGSGYYHRPISINYRT-Хз 12 Fc- 2011B11 fusion protein XI is selected from the hinge region sequences SEQ ID NO: 317-321; X2 is selected from the linker sequences of SEQ ID NOs: 310-314 and 620-634; X3 is optional and, if present, may be a C-terminal tail sequence selected from SEQ ID NO: 380-395 and EIEK (SEQ ID NO: 635) Xi- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYT qkslslspg-x ₂ - VSDVPRDLEVVAATPTSLLISWAPPSDAYGYYR IT YGE T GGN S PVQ E FTVPIGKGTATIS GL KPGV DYTITVYAVEYPYSHAGYYHRPISINYRT-Хз 13 Fc- 2971A03 fusion protein XI is selected from the hinge region sequences SEQ ID NO: 317-321; X2 Xi- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK

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selected from linker sequences SEQ ID NO: 310-314 and 620-634; X3 is optional and, if present, may be a C-terminal tail sequence selected from SEQ ID NO: 380-395 and EIEK (SEQ ID NO: 635) AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT qkslslspg-x ₂ - VSDVPRDLEVVAATPTSLLISWDPPSDDYGYYR IT YGE T GGN S PVQ E FTVPIGKGTATIS GL KPGV DYTITVYAVEFPWPHAGYYHRPISINYRT-Хз 14 Fc- 2971A09 fusion protein XI is selected from the hinge region sequences SEQ ID NO: 317-321; X2 is selected from the linker sequences of SEQ ID NOs: 310-314 and 620-634; X3 is optional and, if present, may be a C-terminal tail sequence selected from SEQ ID NO: 380-395 and EIEK (SEQ ID NO: 635) Xi- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVD KSRWQQGNVFSCSVMHEALHNHYT qkslslspg-x ₂ - VSDVPRDLEVVAATPTSLLISWDAPADDYGYYR IT YGE T GGN S PVQ E FTVPIGKGTATIS GL KPGV DYTITVYAVEFPWPHAGYYHRPISINYRT-Хз 15 Fc- 2971E02 fusion protein XI is selected from the hinge region sequences SEQ ID NO: 317-321; X2 is selected from the linker sequences of SEQ ID NOs: 310-314 and 620-634; Xi- VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYT X3 is optional and, if present, may be a C-terminal tail sequence selected from SEQ ID NO: 380-395 and EIEK (SEQ ID NO: 635) qkslslspg-x ₂ - VSDVPRDLEVVAATPTSLLISWDAPSDDYGYYR IT YGE T GGN S PVQ E FTVPIGKGTATIS GL KPGV DYTITVYAVEFPWPHAGYYHRPISINYRT-Хз

In some embodiments, aHTU-PCSK9 adnectin contains an Fn3 domain and a PK group. In some embodiments, the Fn3 domain is Fn3 domain ¹⁰ . In some embodiments, the RK group increases the half-life of the peptide in serum by more than 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 150, 200, 400, 600, 800, 1000% or more relative to the Fn3 domain without the PK group.

In some embodiments, the RK group is a polymer sugar. In some embodiments, the RA group is polyethylene glycol. In some embodiments, the PK group is human serum albumin. In some embodiments, the PK group is a serum immunoglobulin-binding protein. In some versions

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The RK group is transferrin.

In some embodiments, the PK group is another adnectin that is specific to a whey protein, such as HSA. Disclosed herein are the specific serum albumin binding molecules adnectin (or SABA). In certain embodiments, the sequence of the PCSK9 adnectin SABA fusion protein may be generally represented as the PCSK9 adnectin X ₂ -X3-X4 core SABA-X ₅ (SEQ ID NO: 618) or the PCSK9 core 8ABA-X ₂ -X ₃ -X ₄ core part of PCSK9 adnectin-X ₅ (SEQ ID NO: 619), where X ₃ and X ₄ are optional N-terminal extensions, X ₂ and X ₅ are optional C- terminal extensions, X ₃ is a linker. In one embodiment, the adnectins (either PCK9 or serum albumin binding) of SEQ ID NOs: 618 and 619 include the core region of adnectin, i.e. the core sequence of PCSK9 adnectin can be any of the PCSK9 adnectin sequences presented in table. 4, in which the sequence data begins with an amino acid residue corresponding to residue E8 of SEQ ID NO: 1 and ends with an amino acid residue corresponding to residue T94 of SEQ ID NO: 1. The SABA core sequence may be selected from any of the SABA core sequences presented in table 6.

In some embodiments, X ₃ and X ₄ are independently optional and, if present, are independently selected from the sequences of SEQ ID NOs: 371-379 given in table. 6. These sequences may contain an M, G or MG sequence at the N-terminus if such amino acid residues are not included in the sequence. For expression in mammalian cells, the fusion proteins may also contain at the N-terminus a leader signal sequence, such as METDTLLLWVLLLWVPGSTG (SEQ ID NO: 326). In some embodiments, X ₂ and X ₅ are independently optional and, if present, are independently selected from the sequences of SEQ ID NO: 380-395 given in table. 6. In certain embodiments, X ₃ is a linker sequence selected from the sequences of SEQ ID NO: 396-419 given in table. 6. Sequences in table. 2 are examples of anti-PC8K9 and SABA fusion proteins. It should be understood that any PCSK9 adnectin and SABA sequence described herein may be included in such fusion proteins.

table 2

Example sequences of the PCSK9 adnectin-SABA fusion protein

EQ ID Clone or title Description Subsequence 16 PCSK9 AdnectinSABA fusion protein The PCSK9 Adnectin sequence is underlined; the SABA sequence is shown in bold. The Adnectin PCSK9 sequence represents the core region from clone 2013E01; X1- EVVAATPTSLLISWVPPSDDYGYYRITY GETGGNSPVQEFTVPIGKGTATISGLKP GVDYTITVYAVE FPWPHAGYYHRPISIN YRT-X2-X3-X4- EWAATPTSLLISWHSYYEQNSYYRITY GETGGNSPVQEFTVPYSQTTATISGLKP GVDYTITVYAVYGSKYYYPISINYRT- X5

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SABA sequence taken from SABA 1 (SEQ ID NO: 330) 17 SABA- PCSK9 Adnectin fusion protein The SABA sequence is in bold, the PCSK9 Adnectin sequence is underlined. The SABA sequence is taken from SABA 1 (SEQ ID NO: 330); Adnectin PCSK9 sequence represents the core region from clone 2013E01 Χι- Ε WAATPTSLLISWH SYYEQNS YYRIΤΥ GETGGNSPVQEFTVPYSQTTATISGLKP GVDYTITVYAVYGSKYYYPISINYRT- Х ₂ -Хз-Х ₄ - EVVAATPTSLLISWVPPSDDYGYYRITY GETGGNSPVQEFTVPIGKGTATISGLKP GVDYTITVYAVEFPWPHAGYYHRPISIN YRT~X ₅

Biophysical and biochemical characteristics.

This application describes adnectin containing an Fn3 domain that binds PCSK9. The binding of a polypeptide to a target molecule can be assessed using equilibrium constants (eg, dissociation constant, KD) and kinetic constants (eg, forward and reverse reaction rate constants, K _on and K _off , respectively). Adnectin typically binds to the target molecule with a KD of less than 500, 100, 10, 1 nM, 500, 200, 100 pM. However, a higher KD may be acceptable if Koff is _low enough or _Kon high enough.

The sequences of the BC, DE and FG loops of the anti-PCSK9 adnectins (SEQ ID NO) described in the present invention are presented in table. 3.

Table 3

Loops BC, DE and FG of anti-PCSK9 adnectins

Clone ID NITY (Kp, nM) EU SK9- EGFA FRET (EC™.nM) Reduced quantity LDLR (% inhibition Sun loop S.E. QIDNO Loop I'm DE SEQ ID NO Hinge FG SEQ ID NO 1459D05 14.4f 1.58* 26 ^L 4 66.8, >200 SWPPPSH GYG 2 PPG KGT 18 EYPYKHSGY YHRP 28 1784F03 3.8 ^L 2 150.2, 26±13 SWRPPIH AYG 3 PIV EGT 19 EYTFKHSGY YHRP 29 1784F03 -ml nd nd VD, >2000 SWDAPIH AYG 4 P.G.S. EGT 20 EYTFKHSGY YHRP 29 1784F03 -m2 ND ND VD, >2000 SWDAPAH AYG 5 P.G.S. KGT 21 EYTFKHSGY YHRP 29 1784F03 -m3 ND ND VD, >2000 SWDAPAV TYG 6 P.G.S. KST 22 EYTFKHSGY YHRP 29 1813E02 < ₂ L 1.3 nd,16 SWSPPAN GYG 7 PVG RGT 23 EYTYKGSGY YHRP thirty 1923B02 0.173* 2.3 178.0, 23±7 SWTPPPK GYG 8 PVG EGT 24 EYTYNGAGY YHRP 31

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1923B02 (N82I) nd nd nd, 14 SWTPPPK GYG 8 PVG EGT 24 EYTYIGAGY YHRP 32 1923B02 (N82E) nd ND nd, 28 SWTPPPK GYG 8 PVG EGT 24 EYTYEGAGY YHRP 33 1923B02 (T80A) nd ND nd, 42 SWTPPPK GYG 8 PVG EGT 24 EYAYNGAGY YHRP 34 1922G04 0.09* 1.2 105.1, 10±2 SWRPPSH AYG 9 PIG KGT 25 EYPWKGSGY YHRP 35 1922G04 (R25D) nd 2.5 nd, 29±8 SWDPPSH AYG 10 PIG KGT 25 EYPWKGSGY YHRP 35 1922G04 (R25E) nd 3.5 VD, 29±18 SWEPPSH AYG eleven PIG KGT 25 EYPWKGSGY YHRP 35 1922G04 (R25S) nd nd nd, 21 SWSPPSH AYG 12 PIG KGT 25 EYPWKGSGY YHRP 35 2012A04 0.25* 2.1 144.5, 12±6 SWRPPSN GHG 13 PVN EGT 26 EFPFKWSGY YHRP 36 2013E01 E51| 0.29* 1.6 165.5, 10±4 SWVPSD DYG 14 PIG KGT 25 EFPWPHAGY YHRP 37 2011H05 0.08* 2.7 197.6, 12±5 SWVPSSH AYG 15 PVG VGT 27 EYAFEGAGY YHRP 38 2011H05 (V23D) nd 5.5 nd, 18±3 SWDPSSH AYG 16 PVG VGT 27 EYAFEGAGY YHRP 38 2011H05 (V23E) nd 7.4 nd, 12±3 SWEPSH AYG 17 PVG VGT 27 EYAFEGAGY YHRP 38 2381B02 (1) 3.29f 2.5 125.4, n.d. SWEPFSR LPGGGE 10 6 P.G.S. KGT 21 EYPYDYSGY YHRP 142 2381B04 (1) 0.527f 2.4 121.6, n.d. SWEPFSR LPGGGE 10 6 P.G.S. KGT 21 EYPYEHSGY YHRP 143 2381B06 (1) ND 3.5 I9.7, nd SWEPFSR LPGGGE 10 6 P.G.S. KGT 21 EYPYPHSGY YHRP 144 2381B08 4.Ilf 2.6 124.8, n.d. SWDAPAD GGYG 10 7 P.S.S. KGT 136 EYTFPGAGY YHRP 145 2381D02 (1) nd 3.1 185.0, n.d. SWEPFSR LPGGGE 10 6 P.G.S. KGT 21 EYPYDHSGY YHRP 146 2381D04 (1) 0.237f 2.9 119.2, n.d. SWEPFSR LPGGGE 10 6 P.G.S. KGT 21 EFPYDHSGY YHRP 147 2381F11 E59f 4 110.2, n.d. SWDAPAD GGYG 10 7 PVS KST 137 EYTFPGAGY YHRP 145 2381G03 (1) nd 3.4 70.2, n.d. SWEPFSR LPGGGE 10 6 P.G.S. KGT 21 EFPYAHSGY YHRP 148 2381G09 E12f 3.1 133.0, n.d. SWDAPAG DGYG 10 8 PVS KGT 138 EFTFPGAGY YHRP 149 2381H03 (1) nd 3.4 89.8, n.d. SWEPFSR LPGGGE 10 6 P.G.S. KGT 21 EYPYAHSGY FHRP 150 2382A01 nd 12. 9 119.8, n.d. SWAAPAG GGYG 10 9 PVS KGT 138 EYDFPGAGY YHRP 151 2382B10 2.35f 3 100.2, n.d. SWDAPAD AYG eleven 0 P.S.S. KGT 136 EYDFPGSGY YHRP 152

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2382B09 0.656f 3.8 105.0, n.d. SWDAPAD AYG 0 eleven PVS KGT 138 EFDYPGSGY YHRP 153 2382C05 2.49f 4 105.3, n.d. SWDAPAD GAYG 1 eleven PVS KGT 138 EYSFPGAGY YHRP 154 2382C09 0.757f 3.5 121.7, n.d. SWDAPAE G.Y.G. 2 eleven PVS KGT 138 EFDFPGSGY YHRP 155 2382D03 1.53f 3.3 80.4, n.d. SWDAPAD EAYG 3 eleven PVS KGT 138 EFDFPGAGY YHRP 156 2382D05 0.3141 2.6 140.5, n.d. SWDAPAD GGYG 7 10 PVS KGT 138 EFDFPGAGY YHRP 156 2382D08 VD 3.1 106.6, n.d. SWDAPAD GYG 4 eleven PVS KGT 138 EFPFPGSGY YHRP 157 2382D09 0.304f 2.6 109.1, n.d. SWDAPAE GYG 2 eleven PVS KGT 138 EFDFPGAGY YHRP 156 2382F02 VD 2.6 -6.3, nd SWDAPAG GGYG 5 eleven PVS KGT 138 EFDFPGSGY YHRP 155 2382F03 ND 2.7 88.6, n.d. SWDAPAA DAYG 6 eleven PVS KGT 138 EFNFPGAGY YHRP 158 2382F05 4.54f 2.4 72.2, n.d. SWDAPAE AGKHYG 7 eleven PVS KGT 138 EFDFPGAGY YHRP 156 2382F08 VD 2.5 105.0, n.d. SWDAPAE AYG 8 eleven PVS KGT 138 EFTYPGSGY YHRP 159 2382F09 VD 3.1 109.7, n.d. SWDAPAA AYG 9 eleven PVS KGT 138 EYDFPGSGY YHRP 152 2382G04 lilt 2.9 146.1, n.d. SWDAPAG GGYG 5 eleven P.S.S. KGT 136 EFDFPGAGY YHRP 156 2382H10 1.40f 2.6 118.6, n.d. SWDAPAG GYG 0 12 PVS KGT 138 EFDFPGSGY YHRP 155 2382H11 VD 2.9 117.2, n.d. SWDAPAD GYG 4 eleven PVF KGT 139 EFDYPGSGY YHRP 153 2382H04 ND 3.2 68.2, n.d. SWDAPAA GGYG 1 12 P.S.S. KGT 136 EYDFPGAGY YHRP 151 2382H07 ND 2.7 86.2, n.d. SWDAPAD AYG 0 eleven P.G.S. KGT 21 EFDFPGSGY YHRP 155 2382H09 1.86t 0.9 101.2, n.d. SWDAPAA AYG 9 eleven P.S.S. KGT 136 EFDFPGSGY YHRP 155 2451A02 ND 3.2 106.4, n.d. SWDAPAA GYG 2 12 PVS KGT 138 EFPFPGSGY YHRP 157 2451B05 ND 6.3 91.7, n.d. SWDAPAG GYG 0 12 P.S.S. KGT 136 EFDYPGSGY YHRP 153 2451B06 ND 4.5 92.2, n.d. SWDAPAD GGYG 7 10 PVS KGT 138 EFDFPGAGY YHRP 156 2451C06 1.27f 1.2 89.4, n.d. SWDAPAG AASYG 3 12 PVS KGT 138 EFPFPGAGY YHRP 160 2451D05 ND 2.8 115.0, n.d. SWDAPAG AYG 4 12 PVS KGT 138 EFDFPGSGY YHRP 155 2451F03 ND 2.8 113.2, n.d. SWDPPAE GYG 5 12 PVS KGT 138 EFNFPGSGY YHRP 161

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2451 GO 1 nd 3.8 90.8, n.d. SWDAPAG GYG 0 12 P.S.S. KGT 136 EFDFPGSGY YHRP 155 2451Н07 (2) 2.08f 0.2 88.8, n.d. SWNPPDV NYG 6 12 PVS KGT 138 EYPYAHAGY YHRP 162 2382E03 2.94| 2.4 89.5, n.d. SWDAPAG DGYG 8 10 PVS KGT 138 EFDFPGAGY YHRP 156 2382E04 nd 3 61.5, n.d. SWDAPAG GGYG 5 eleven PVS KGT 138 EFTFPGAGY YHRP 149 2382E05 0.604f 2.8 103.5, n.d. SWDAPAE GGYG 7 12 PVS KGT 138 EFDFPGAGY YHRP 156 2382E09 nd 6.2 97.2, n.d. SWDAPAE AYG 8 eleven PVS KGT 138 EYDFPGSGY YHRP 152 2381A04 (1) nd 3.3 100.1, n.d. SWEPFSR LPGGGE 6 10 P.G.S. KGT 21 EYPYPFSGY YHRP 163 2381A08 nd 3.6 91.4, n.d. SWDAPAD GGYG 7 10 P.G.S. KGT 21 EYDFPGAGY YHRP 151 2381В10 nd 7.3 96.4, n.d. SWDAPAG GGYG 5 eleven PVS KGT 138 EYNFIGAGY YHRP 164 2381С08 ND 0.7 15.3, n.d. SWDAPAD GAYG 1 eleven PVS KGT 138 EFPYPFAGY YHRP 165 2381G06 (3) ND 9 57.7, n.d. SWSEKLD GKARRG 8 12 P.G.S. KGT 21 EFPYDHSGY YHRP 147 2381N01 (3) ND 4 22.2, n.d. SWSPRDS TGLVRRG 9 12 P.G.S. KGT 21 EYPYDHSGY YHRP 146 2381N06 (4) ND 5 53.4, n.d. SWGDVRT NEARQG 0 13 P.G.S. KGT 21 EYTYEHSGY YHRP 166 2381Н09 3.23f 3.4 94.4, n.d. SWDAPAG GGYG 5 eleven PVS KGT 138 EFDFVGAGY YHRP 167 2382B11 nd 2.9 88.8, n.d. SWDAPAA AYG 9 eleven PVS KGT 138 EYDFAGSGY YHRP 168 2382В08 nd 2.9 107.2, n.d. SWDAPAD AYG 0 eleven P.S.S. KGT 136 EFAFPGAGY YHRP 169 2382С11 nd 3.7 82.9, n.d. SWDAPAG GYG 0 12 PVS KGT 138 EYDFAGSGY YHRP 168 2382G03 ND 2.7 77.8, n.d. SWDAPAE AEAYG 1 13 PVS KGT 138 EYVFPGAGY YHRP 170 2382N03 0.677| 3.4 102.1, n.d. SWDAPAE GAYG 2 13 PVS KGT 138 EYPYPFAGY YHRP 171 2451A10 (5) ND 10. 9 53.7, n.d. SWQPPAV TYG 3 13 PVY KGT 140 EYPYDHSGY YHRP 146 2451В02 ND 5.3 71.4, n.d. SWDAPAA AYG 9 eleven PVS KGT 138 EFDYPGSGY YHRP 153 2451С11 (6) ND 9.7 70.3, n.d. SWDPPAG AYG 4 13 P.G.Y. KGT 141 EYPYDHSGY YHRP 146 2451N01 nd 2.8 95.8, n.d. SWDAPAA GYG 2 12 PVS KGT 138 EYDFPGSGY YHRP 152 2011В11 nd 1.7 144.5, n.d. SWAPPSD AYG 5 13 PIG KGT 25 EYPYSHAGY YHRP 172 2971A03 0.806f nd 120.1, n.d. SWDPPSD DYG 1 thirty PIG KGT 25 EFPWPHAGY YHRP 37 2971A09 2.79| nd 132.3, n.d. SWDAPAD DYG 2 thirty PIG KGT 25 EFPWPHAGY YHRP 37 2971E02 1.781 VD 126.2, n.d. SWDAPSD DYG 3 thirty PIG KGT 25 EFPWPHAGY YHRP 37

t KD determined using Octet Red at 37°C;

*KD determined using ProteOn at 25°C;

^L KD determined using ITC at 37°C;

(1) In addition to the loop mutations, these clones also contain the V45L and E47Q mutations;

(2) In addition to the loop mutations, these clones also contain the V1I, S2T, and V45L mutations;

(3) In addition to the loop mutations, these clones also contain the E47Q mutation;

- 87 044608 (4) In addition to the loop mutations, these clones also contain the V45L and E47G mutations;

(5) In addition to the loop mutations, these clones also contain the S2T, L8M, P44T, V45L, and E47Q mutations;

(6) In addition to the loop mutations, these clones also contain the V1I, S2V, V45K, and E47Q mutations.

SEQ ID NO of the sequences of the anti-PCSK9 adnectin family described in the present invention are presented in table. 4

Table 4

Anti-PCSK9 adnectin family

Clone Subsequence Amino acid Nucleotide 1459D05 also designated AT1000891 or ATI-891 MGVSDVPRDLEVVAAT PTSLLISWPPPSHGYGYYRITY GETGGNSPVQEFTVPPGKGTAT ISGLKPGVDYTITVYAVEYPYK HSGYYHRPISINYRTEIDKPSQ NNNNNNN (SEQ ID NO:39) ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTG ATCAGCTGGCCGCCGCCGTCTCATGGTTACGGTTATTACCGCATCACTTACGGCGAAACAGGAGGCAA TAGCCCTGTCCAGGAGTTCACTGTGCCGCCTGGTAAAGGTACAGCTACCATCAGCGGCCTTAAACCTG GCGTTGATTATACCATCACTGTGTATGCTGTCGAATACC CGTACAAACATTCTGGTTACTACCATCGT CCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCACCACCACCAC (SEQ ID NQ:40) 1784F03 MGVSDVPRDLEVVAAT PTSLLISWRPPIHAYGYYRITY GETGGNSPVQEFTVPIVEGTAT ISGLKPGVDYTITVYAVEYTFK HSGYYHRPISINYRTEIDKPSQ HHHHHH (SEQ ID NO :41) ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCACCAGCCTGCTG ATCAGCTGGAGGCCGCCGATTCATGCTTACGGGTATTACCGCATCACTTACGGCGAAACAGGAGGCAA TAGCCCTGTCCAGGAGTTCACTGTGCCTATTGTTGAAGGTACAGCTACCATCAGCGGCCTTAAACCTG GCGTTGATTATACCATCACTGTGTATGCTGTCGAATA TACATTTAAACATTCCGGTTACTACCATCGT CCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCACCACCACCAC (SEQ ID NO:42) 1784F03- ml MGVSDVPRDLEVVAAT PTSLLISWDAPIHAYGYYRITY GETGGNSPVQEFTVPGSEGTAT ISGLKPGVDYTITVYAVEYTFK HSGYYHRPISINYRTEIDKPSQ HHHHHH (SEQ ID NO:43) ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCACAGCCTGCTG ATCAGCTGGGACGCTCCGATTCATGCTTACGGGTATTACCGCATCACTTACGGCGAAACAGGAGGCAA TAGCCCTGTCCAGGAGTTCACTGTGCCTGGTTCTGAAGGTACAGCTACCATCAGCGGCCTTAAACCTG GCGTTGATTATACCATCACTGTGTATGCTGTCGAATA TACATTTAAACATTCCGGTTACTACCATCGT CCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCACCACCACCAC (SEQ ID NO:44) 1784F03- m2 MGVSDVPRDLEVVAAT PTSLLISWDAPAHAYGYYRITY GETGGNSPVQEFTVPGSKGTAT ISGLKPGVDYTITVYAVEYTFK HSGYYHRPISINYRTEIDKPSQ HHHHHH (SEQ ID NO:45) ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCACCAGCCTGCTG ATCAGCTGGGACGCTCCGGCTCATGCTTACGGGTATTACCGCATCACTTACGGCGAAACAGGAGGCAA TAGCCCTGTCCAGGAGTTCACTGTGCCTGGTTCTAAAGGTACAGCTACCATCAGCGGCCTTAAACCTG GCGTTGATTATACCATCACTGTGTATGCTGTCGAATATA CATTTAAACATTCCGGTTACTACCATCGT CCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCACCACCACCAC (SEQ ID NO:46) 1784F03- m3 MGVSDVPRDLEVVAAT PTSLLISWDAPAVTYGYYRITY GETGGNSPVQEFTVPGSKSTAT ISGLKPGVDYTITVYAVEYTFK HSGYYHRPISINYRTEIDKPSQ HHHHHH (SEQ ID NO:47) ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTG ATCAGCTGGGACGCTCCGGCTGTTACTTACGGGTATTACCGCATCACTTACGGCGAAACAGGAGGCAA TAGCCCTGTCCAGGAGTTCACTGTGCCTGGTTCTAAATCTACAGCTACCATCAGCGGCCTTAAACCTG GCGTTGATTATACCATCACTGTGTATGCTGTCGAATATACAT TTAAACATTCCGGTTACTACCATCGT CCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCACCACCACCAC (SEQ ID NO:48) 1813E02 MGVSDVPRDLEVVAAT PTSLLISWSPPANGYGYYRITY GETGGNSPVQEFTVPVGRGTAT ISGLKPGVDYTITVYAVEYTYK GSGYYHRPISINYRTEIDKPSQ HHHHHH (SEQ ID NO :49) ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCACAGCCTGCTG ATCAGCTGGTCCCCACCGGCTAACGGTTACGGTTATTACCGCATCACTTACGGCGAAACAGGAGGCAA TAGCCTGTCCAGGAGTTCACTGTGCCTGTTGGTAGAGGTACAGCTACCATCAGCGGCCTTAAACCTG GCGTTGATTATACCATCACTGTGTATGCTGTCGAG TATACCTACAAAGGCTCTGGTTACTACCATCGC CCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCACCACCACCAC (SEQ ID NQ:50)

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1923B02 MGVSDVPRDLEVVAAT PTSLLISWTPPPKGYGYYRITY GETGGNSPVQEFTVPVGEGTAT ISGLKPGVDYTITVYAVEYTYN GAGYYHRPISINYRTEIDKPSQ HHHHHH (SEQ ID NO:51) ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCACAGCCTGCTG ATCAGCTGGACGCCTCCCCCTAAAGGGTATGGTTATTACCGCATCACTTACGGCGAAACAGGAGGCAA TAGCCCTGTCCAGGAGTTCACTGTGCCTGTTGGTGAAGGTACAGCTACCATCAGCGGCCTTAAACCTG GCGTTGATTATACCATCACTGTGTATGCTGTCGAATA CACGTACAACGGTGCCGGTTACTACCACCGG CCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCACCACCACCAC (SEQ ID NO:52) 1923B02( N82I) MGVSDVPRDLEVVAAT PTSLLISWTPPPKGYGYYRITY GETGGNSPVQEFTVPVGEGTAT ISGLKPGVDYTITVYAVEYTYI GAGYYHRPISINYRTGSGSHHH HHH (SEQ ID NO:53) ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTG ATCAGCTGGACGCCTCCCCCTAAAGGGTATGGTTATTACCGCATCACTTACGGCGAAACAGGAGGCAA TAGCCCTGTCCAGGAGTTCACTGTGCCTGTTGGTGAAGGTACAGCTACCATCAGCGGCCTTAAACCTG GCGTTGATTATACCATCACTGTGTATGCTGTCGAATACACGTACATTGGTGCCGGTTACTACCACCGG CCAATTCCATTAATTACCGCACAGGTAGCGGTTCCCACCATCACCACCATCAC (SEQ ID NO:54) 1923В02( N82E) MGVSDVPRDLEVVAAT PTSLLISWTPPPKGYGYYRITY GETGGNSPVQEFTVPVGEGTAT ISGLKPGVDYTITVYAVEYTYE GAGYYHRPISINYRTGSGSHHH HHH (SEQ ID NO:55) ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCACAGCCTGCTG ATCAGCTGGACGCCTCCCCCTAAAGGGTATGGTTATTACCGCATCACTTACGGCGAAACAGGAGGCAA TAGCCCTGTCCAGGAGTTCACTGTGCCTGTTGGTGAAGGTACAGCTACCATCAGCGGCCTTAAACCTG GCGTTGATTATACCATCACTGTGTATGCTGTCGAATA CACGTACGAAGGTGCCGGTTACTACCACCGG CCAATTTCCATTAATTACCGCACAGGTAGCGGTTCCCACCATCACCACCATCAC (SEQ ID NO:5 6) 1923В02( T80A) MGVSDVPRDLEVVAAT PTSLLISWTPPPKGYGYYRITY GETGGNSPVQEFTVPVGEGTAT ISGLKPGVDYTITVYAVEYAYN GAGYYHRPISINYRTGSGSHHH HHH (SEQ ID NO:57) ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTG ATCAGCTGGACGCCTCCCCCTAAAGGGTATGGTTATTACCGCATCACTTACGGCGAAACAGGAGGCAA TAGCCCTGTCCAGGAGTTCACTGTGCCTGTTGGTGAAGGTACAGCTACCATCAGCGGCCTTAAACCTG GCGTTGATTATACCATCACTGTGTATGCTGTCGAATACGCGTACAACGGTGCCGGTTACTACCACCGG CCAATTCCATTAATTACCGCACAGGTAGCGGTTCCCACCATCACCACCATCAC (SEQ ID NO:58) 1922G04 Also designated here's how ATyuO1057 or ATI-105 7 MGVSDVPRDLEVVAAT PTSLLISWRPPSHAYGYYRITY GETGGNSPVQEFTVPIGKGTAT ISGLKPGVDYTITVYAVEYPWK GSGYYHRPISINYRTEIDKPSQ HHHHHH (SEQ ID NO :59) ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCACAGCCTGCTG ATCAGCTGGCGGCCGCCATCTCATGCTTATGGTTATTACCGCATCACTTACGGCGAAACAGGAGGCAA TAGCCCTGTCCAGGAGTTCACTGTGCCTATTGGGAAAGGTACAGCTACCATCAGCGGCCTTAAACCTG GCGTTGATTATACCATCACTGTGTATGCTGTCGAATACCCG TGGAAAGGTTCTGGTTACTACCATCGG CCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCACCACCACCAC (SEQ ID NO:60) 1922G04( R25D) MGVSDVPRDLEVVAAT PTSLLISWDPPSHAYGYYRITY GETGGNSPVQEFTVPIGKGTAT ISGLKPGVDYTITVYAVEYPWK GSGYYHRPISINYRTGSGSHHH HHH (SEQ ID NO:61) ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTG ATCAGCTGGGACCCGCCATCTCATGCTTATGGTTATTACCGCATCACTTACGGCGAAACAGGAGGCAA TAGCCCTGTCCAGGAGTTCACTGTGCCTATTGGGGAAAGGTACAGCTACCATCAGCGGCCTTAAACCTG GCGTTGATTATACCATCACTGTGTATGCTGTCGAATACCCGTGGAAAGGTTCTGGTTACTACCATCGG CCAATTCCATTAATTACCGCACAGGTAGCGGTTCCCACCATCACCACCATCAC (SEQ ID NO:62) 1922G04( R25E) MGVSDVPRDLEVVAAT PTSLLISWEPPSHAYGYYRITY GETGGNSPVQEFTVPIGKGTAT ISGLKPGVDYTITVYAVEYPWK GSGYYHRPISINYRTGSGSHHH HHH (SEQ ID NO:63) ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTG ATCAGCTGGGAACCGCCATCTCATGCTTATGGTTATTACCGCATCACTTACGGCGAAACAGGAGGCAA TAGCCCTGTCCAGGAGTTCACTGTGCCTATTGGGGAAAGGTACAGCTACCATCAGCGGCCTTAAACCTG GCGTTGATTATACCATCACTGTGTATGCTGTCGAATACCCGTGGAAAGGTTCTGGTTACTACCATCGG CCAATTCCATTAATTACCGCACAGGTAGCGGTTCCCACCATCACCACCATCAC (SEQ ID NO:64) 1922G04( R25S) MGVSDVPRDLEVVAAT PTSLLISWSPPSHAYGYYRITY GETGGNSPVQEFTVPIGKGTAT ISGLKPGVDYTITVYAVEYPWK GSGYYHRPISINYRTGSGSHHH HHH (SEQ ID NO:65) ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTG ATCAGCTGGAGCCCGCCATCTCATGCTTATGGTTATTACCGCATCACTTACGGCGAAACAGGAGGCAA TAGCCCTGTCCAGGAGTTCACTGTGCCTATTGGGGAAAGGTACAGCTACCATCAGCGGCCTTAAACCTG GCGTTGATTATACCATCACTGTGTATGCTGTCGAATACCCGTGGAAAGGTTCTGGTTACTACCATCGG CCAATTCCATTAATTACCGCACAGGTAGCGGTTCCCACCATCACCACCATCAC (SEQ ID NO:66) 2012A04 MGVSDVPRDLEVVAAT PTSLLISWRPPSNGHGYYRITY GETGGNSPVQEFTVPVNEGTAT ISGLKPGVDYTITVYAVEFPFK WSGYYHRPISINYRTEIDKPSQ HHHHHH (SEQ ID NO :67) ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCACAGCCTGCTG ATCAGCTGGCGGCCCCCCTCTAATGGTCACGGTTATTACCGCATCACTTACGGCGAAACAGGAGGCAA TAGCCCTGTCCAGGAGTTCACTGTGCCTGTTAATGAAGGTACAGCTACCATCAGCGGCCTTAAACCTG GCGTTGATTATACCATCACTGTGTATGCTGTCGAATTC CCCTTCAAGTGGTCGGGCTACTACCATCGA CCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCACCACCACCAC (SEQ ID NO:68)

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2013E01 Also designated as ATI001081 or ATI-1081 MGVSDVPRDLEVVAAT PTSLLISWVPPSDDYGYYRITY GETGGNSPVQEFTVPIGKGTAT ISGLKPGVDYTITVYAVEFPWP HAGYYHRPISINYRTEIDKPSQ NNNNNNN (SEQ ID NO:69) ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCACAGCCTGCTG ATCAGCTGGGTCCCGCCTTCAGATGATTACGGTTATTACCGCATCACTTACGGCGAAACAGGAGGCAA TAGCCCTGTCCAGGAGTTCACTGTGCCTATTGGTAAAGGAACAGCTACCATCAGCGGCCTTAAACCTG GCGTTGATTATACCATCACTGTGTATGCTGTCGAGTT TCCGTGGCCACATGCTGGTTACTATCATCGG CCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCACCACCACCAC (SEQ ID NO:70) 2011Н05 Also designated as ATI001091 or ATI-1091 MGVSDVPRDLEVVAAT PTSLLISWVPSSHAYGYYRITY GETGGNSPVQEFTVPVGVGTAT ISGLKPGVDYTITVYAVEYAFE GAGYYHRPISINYRTEIDKPSQ HHHHHH (SEQ ID NO :71) ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCACCAGCCTGCTG ATCAGCTGGGTTCCGTCGTCTCATGCCTACGGTTATTACCGCATCACTTACGGCGAAACAGGAGGCAA TAGCCTGTCCAGGAGTTCACTGTGCCTGTGGGGGTAGGTACAGCTACCATCAGCGGCCTTAAACCTG GCGTTGATTATACCATCACTGTGTATGCTGTCGAATA CGCTTTCGAAGGGGCTGGTTACTACCATCGT CCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCACCACCACCAC (SEQ ID NO:72) 2011H05( V23D) MGVSDVPRDLEVVAAT PTSLLISWDPSSHAYGYYRITY GETGGNSPVQEFTVPVGVGTAT ISGLKPGVDYTITVYAVEYAFE GAGYYHRPISINYRTEIDKPSQ HHHHHH (SEQ ID NO:73) ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCACCAGCCTGCTG ATCAGCTGGGACCCGTCGTCTCATGCCTACGGTTATTACCGCATCACTTACGGCGAAACAGGAGGCAA TAGCCTGTCCAGGAGTTCACTGTGCCTGTGGGGGTAGGTACAGCTACCATCAGCGGCCTTAAACCTG GCGTTGATTATACCATCACTGTGTATGCTGTCGAATA CGCTTTCGAAGGGGCTGGTTACTACCATCGT CCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCACCACCACCAC (SEQ ID NO:74) 2011H05( V23E) MGVSDVPRDLEVVAAT PTSSLLISWEPSSHAYGYYRITY GETGGNSPVQEFTVPVGVGTAT ISGLKPGVDYTITVYAVEYAFE GAGYYHRPISINYRTEIDKPSQ HHHHHH (SEQ ID NO:75) ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCACCAGCCTGCTG ATCAGCTGGGAACCGTCGTCTCATGCCTACGGTTATTACCGCATCACTTACGGCGAAACAGGAGGCAA TAGCCTGTCCAGGAGTTCACTGTGCCTGTGGGGGTAGGTACAGCTACCATCAGCGGCCTTAAACCTG GCGTTGATTATACCATCACTGTGTATGCTGTCGAATA CGCTTTCGAAGGGGCTGGTTACTACCATCGT CCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCACCACCACCAC (SEQ ID NO:76) 2381В02 (SEQ ID NO:173) ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCACCAGCCTGCTG ATCAGCTGGGAGCCGTTCAGCCGGTTGCCCGGGGGCGGCGAGTATTACCGGATCACTTACGGCGAAAC AGGAGGCAATAGCCCTCTGCAGCAGTTCACTGTGCCTGGTTCTAAAGGTACAGCTACCATCAGCGGCC TTAAACCTGGCGTTGATTATACCATCACTGTGTATG CTGTCGAATACCCGTACGACTATTCTGGTTAC TACCATCGCCCCATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCACCACCA CCAC (SEQ ID NO :174) 2381В04 (SEQ ID NO:175) ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCACCAGCCTGCTG ATCAGCTGGGAGCCGTTCAGCCGGTTGCCCGGGGGCGGCGAGTATTACCGGATCACTTACGGCGAAAC AGGAGGCAATAGCCCTCTGCAGCAGTTCACTGTGCCTGGTTCTAAAGGTACAGCTACCATCAGCGGCC TTAAACCTGGCGTTGATTATACCATCACTGTGTATG CTGTCGAATACCCGTACGAGCATTCTGGGTAC TATCATCGTCCGATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCACCACCA CCAC (SEQ ID NO :176) 2381В06 (SEQ ID NO:177) ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCACCAGCCTGCTG ATCAGCTGGGAGCCGTTCAGCCGGTTGCCCGGGGGCGGCGAGTATTACCGGATCACTTACGGCGAAAC AGGAGGCAATAGCCCTCTGCAGCAGTTCACTGTGCCTGGTTCTAAAGGTACAGCTACCATCAGCGGCC TTAAACCTGGCGTTGATTATACCATCACTGTGTATG CTGTCGAATACCCGTACCCGCATTCTGGTTAC TACCATCGACCGATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCACCACCA CCAC (SEQ ID NO :178) 2381В08 MGVSDVPRDLEVVAAT PTSLLISWDAPADGGYGYYRIT YGETGGNSPVQEFTVPSSKGTA TISGLKPGVDYTITVYAVEYTF PGAGYYHRPISINYRTEIDKPS QHHHHHH (SEQ ID NO:179) ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCACAGCCTGCTG ATCAGCTGGGACGCTCCGGCTGATGGAGGGTACGGTTATTACCGCATCACTTACGGCGAAACAGGAGG CAATAGCCCTGTCCAGGAGTTCACTGTGCCTAGTTCTAAAGGTACAGCTACCATCAGCGGCCTTAAAC CTGGCGTTGATTATACCATCACTGTGTATGCTGTCGA ATACACCTTCCCGGGCGCCGGTTACTACCAT CGTCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCACCACCACCAC (SEQ ID NO:180) 2381D02 MGVSDVPRDLEVVAAT PTSLLISWEPFSRLPGGGEYYR ITYGETGGNSPLQQFTVPGSKG TATISGLKPGVDYTITVYAVEY PYDHSGYYHRPISINYRTEIDK PSQHHHHHH (SEQ ID NO:181) ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCACCAGCCTGCTG ATCAGCTGGGAGCCGTTCAGCCGGTTGCCCGGGGGCGGCGAGTATTACCGGATCACTTACGGCGAAAC AGGAGGCAATAGCCCTCTGCAGCAGTTCACTGTGCCTGGTTCTAAAGGTACAGCTACCATCAGCGGCC TTAAACCTGGCGTTGATTATACCATCACTGTGTATG CTGTCGAATACCCGTACGACCATTCTGGTTAC TACCATCGTCCCATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCACCACCA CCAC (SEQ ID NO :182)

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2381D04 MGVSDVPRDLEVVAAT PTSLLISWEPFSRLPGGGEYYR ITYGETGGNSPLQQFTVPGSKG TATISGLKPGVDYTITVYAVEF PYDHSGYYHRPISINYRTEIDK PSQHHHHHH (SEQ ID NO:183) ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCACCAGCCTGCTG ATCAGCTGGGAGCCGTTCAGCCGGTTGCCCGGGGGCGGCGAGTATTACCGGATCACTTACGGCGAAAC AGGAGGCAATAGCCCTCTGCAGCAGTTCACTGTGCCTGGTTCTAAAGGTACAGCTACCATCAGCGGCC TTAAACCTGGCGTTGATTATACCATCACTGTGTATG CTGTCGAATTCCCGTACGACCATTCTGGTTAC TACCATCGGCCCATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCACCACCA CCAC (SEQ ID NO:184) 2381F11 MGVSDVPRDLEVVAAT PTSLLISWDAPADGGYGYYRIT YGETGGNSPVQEFTVPVSKSTA TISGLKPGVDYTITVYAVEYTF PGAGYYHRPISINYRTEIDKPS QHHHHHH (SEQ ID NO:185) ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTG ATCAGCTGGGACGCTCCGGCTGACGGGGGGTACGGTTATTACCGCATCACTTACGGCGAAACAGGAGG CAATAGCCCTGTCCAGGAGTTCACTGTGCCTGTTTCTAAAAGTACAGCTACCATCAGCGGCCTTAAAC CTGGCGTTGATTATACCATCACTGTGTATGCTGTCGA ATACACCTTCCCCGGCGCCGGTTACTACCAT CGTCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCACCACCACCAC (SEQ ID NO:186) 2381G03 (SEQ ID NO:187) ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCACCAGCCTGCTG ATCAGCTGGGAGCCGTTCAGCCGGTTGCCCGGGGGCGGCGAGTATTACCGGATCACTTACGGCGAAAC AGGAGGCAATAGCCCTCTGCAGCAGTTCACTGTGCCTGGTTCTAAAGGTACAGCTACCATCAGCGGCC TTAAACCTGGCGTTGATTATACCATCACTGTGTATG CTGTCGAATTCCCGTACGCGCATTCTGGGTAC TACCATCGTCCGATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCACCACCA CCAC (SEQ ID NO:188) 2381G09 MGVSDVPRDLEVVAAT PTSLLISWDAPAGDGYGYYRIT YGETGGNSPVQEFTVPVSKGTA TISGLKPGVDYTITVYAVE FT F PGAGYYHRPISINYRTEIDKPS QHHHHHH (SEQ ID NO:189) ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCACCAGCCTGCTG ATCAGCTGGGACGCTCCGGCTGGGGACGGTTACGGTTATTACCGCATCACTTACGGCGAAACAGGAGG CAATAGCCCTGTCCAGGAGTTCACTGTGCCCGTTTCTAAAGGTACAGCTACCATCAGCGGCCTTAAAC CTGGCGTTGATTATACCATCACTGTGTATGCTGTCGA ATTCACCTTCCCGGGCGCCGGTTACTACCAT CGTCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCACCACCACCAC (SEQ ID NO:190) 2381H03 MGVSDVPRDLEVVAAT PTSLLISWEPFSRLPGGGEYYR ITYGETGGNSPLQQFTVPGSKG TATISGLKPGVDYTITVYAVEY PYAHSGYFHRPISINYRTEIDK PSQHHHHHH (SEQ ID NO:191) ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCACCAGCCTGCTG ATCAGCTGGGAGCCGTTCAGCCGGTTGCCCGGGGGCGGCGAGTATTACCGGATCACTTACGGCGAAAC AGGAGGCAATAGCCCTCTGCAGCAGTTCACTGTGCCTGGTTCTAAAGGTACAGCTACCATCAGCGGCC TTAAACCTGGCGTTGATTATACCATCACTGTGTATG CTGTCGAATACCCGTACGCGCATTCTGGTTAC TTCCATCGTCCGATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCACCACCA CCAC (SEQ ID NO :192) 2382A01 MGVSDVPRDLEVVAAT PTSLLISWAAPAGGGYGYYRIT YGETGGNSPVQEFTVPVSKGTA TISGLKPGVDYTITVYAVEYDF PGAGYYHRPISINYRTEIDKPS QHHHHHH (SEQ ID NO:193) ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCACAGCCTGCTG ATCAGCTGGGCCGCTCCGGCTGGTGGTGGCTACGGTTATTACCGCATCACTTACGGCGAAACAGGAGG CAATAGCCCTGTCCAGGAGTTCACTGTGCCTGTTTCTAAAGGTACAGCTACCATCAGCGGCCTTAAAC CTGGCGTTGATTATACCATCACTGTGTATGCTGTC GAATACGACTTCCCGGGCGCCGGTTACTACCAT CGTCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCACCACCACCAC (SEQ ID NO:194)

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23 82В10 MGVSDVPRDLEVVAAT ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTG PTSLLISWDAPADAYGYYRITY GETGGNSPVQEFTVPSSKGTAT ISGLKPGVDYTITVYAVEYDFP GSGYYHRPISINYRTEIDKPSQ HHHHHH (SEQ ID NO:195) ATAAGCTGGGACGCTCCGGCTGACGCGTACGGTTATTACCGCATCACTTACGGCGAAACAGGAGGCAA TAGCCCTGTCCAGGAGTTCACTGTGCCTAGTTCTAAAGGTACAGCTACCATCAGCGGCCTTAAACCTG GCGTTGATTATACCATCACTGTGTATGCTGTCGAATACGACTTCCCCGGCAGCGGTTACTACCATCGT CCAATTTCCATTAATTACCGCACAGAAATTGACAAAC CATCCCAGCACCATCACCACCACCAC (SEQ ID NO:196) 2382В09 MGVSDVPRDLEVVAAT PTSSLLISWDAPADAYGYYRITY GETGGNSPVQEFTVPVSKGTAT ISGLKPGVDYTITVYAVEFDYP GSGYYHRPISINYRTEIDKPSQ HHHHHH (SEQ ID NO:197) ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTG ATCAGCTGGGACGCTCCGGCTGACGCGTACGGTTATTACCGCATCACTTACGGCGAAACAGGAGGCAA TAGCCTGTCCAGGAGTTCACTGTGCCTGTTTCTAAAGGTACAGCTACCATCAGCGGCCTTAAACCTG GCGTTGATTATACCATCACTGTGTATGCTGTCGAAT TCGACTACCCCGGCTCCGGTTACTACCATCGT CCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCACCACCACCAC (SEQ ID NO:198) 2382С05 MGVSDVPRDLEVVAAT PTSLLISWDAPADGAYGYYRIT YGETGGNSPVQEFTVPVSKGTA TISGLKPGVDYTITVYAVEY S F PGAGYYHRPISINYRTEIDKPS QHHHHHH (SEQ ID NO:19 9) ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCACCAGCCTGCTG ATCAGCTGGGACGCTCCGGCTGATGGGGCATACGGTTATTACCGCATCACTTACGGCGAAACAGGAGG CAATAGCCCTGTCCAGGAGTTCACTGTGCCTGTTTCTAAGGGTACAGCTACCATCAGCGGCCTTAAAC CTGGCGTTGATTATACCATCACTGTGTATGCTGTCGAAT ACTCCTTCCCCGGCGCCGGTTACTACCAT CGTCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCACCACCACCAC (SEQ ID NQ:200) 2382С09 MGVSDVPRDLEVVAAT PTSLLISWDAPAEGYGYYRITY GETGGNSPVQEFTVPVSKGTAT ISGLKPGVDYTITVYAVEFDFP GSGYYHRPISINYRTEIDKPSQ HHHHHH (SEQ ID NQ:201) ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCACAGCCTGCTG ATCAGCTGGGACGCTCCGGCTGAGGGTTACGGTTATTACCGCATCACTTACGGCGAAACAGGAGGCAA TAGCCCTGTCCAGGAGTTCACTGTGCCTGTTTCTAAAGGTACAGCTACCATCAGCGGCCTTAAACCTG GCGTTGATTATACCATCACTGTGTATGCTGTCGAATTC GACTTCCCCGGCTCCGGTTACTACCATCGT CCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCACCACCACCAC (SEQ ID NQ:202) 2382D03 MGVSDVPRDLEVVAAT PTSLLISWDAPADEAYGYYRIT YGETGGNSPVQEFTVPVSKGTA TISGLKPGVDYTITVYAVEFDF PGAGYYHRPISINYRTEIDKPS QHHHHHH (SEQ ID NQ:203) ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCACCAGCCTGCTG ATCAGCTGGGACGCTCCGGCTGACGAGGCGTACGGTTATTACCGCATCACTTACGGCGAAACAGGAGG CAATAGCCCTGTCCAGGAGTTCACTGTGCCTGTTTCTAAAGGTACAGCTACCATCAGCGGCCTTAAAC CTGGTGTTGATTATACCATCACTGTGTATGCTGTCGA ATTCGACTTCCCCGGCGCCGGTTACTACCAT CGTCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCACCACCACCAC (SEQ ID NQ:204) 2382D05 MGVSDVPRDLEVVAAT PTSLLISWDAPADGGYGYYRIT YGETGGNSPVQEFTVPVSKGTA TISGLKPGVDYTITVYAVEFDF PGAGYYHRPISINYRTEIDKPS QHHHHHH (SEQ ID NO:205) ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTG ATCAGCTGGGACGCTCCGGCTGATGGTGGTTACGGTTATTACCGCATCACTTACGGCGAAACAGGAGG CAATAGCCCTGTCCAGGAGTTCACTGTGCCTGTTTCTAAAGGTACAGCTACCATCAGCGGCCTTAAAC CTGGCGTTGATTATACCATCACTGTGTATGCTGTCGA ATTCGACTTCCCGGGCGCCGGTTACTACCAT CGTCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCACCACCACCAC (SEQ ID NQ:206) 2382D08 MGVSDVPRDLEVVAAT PTSLLISWDAPADGYGYYRITY GETGGNSPVQEFTVPVSKGTAT ISGLKPGVDYTITVYAVEFDFP GSGYYHRPISINYRTEIDKPSQ HHHHHH (SEQ ID NQ:207) ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCACCAGCCTGCTG ATCAGCTGGGACGCTCCGGCTGATGGCTACGGTTATTACCGCATCACTTACGGCGAAACAGGAGGCAA TAGCCCTGTCCAGGAGTTCACTGTGCCTGTTTCTAAAGGTACAGCTACCATCAGCGGCCTTAAACCTG GCGTTGATTATACCATCACTGTGTATGCTGTCGAATTC CCCTTCCCCGGCTCCGGTTACTACCATCGT CCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCACCACCACCAC (SEQ ID NQ:208) 2382D09 MGVSDVPRDLEVVAAT PTSLLISWDAPAEGYGYYRITY GETGGNSPVQEFTVPVSKGTAT ISGLKPGVDYTITVYAVEFDFP GAGYYHRPISINYRTEIDKPSQ HHHHHH (SEQ ID NO:209) ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCACAGCCTGCTG ATCAGCTGGGACGCTCCGGCTGAAGGGTACGGTTATTACCGCATCACTTACGGCGAAACAGGAGGCAA TAGCCCTGTCCAGGAGTTCACTGTGCCTGTTTCTAAAGGTACAGCTACCATCAGCGGCCTTAAACCTG GCGTTGATTATACCATCACTGTGTATGCTGTCGAAT TCGACTTCCCCGGCGCGGTTACTACCATCGT CCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCACCACCACCAC (SEQ ID NO:210) 2382F02 MGVSDVPRDLEVVAAT PTSLLISWDAPAGGGYGYYRIT YGETGGNSPVQEFTVPVSKGTA TISGLKPGVDYTITVYAVEFDF PGSGYYHRPISINYRTEIDKPS QHHHHHH (SEQ ID NO:211) ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCACCAGCCTGCTG ATCAGCTGGGACGCTCCGGCTGGCGGGGGGTACGGTTATTACCGCATCACTTACGGCGAAACAGGAGG CAATAGCCCTGTCCAGGAGTTCACTGTGCCTGTTTCTAAAGGTACAGCTACCATCAGCGGCCTTAAAC CTGGCGTTGATTATACCATCACTGTGTATGCTGTCGA ATTCGACTTCCCGGGCTCCGGTTACTACCAT CGTCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCACCACCACCAC (SEQ ID NO:212)

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2382F03 MGVSDVPRDLEVVAAT ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTG PTSLLISWDAPAADAYGYYRIT YGETGGNSPVQEFTVPVSKGTA TISGLKPGVDYTITVYAVE FNF PGAGYYHRPISINYRTEIDKPS QHHHHHH (SEQ ID NO:213) ATCAGCTGGGACGCTCCGGCTGCCGATGCTTACGGTTATTACCGCATCACTTACGGCGAAACAGGAGG CAATAGCCCTGTCCAGGAGTTCACTGTGCCTGTTTCTAAAGGTACAGCTACCATCAGCGGCCTTAAAC CTGGCGTTGATTATACCATCACTGTGTATGCTGTCGAATTCAACTTCCCGGGCGCCGGTTACTACCAT CGTCCAATTTCCATTAATTACCGCACAGAAATTGA CAAACCATCCCAGCACCATCACCACCACCAC (SEQ ID NO:214) 2382F05 (SEQ ID NO:215) ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTG ATCAGCTGGGACGCTCCGGCTGAAGCAGGTAAGCACTACGGTTATTACCGCATCACTTACGGCGAAAC AGGAGGCAATAGCCCTGTCCAGGAGTTCACTGTGCCTGTTTCTAAAGGTACAGCTACCATCAGCGGCC TTAAACCTGGCGTTGATTATACCATCACTGTGTATG CTGTCGAATTCGACTTCCCGGGCGCCGGTTAC TACCATCGTCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCACCACCA CCAC (SEQ ID NO :216) 2382F08 MGVSDVPRDLEVVAAT PTSLLISWDAPAEAYGYYRITY GETGGNSPVQEFTVPVSKGTAT ISGLKPGVDYTITVYAVEFTYP GSGYYHRPISINYRTEIDKPSQ HHHHHH (SEQ ID NO:217) ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCACAGCCTGCTG ATCAGCTGGGACGCTCCGGCTGAAGCATACGGTTATTACCGCATCACTTACGGCGAAACAGGAGGCAA TAGCCCTGTCCAGGAGTTCACTGTGCCTGTTTCTAAAGGTACAGCTACCATCAGCGGCCTTAAACCTG GCGTTGATTATACCATCACTGTGTATGCTGTCGAATTC ACCTACCCCGGCTCCGGTTACTACCATCGT CCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCACCACCACCAC (SEQ ID NO:218) 2382F09 MGVSDVPRDLEVVAAT PTSLLISWDAPAAAYGYYRITY GETGGNSPVQEFTVPVSKGTAT ISGLKPGVDYTITVYAVEYDFP GSGYYHRPISINYRTEIDKPSQ HHHHHH (SEQ ID NO :219) ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCACCAGCCTGCTG ATCAGCTGGGACGCTCCGGCTGCAGCCTACGGTTATTACCGCATCACTTACGGCGAAACAGGAGGCAA TAGCCCTGTCCAGGAGTTCACTGTGCCTGTTTCTAAAGGTACAGCTACCATCAGCGGCCTTAAACCTG GCGTTGATTATACCATCACTGTGTATGCTGTCGAATACG ACTTCCCCGGCTCCGGTTACTACCATCGT CCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCACCACCACCAC (SEQ ID NO:220) 2382G04 MGVSDVPRDLEVVAAT PTSLLISWDAPAGGGYGYYRIT YGETGGNSPVQEFTVPSSKGTA TISGLKPGVDYTITVYAVEFDF PGAGYYHRPISINYRTEIDKPS QHHHHHH (SEQ ID NO:221) ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCACCAGCCTGCTG ATCAGCTGGGACGCTCCGGCTGGTGGGGGATACGGTTATTACCGCATCACTTACGGCGAAACAGGAGG CAATAGCCCTGTCCAGGAGTTCACTGTGCCTAGTTCTAAAGGTACAGCTACCATCAGCGGCCTTAAAC CTGGCGTTGATTATACCATCACTGTGTATGCTGTCGA ATTCGACTTCCCGGGCGCCGGTTACTACCAT CGTCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCACCACCACCAC (SEQ ID NO:222) 2382H10 MGVSDVPRDLEVVAAT PTSLLISWDAPAGGYGYYRITY GETGGNSPVQEFTVPVSKGTAT ISGLKPGVDYTITVYAVEFDFP GSGYYHRPISINYRTEIDKPSQ HHHHHH (SEQ ID NO:223) ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCACCAGCCTGCTG ATCAGCTGGGACGCTCCGGCTGGGGGCTACGGTTATTACCGCATCACTTACGGCGAAACAGGAGGCAA TAGCCCTGTCCAGGAGTTCACTGTGCCTGTTTCTAAAGGTACAGCTACCATCAGCGGCCTTAAACCTG GCGTTGATTATACCATCACTGTGTATGCTGTCGAATTC GACTTCCCCGGCTCCGGTTACTACCATCGT CCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCACCACCACCAC (SEQ ID NO:224) 2382H11 MGVSDVPRDLEVVAAT PTSLLISWDAPADGYGYYRITY GETGGNSPVQEFTVPVFKGTAT ISGLKPGVDYTITVYAVEFDYP GSGYYHRPISINYRTEIDKPSQ HHHHHH (SEQ ID NO:225) ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTG ATCAGCTGGGACGCTCCGGCTGATGGTTACGGTTATTACCGCATCACTTACGGCGAAACAGGAGGCAA TAGCCCTGTCCAGGAGTTCACTGTGCCTGTTTTTTAAAGGTACAGCTACCATCAGCGGCCTTAAACCTG GCGTTGATTATACCATCACTGTGTATGCTGTCGAATTC GACTACCCCGGCTCCGGTTACTACCATCGT CCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCACCACCACCAC (SEQ ID NO:226) 2382H04 MGVSDVPRDLEVVAAT PTSLLISWDAPAAGGYGYYRIT YGETGGNSPVQEFTVPSSKGTA TISGLKPGVDYTITVYAVEYDF PGAGYYHRPISINYRTEIDKPS QHHHHHH (SEQ ID NO:227) ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCACCAGCCTGCTG ATCAGCTGGGACGCTCCGGCTGCGGGGGGGTACGGTTATTACCGCATCACTTACGGCGAAACAGGAGG CAATAGCCCTGTCCAGGAGTTCACTGTGCCTAGTTCTAAAGGTACAGCTACCATCAGCGGCCTTAAAC CTGGCGTTGATTATACCATCACTGTATATGCTGTCGA ATACGACTTCCCCGGCGCCGGTTACTACCAT CGTCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCACCACCACCAC (SEQ ID NO:228) 2382H07 MGVSDVPRDLEVVAAT PTSLLISWDAPADAYGYYRITY GETGGNSPVQEFTVPGSKGTAT ISGLKPGVDYTITVYAVEFDFP GSGYYHRPISINYRTEIDKPSQ HHHHHH (SEQ ID NO:229) ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTG ATCAGCTGGGACGCTCCGGCTGATGCTTACGGTTATTACCGCATCACTTACGGCGAAACAGGAGGCAA TAGCCCAGTCCAGGAGTTCACTGTGCCTGGTTCTAAAGGTACAGCTACCATCAGCGGCCTTAAACCTG GCGTTGATTATACCATCACTGTGTATGCTGTCGAATTC GACTTCCCCGGCTCCGGTTACTACCATCGT CCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCACCACCACCAC (SEQ ID NQ:230)

- 93 044608

2382H09 MGVSDVPRDLEVVAAT PTSLLISWDAPAAAYGYYRITY GETGGNSPVQEFTVPSSKGTAT ISGLKPGVDYTITVYAVEFDFP GSGYYHRPISINYRTEIDKPSQ HHHHHH (SEQ ID NO:231) ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTG ATCAGCTGGGACGCTCCGGCTGCGGCTTACGGTTATTACCGCATCACTTACGGCGAAACAGGAGGCAA TAGCCCTGTCCAGGAGTTCACTGTGCCTAGTTCTAAAGGTACAGCTACCATCAGCGGCCTTAAACCTG GCGTTGATTATACCATCACTGTGTATGCTGTCGAATTC GACTTCCCCGGCTCCGGTTACTACCATCGC CCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCACCACCACCAC (SEQ ID NO:232) 2451A02 MGVSDVPRDLEVVAAT PTSLLISWDAPAAGYGYYRITY GETGGNSPVQEFTVPVSKGTAT ISGLKPGVDYTITVYAVEFPFP GSGYYHRPISINYRTEIDKPSQ HHHHHH (SEQ ID NO:233) ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCACAGCCTGCTG ATCAGCTGGGACGCTCCGGCTGCGGGTTACGGTTATTACCGCATCACTTACGGCGAAACAGGAGGCAA TAGCCCTGTCCAGGAGTTCACTGTGCCTGTTTCTAAAGGTACAGCTACCATCAGCGGCCTTAAACCTG GCGTTGATTATACCATCACTGTGTATGCTGTCGAATTC CCCTTCCCCGGCTCCGGTTACTACCATCGT CCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCACCACCACCAC (SEQ ID NO:234) 2451B05 MGVSDVPRDLEVVAAT PTSLLISWDAPAGGYGYYRITY GETGGNSPVQEFTVPSSKGTAT ISGLKPGVDYTITVYAVEFDYP GSGYYHRPISINYRTEIDKPSQ HHHHHH (SEQ ID NO:235) ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCACAGCCTGCTG ATCAGCTGGGACGCTCCGGCTGGGGGATACGGTTATTACCGCATCACTTACGGCGAAACAGGAGGCAA TAGCCCTGTCCAGGAGTTCACTGTGCCTAGTTCTAAAGGTACAGCTACCATCAGCGGCCTTAAACCTG GCGTTGATTATACCATCACTGTGTATGCTGTCGAATTC GACTACCCCGGCTCCGGTTACTACCATCGT CCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCACCACCACCAC (SEQ ID NO:236) 2451B06 (equivalent to 2382D05) MGVSDVPRDLEVVAAT PTSLLISWDAPADGGYGYYRIT YGETGGNSPVQEFTVPVSKGTA TISGLKPGVDYTITVYAVEFDF PGAGYYHRPISINYRTEIDKPS QHHHHHH (SEQ ID NO:205) ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTG ATCAGCTGGGACGCTCCGGCTGATGGTGGTTACGGTTATTACCGCATCACTTACGGCGAAACAGGAGG CAATAGCCCTGTCCAGGAGTTCACTGTGCCTGTTTCTAAAGGTACAGCTACCATCAGCGGCCTTAAAC CTGGCGTTGATTATACCATCACTGTGTATGCTGTCGA ATTCGACTTCCCGGGCGCCGGTTACTACCAT CGTCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCACCACCACCAC (SEQ ID NO:206) 2451С06 (SEQ ID NO:237) ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCACAGCCTGCTG ATCAGCTGGGACGCTCCGGCTGGGGCAGCGTCCTACGGTTATTACCGCATCACTTACGGCGAAACAGG AGGCAATAGCCCTGTCCAGGAGTTCACTGTGCCTGTTTCTAAAGGTACAGCTACCATCAGCGGCCTTA AACCTGGCGTTGATTATACCATCACTGTGTATGCTGTC GAATTCCCCTTCCCCGGCGCCGGTTACTAC CATCGTCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCACCACCACCA C (SEQ ID NO:238) 2451D05 MGVSDVPRDLEVVAAT PTSLLISWDAPAGAYGYYRITY GETGGNSPVQEFTVPVSKGTAT ISGLKPGVDYTITVYAVEFDFP GSGYYHRPISINYRTEIDKPSQ HHHHHH (SEQ ID NO:239) ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCACAGCCTGCTG ATCAGCTGGGACGCTCCGGCTGGCGCGTACGGTTATTACCGCATCACTTACGGCGAAACAGGAGGCAA TAGCCTGTCCAGGAGTTCACTGTGCCTGTTTCTAAAGGTACAGCTACCATCAGCGGCCTTAAACCTG GCGTTGATTATACCATCACTGTGTATGCTGTCGAAT TCGACTTCCCCGGCTCCGGTTACTACCATCGT CCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCACCACCACCAC (SEQ ID NO:240) 2451F03 MGVSDVPRDLEVVAAT PTSLLISWDPPAEGYGYYRITY GETGGNSPVQEFTVPVSKGTAT ISGLKPGVDYTITVYAVEFNFP GSGYYHRPISINYRTEIDKPSQ HHHHHH (SEQ ID NO :241) ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTG ATCAGCTGGGACCCTCCGGCTGAAGGTTACGGTTATTACCGCATCACTTACGGCGAAACAGGAGGCAA TAGCCCTGTCCAGGAGTTCACTGTGCCTGTTTCTAAAGGTACAGCTACCATCAGCGGCCTTAAACCTG GCGTTGATTATACCATCACTGTGTATGCTGTCGAAT TCAACTTCCCCGGCTCCGGTTACTACCATCGT CCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCACCACCACCAC (SEQ ID NO:242) 2451 GO 1 MGVSDVPRDLEVVAAT PTSLLISWDAPAGGYGYYRITY GETGGNSPVQEFTVPSSKGTAT ISGLKPGVDYTITVYAVEFDFP GSGYYHRPISINYRTEIDKPSQ HHHHHH (SEQ ID NO:243) ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCACCAGCCTGCTG ATCAGCTGGGACGCTCCGGCTGGGGGCTACGGTTATTACCGCATCACTTACGGCGAAACAGGAGGCAA TAGCCCTGTCCAGGAGTTCACTGTGCCTAGTTCTAAAGGTACAGCTACCATCAGCGGCCTTAAACCTG GCGTTGATTATACCATCACTGTGTATGCTGTCGAATTC GACTTCCCGGGCTCCGGTTACTACCATCGT CCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCACCACCACCAC (SEQ ID NO:244) 2451Н07 MGITDVPRDLEVVAAT PTSLLISWNPPDVNYGYYRITY GETGGNSPLQEFTVPVSKGTAT ISGLKPGVDYTITVYAVEYPYA HAGYYHRPISINYRTEIDKPSQ HHHHHH (SEQ ID NO:245) ATGGGTATCACGGATGTGCCGCGAGACTTGGAAGTGGTTGCTGCCACCCCACAGCCTGCTG ATCAGCTGGAACCCGCCGGATGTGAATTACGGTTATTATCGCATCACTTACGGGGAAACAGGAGGCAA TAGCCTTTGCAGGAGTTCACTGTGCCTGTTTCTAAAGGTACAGCTACCATCAGCGGCCTTAAACCTG GCGTTGATTATACCATCACTGTGTATGCTGTCGAATATC CGTACGCGCACGCTGGTTACTACCATCGT CCGATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCACCACCACCAC (SEQ ID NO:246)

- 94 044608

2382E03 (SEQ ID NO:247) ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCACAGCCTGCTG ATCAGCTGGGACGCTCCGGCTGGGGACGGGTACGGTTATTACCGCATCACTTACGGCGAAACAGGAGG CAATAGCCCTGTCCAGGAGTTCACTGTGCCTGTTTCTAAAGGTACAGCTACCATCAGCGGCCTTAAAC CTGGCGTTGATTATACCATCACTGTGTATGCTGTCGA ATTCGACTTCCCCGGCGCCGGTTACTACCAT CGTCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCACCACCACCAC (SEQ ID NO:248) 2382E04 MGVSDVPRDLEVVAAT PTSLLISWDAPAGGGYGYYRIT YGETGGNSPVQEFTVPVSKGTA TISGLKPGVDYTITVYAVE FT F PGAGYYHRPISINYRTEIDKPS QHHHHHH (SEQ ID NO:2 4 9) ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCACAGCCTGCTG ATCAGCTGGGACGCTCCGGCTGGTGGTGGATACGGTTATTACCGCATCACTTACGGCGAAACAGGAGG CAATAGCCCTGTCCAGGAGTTCACTGTGCCTGTTTCTAAAGGTACAGCTACCATCAGCGGCCTTAAAC CTGGCGTTGATTATACCATCACTGTGTATGCTGTC GAATTCACCTTCCCGGGCGCCGGTTACTACCAT CGTCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCACCACCACCAC (SEQ ID NO:250) 2382E05 MGVSDVPRDLEVVAAT PTSLLISWDAPAEGGYGYYRIT YGETGGNSPVQEFTVPVSKGTA TISGLKPGVDYTITVYAVEFDF PGAGYYHRPISINYRTEIDKPS QHHHHHH (SEQ ID NO:251) ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCACAGCCTGCTG ATCAGCTGGGACGCTCCGGCTGAGGGCGGCTACGGTTATTACCGCATCACTTACGGCGAAACAGGAGG CAATAGCCCTGTCCAGGAGTTCACTGTGCCTGTTTCTAAAGGTACAGCTACCATCAGCGGCCTTAAAC CTGGCGTTGATTATACCATCACTGTGTATGCTGTCGA ATTCGACTTCCCCGGCGCCGGTTACTACCAT CGTCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCACCACCACCAC (SEQ ID NO:252) 2382E09 MGVSDVPRDLEVVAAT PTSLLISWDAPAEAYGYYRITY GETGGNSPVQEFTVPVSKGTAT ISGLKPGVDYTITVYAVEYDFP GSGYYHRPISINYRTEIDKPSQ HHHHHH (SEQ ID NO:253) ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCACCAGCCTGCTG ATCAGCTGGGACGCTCCGGCTGAGGCTTACGGTTATTACCGCATCACTTACGGCGAAACAGGAGGCAA TAGCCCTGTCCAGGAGTTCACTGTGCCTGTTTCTAAAGGTACAGCTACCATCAGCGGCCTTAAACCTG GCGTTGATTATACCATCACTGTGTATGCTGTCGAATACG ACTTCCCCGGCTCCGGTTACTACCATCGT CCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCACCACCACCAC (SEQ ID NO:254) 2381A04 (SEQ ID NO:255) ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCACCAGCCTGCTG ATCAGCTGGGAGCCGTTCAGCCGGTTGCCCGGGGGCGGCGAGTATTACCGGATCACTTACGGCGAAAC AGGAGGCAATAGCCCTCTGCAGCAGTTCACTGTGCCTGGTTCTAAAGGTACAGCTACCATCAGCGGCC TTAAACCTGGCGTTGATTATACCATCACTGTGTATG CTGTCGAATACCCGTACCCGTTTTCTGGTTAC TACCATCGTCCCATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCACCACCA CCAC (SEQ ID NO :256) 2381A08 MGVSDVPRDLEVVAAT PTSLLISWDAPADGGYGYYRIT YGETGGNSPVQEFTVPGSKGTA TISGLKPGVDYTITVYAVEYDF PGAGYYHRPISINYRTEIDKPS QHHHHHH (SEQ ID NO:257) ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTG ATCAGCTGGGACGCTCCGGCTGACGGCGGGTACGGTTATTACCGCATCACTTACGGCGAAACAGGAGG CAATAGCCCTGTCCAGGAGTTCACTGTGCCTGGTTCTAAAGGTACAGCTACCATCAGCGGCCTTAAAC CTGGCGTTGATTATACCATCACTGTGTATGCTGTCGA ATACGACTTCCCGGGCGCCGGTTACTACCAT CGTCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCACCACCACCAC (SEQ ID NO:258)

- 95 044608

2381B10 MGVSDVPRDLEVVAAT PT SLLISWDAPAGGGYGYYRIT YGETGGNSPVQEFTVPVSKGTA TISGLKPGVDYTITVYAVEYNF IGAGYYHRPISINYRTEIDKPS QHHHHHH (SEQ ID NO:259) ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTG ATCAGCTGGGACGCTCCGGCTGGGGGTGGATACGGTTATTACCGSATCACTTACGGCGAAACAGGAGG CAATAGCCCTGTCCAGGAGTTCACTGTGCCTGTTTCTAAAGGTACAGCTACCATCAGCGGCCTTAAAC CTGGCGTTGATTATACCATCACTGTGTATGCTGTCGA ATACAACTTCATCGGCGCCGGTTACTACCAT CGTCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCACCACCACCAC (SEQ ID NO:260) 2381C08 MGVSDVPRDLEVVAAT PT SLLISWDAPADGAYGYYRIT YGETGGNSPVQEFTVPVSKGTA TISGLKPGVDYTITVYAVEFPY PFAGYYHRPISINYRTEIDKPS QHHHHHH (SEQ ID NO:2 61) ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCACAGCCTGCTG ATCAGCTGGGACGCTCCGGCTGACGGTGCCTACGGTTATTACCGCATCACTTACGGCGAAACAGGAGG CAATAGCCCTGTCCAGGAGTTCACTGTGCCTGTTTCTAAAGGTACAGCTACCATCAGCGGCCTTAAAC CTGGCGTTGATTATACCATCACTGTGTATGCTGTCGA ATTCCCCTACCCCTTCGCCGGTTACTACCAT CGTCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCACCACCACCAC (SEQ ID NO:262) 2381G06 (SEQ ID NO:263) ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCACCAGCCTGCTG ATCAGCTGGTCGGAGAAGTTGGACGGGAAGGCGCGCCGCGGGTATTACCGCATCACATACGGCGAAAC AGGAGGCAATAGCCCTGTCCAGCAGTTCACTGTGCCTGGTTCTAAAGGTACAGCTACCATCAGCGGCC TTAAACCTGGCGTTGATTATACCATCACTGTGTATGCT GTCGAATTCCCGTACGACCATTCTGGTTAC TACCATCGTCCCATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCACCACCA CCAC (SEQ ID NO :264) 2381H01 MGVSDVPRDLEVVAAT PTSLLISWSPRDSTGLVRRGYY RITYGETGGNSPVQQFTVPGSK GTATISGLKPGVDYTITVYAVE YPYDHSGYYHRPISINYRTEID KPSQHHHHHH (SEQ ID NO :265) ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCACCAGCCTGCTG ATCAGCTGGAGCCCGCGGGACTCCACCGGCTTGGTGAGGCGGGGGTATTACCGCATCACTTACGGCGA AACAGGAGGCAATAGCCCTGTTCAGCAGTTCACTGTGCCTGGTTCTAAAGGTACAGCTACCATCAGCG GCCTTAAACCTGGCGTTGATTATACCATCACTGTGTATG CTGTCGAATACCCGTACGACCATTCTGGT TACTACCATCGGCCCATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCACCA CCACCAC (SEQ ID NO :266) 2381H06 MGVSDVPRDLEVVAAT PT SLLISWGDVRTNEARQGYYR ITYGETGGNSPLQGFTVPGSKG TATISGLKPGVDYTITVYAVEY TYEHSGYYHRPISINYRTEIDK PSQHHHHHH (SEQ ID NO:267) ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCACCAGCCTGCTG ATCAGCTGGGGCGACGTCCGGACGAACGAGGCGCGGCAGGGCTATTACCGGATCACTTACGGCGAAAC AGGAGGCAATAGCCCTCTCCAGGGGTTCACTGTGCCTGGTTCTAAAGGTACAGCTACCATCAGCGGCC TTAAACCTGGCGTTGATTATACCATCACTGTGTATGCT GTCGAGTATACGTACGAGCATTCTGGTTAC TACCATCGTCCGATTTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCACCACCA CCAC (SEQ ID NO :268) 2381H09 MGVSDVPRDLEVVAAT PTSLLISWDAPAGGGYGYYRIT YGETGGNSPVQEFTVPVSKGTA TISGLKPGVDYTITVYAVEFDF VGAGYYHRPISINYRTEIDKPS QHHHHHH (SEQ ID NO:2 6 9) ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCACAGCCTGCTG ATCAGCTGGGACGCTCCGGCTGGGGGGGGCTACGGTTATTACCGCATCACTTACGGCGAAACAGGAGG CAATAGCCCTGTCCAGGAGTTCACTGTGCCTGTTTCTAAAGGTACAGCTACCATCAGCGGCCTTAAAC CTGGCGTTGATTATACCATCACTGTGTATGCTGTCGA ATTCGACTTCGTCGGCGCCGGTTACTACCAT CGTCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCACCACCACCAC (SEQ ID NQ:270) 23 82В11 MGVSDVPRDLEVVAAT PTSLLISWDAPAAAYGYYRITY GETGGNSPVQEFTVPVSKGTAT ISGLKPGVDYTITVYAVEYDFA GSGYYHRPISINYRTEIDKPSQ HHHHHH (SEQ ID NO:271) ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCACCAGCCTGCTG ATCAGCTGGGACGCTCCGGCTGCGGCCTACGGTTATTACCGCATCACTTACGGCGAAACAGGAGGCAA TAGCCCTGTCCAGGAGTTCACTGTGCCTGTTTCTAAAGGTACAGCTACCATCAGCGGCCTTAAACCTG GCGTTGATTATACCATCACTGTGTATGCTGTCGAATACG ACTTCGCGGGCTCCGGTTACTACCATCGT CCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCACCACCACCAC (SEQ ID NO:272) 2382В08 MGVSDVPRDLEVVAAT PT SLLISWDAPADAYGYYRITY GETGGNSPVQEFTVPSSKGTAT ISGLKPGVDYTITVYAVEFAFP GAGYYHRPISINYRTEIDKPSQ HHHHHH (SEQ ID NO:273) ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCACAGCCTGCTG ATCAGCTGGGACGCTCCGGCTGACGCGTACGGTTATTACCGCATCACTTACGGCGAAACAGGAGGCAA TAGCCTGTCCAGGAGTTCACTGTGCCTAGTTCTAAAGGTACAGCTACCATCAGCGGCCTTAAACCTG GCGTTGATTATACCATCACTGTATATGCTGTCGAAT TCGCCTTCCCCGGCGCCGGTTACTACCATCGT CCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCACCACCACCAC (SEQ ID NO:274) 2382С11 MGVSDVPRDLEVVAAT PT SLLISWDAPAGGYGYYRITY GETGGNSPVQEFTVPVSKGTAT ISGLKPGVDYTITVYAVEYDFA GSGYYHRPISINYRTEIDKPSQ HHHHHH (SEQ ID NO:275) ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCACAGCCTGCTG ATCAGCTGGGACGCTCCGGCTGGAGGTTACGGTTATTACCGCATCACTTACGGCGAAACAGGAGGCAA TAGCCCTGTCCAGGAGTTCACTGTGCCTGTTTCTAAAGGTACAGCTACCATCAGCGGCCTTAAACCTG GCGTTGATTATACCATCACTGTGTATGCTGTCGAATACG ACTTCGCGGGCTCCGGTTACTACCATCGT CCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCACCACCACCAC (SEQ ID NO:276)

- 96 044608

2382G03 MGVSDVPRDLEVVAAT PTSLLISWDAPAEAEAYGYYRI TYGETGGNSPVQEFTVPVSKGT ATISGLKPGVDYTITVYAVEYV FPGAGYYHRPISINYRTEIDKP SQHHHHHH (SEQ ID NO:277) ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCACCAGCCTGCTG ATCAGCTGGGACGCTCCGGCTGAAGCAGAAGCGTACGGTTATTACCGCATCACTTACGGCGAAACAGG AGGCAATAGCCCTGTCCAGGAGTTCACTGTGCCTGTTTCTAAAGGTACAGCTACCATCAGCGGCCTTA AACCTGGCGTTGATTATACCATCACTGTGTATGCTG TCGAATACGTCTTCCCCGGCGCCGGTTACTAC CATCGTCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCACCACCACCA C (SEQ ID NO:278) 2382H03 MGVSDVPRDLEVVAAT PTSLLISWDAPAEGAYGYYRIT YGETGGNSPVQEFTVPVSKGTA TISGLKPGVDYTITVYAVEY PY PFAGYYHRPISINYRTEIDKPS QHHHHHH (SEQ ID NO:279) ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCACCAGCCTGCTG ATCAGCTGGGACGCTCCGGCTGAGGGCGCTTACGGTTATTACCGCATCACTTACGGCGAAACAGGAGG CAATAGCCCTGTCCAGGAGTTCACTGTGCCTGTTTCTAAAGGTACAGCTACCATCAGCGGCCTTAAAC CTGGCGTTGATTATACCATCACTGTGTATGCTGTCGA ATACCCCTACCCCTTCGCCGGTTACTACCAT CGTCCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCACCACCACCAC (SEQ ID NQ:280) 2451A10 MGVTDVPRDMEVVAAT PTSLLISWQPPAVTYGYYRITY GETGGNSTLQQFTVPVYKGTAT ISGLKPGVDYTITVYAVEYPYD HSGYYHRPISINYRTEIDKPSQ HHHHHH (SEQ ID NO :281) ATGGGTGTCACCGATGTGCCGCGCGACATGGAAGTGGTTGCTGCCACCCACCAGCCTGCTG ATCAGCTGGCAGCCGCCGGCTGTTACTTACGGTTATTATCGCATCACTTACGGCGAAACAGGAGGCAA TAGCACTCTCCAGCAGTTCACTGTGCCTGTTTATAAAGGTACAGCTACCATCAGCGGCCTTAAACCTG GCGTTGATTATACCATCACTGTGTATGCTGTCGAATACC CGTACGACCATTCTGGGTACTACCATCGG CCGATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCACCACCACCAC (SEQ ID NO:282) 2451B02 MGVSDVPRDLEVVAAT PTSLLISWDAPAAAYGYYRITY GETGGNSPVQEFTVPVSKGTAT ISGLKPGVDYTITVYAVEFDYP GSGYYHRPISINYRTEIDKPSQ HHHHHH (SEQ ID NO:283) ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTG ATCAGCTGGGACGCTCCGGCTGCTGCTTACGGTTATTACCGCATCACTTACGGCGAAACAGGAGGCAA TAGCCCTGTCCAGGAGTTCACTGTGCCTGTTTCTAAAGGTACAGCTACCATCAGCGGCCTTAAACCTG GCGTTGATTATACCATCACTGTGTATGCTGTCGAATTC GACTACCCCGGCTCCGGTTACTACCATCGT CCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCACCACCACCAC (SEQ ID NO:284) 2451C11 MGIVDVPRDLEVVAAT PTSLLISWDPPAGAYGYYRITY GETGGNSPKQQFTVPGYKGTAT ISGLKPGVDYTITVYAVEYPYD HSGYYHRPISINYRTEIDKPSQ HHHHHH (SEQ ID NO:285) ATGGGTATCGTGGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCACCAGCCTGCTG ATCAGCTGGGACCCGCCGGCTGGTGCTTACGGTTATTATCGCATCACTTACGGCGAAACAGGAGGCAA TAGCCCAAAGCAGCAGTTCACTGTGCCTGGTTATAAAGGTACAGCTACCATCAGCGGCCTTAAACCTG GCGTTGATTATACCATCACTGTGTATGCTGTCGAATA CCCGTACGACCATTCTGGTTACTACCATCGG CCGATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCACCACCACCAC (SEQ ID NO:286) 2451H01 MGVSDVPRDLEVVAAT PTSLLISWDAPAAGYGYYRITY GETGGNSPVQEFTVPVSKGTAT ISGLKPGVDYTITVYAVEYDFP GSGYYHRPISINYRTEIDKPSQ HHHHHH (SEQ ID NO:287) ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCACAGCCTGCTG ATCAGCTGGGACGCTCCGGCTGCGGGGTACGGTTATTACCGCATCACTTACGGCGAAACAGGAGGCAA TAGCCCTGTCCAGGAGTTCACTGTGCCTGTTTCTAAAGGTACAGCTACCATCAGCGGCCTTAAACCTG GCGTTGATTATACCATCACTGTGTATGCTGTCGAATA CGACTTCCCCGGCTCCGGTTACTACCATCGT CCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCACCACCACCAC (SEQ ID NO:288) 2011B11 MGVSDVPRDLEVVAAT PTSLLISWAPPSDAYGYYRITY GETGGNSPVQEFTVPIGKGTAT ISGLKPGVDYTITVYAVEYPYS HAGYYHRPISINYRTEIDKPSQ HHHHHH (SEQ ID NO:289) ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCACCAGCCTGCTG ATCAGCTGGGCGCCGCCTTCTGATGCGTACGGTTATTACCGCATCACTTACGGCGAAACAGGAGGCAA TAGCCCTGTCCAGGAGTTCACTGTGCCTATTGGTAAAGGTACAGCTACCATCAGCGGCCTTAAACCTG GCGTTGATTATACCATCACTGTGTATGCTGTCGAATA CCCGTATTCACATGCTGGTTACTACCATCGT CCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCACCACCACCAC (SEQ ID NO:290) 2971A03 MGVSDVPRDLEVVAAT PTSLLISWDPPSDDYGYYRITY GETGGNSPVQEFTVPIGKGTAT ISGLKPGVDYTITVYAVEFPWP HAGYYHRPISINYRTEIDKPSQ HHHHHH (SEQ ID NQ:304) ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTG ATCAGCTGGGACCCGCCTTCGGATGATTACGGTTATTACCGCATCACTTACGGCGAAACAGGAGGCAA TAGCCCTGTCCAGGAGTTCACTGTGCCTATTGGTAAAGGAACAGCTACCATCAGCGGCCTTAAACCTG GCGTTGATTATACCATCACTGTGTATGCTGTCGAGTT TCCGTGGCCACATGCTGGTTACTATCATCGG CCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCACCACCACCAC (SEQ ID NQ:305) 2971A09 MGVSDVPRDLEVVAAT PTSLLISWDAPADDYGYYRITY GETGGNSPVQEFTVPIGKGTAT ISGLKPGVDYTITVYAVEFPWP HAGYYHRPISINYRTEIDKPSQ HHHHHH (SEQ ID NO:306) ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCACCAGCCTGCTG ATCAGCTGGGACGCGCCTGCGGATGATTACGGTTATTACCGCATCACTTACGGCGAAACAGGAGGCAA TAGCCCTGTCCAGGAGTTCACTGTGCCTATTGGTAAAGGAACAGCTACCATCAGCGGCCTTAAACCTG GCGTTGATTATACCATCACTGTGTATGCTGTCGAGTT TCCGTGGCCACATGCTGGTTACTATCATCGG CCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCACCACCACCAC (SEQ ID NO:307)

-97044608

2971E02 MGVSDVPRDLEVVAAT PTSLLISWDAPSDDYGYYRITY GETGGNSPVQEFTVPIGKGTAT ISGLKPGVDYTITVYAVEFPWP HAGYYHRPISINYRTEIDKPSQ NNNNNNN (SEQ ID NO:308) ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTG ATCAGCTGGGACGCGCCTTCGGATGATTACGGTTATTACCGCATCACTTACGGCGAAACAGGAGGCAA TAGCCCTGTCCAGGAGTTCACTGTGCCTATTGGTAAAGGAACAGCTACCATCAGCGGCCTTAAACCTG GCGTTGATTATACCATCACTGTGTATGCTGTCGAGTT TCCGTGGCCACATGCTGGTTACTATCATCGG CCAATTTCCATTAATTACCGCACAGAAATTGACAAACCATCCCAGCACCATCACCACCACCAC (SEQ ID NO:309)

SEQ ID NO sequences of the family of pegylated anti-PCSK9 adnectins described in this invention are presented in table. 5.

Table 5

A family of anti-PCSK9 adnectins mutated at a cysteine residue for subsequent pegylation

ATI#/ Clone# [Description] Subsequence Amino acid Nucleotide ATTOO 1170 [2013E01-no His tag, Cys mutant] MGVSDVPRDLEVVA ATPTSLLISWVPPSDDYGY YRITYGETGGNSPVQEFTV PIGKGTATISGLKPGVDYT ITVYAVEFPWPHAGYYHRP ISINYRTEIDKPCQ (SEQ ID NO:78) ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGAT CAGCTGGGTCCCGCCTTCAGATGATTACGGTTATTACCGCATCACTTACGGCGAAACAGGAGGCAATAGCC CTGTCCAGGAGTTCACTGTGCCTATTGGTAAAGGAACAGCTACCATCAGCGGCCTTAAACCTGGCGTTGAT TATACCATCACTGTGTATGCTGTCGAG TTTCCGTGGCCACATGCTGGTTACTATCATCGGCCAATTTCCAT TAATTACCGCACAGAAATTGACAAACCATGCCAGTG (SEQ ID NO :79) ATI001172 [2013E01-without Histag, Cys mutant] MGVSDVPRDLEVVA ATPTSLLISWVPPSDDYGY YRITYGETGGNSPVQEFTV PIGKGTATISGLKPGVDYT ITVYAVEFPWPHAGYYHRP ISINYRTEGSGC (SEQ ID NQ:80) ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGAT CAGCTGGGTCCCGCCTTCAGATGATTACGGTTATTACCGCATCACTTACGGCGAAACAGGAGGCAATAGCC CTGTCCAGGAGTTCACTGTGCCTATTGGTAAAGGAACAGCTACCATCAGCGGCCTTAAACCTGGCGTTGAT TATACCATCACTGTGTATGCTGTCGAGTTTCCGTGGCCACATGCTGGTTACTATCATCGGCCAATTTCCAT TAATTACCGAACAGAAGGTAGCGGTTGCTG (SEQ ID NO:81) ATI001174* £2013E01 - without His tag, Cys mutant designated as ATI-1174 MGVSDVPRDLEVVA ATPTSLLISWVPPSDDYGY YRITYGETGGNSPVQEFTV PIGKGTATISGLKPGVDYT ITVYAVEFPWPHAGYYHRP ISINYRTEIEKPCQ (SEQ ID NO:82) ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGAT CAGCTGGGTCCCGCCTTCAGATGATTACGGTTATTACCGCATCACTTACGGCGAAACAGGAGGCAATAGCC CTGTCCAGGAGTTCACTGTGCCTATTGGTAAAGGAACAGCTACCATCAGCGGCCTTAAACCTGGCGTTGAT TATACCATCACTGTGTATGCTGTCGAGTTTCCGTGGCCACATGCTGGTTACTATCATCGGCCAATTTCCAT TAATTACCGCACAGAAATTGAGAAACCATGCCAGTG (SEQ ID NO:83) ATI001114* £2013E01mu tant according to Cvs1 also designated as ATI-1114 ATI000959* [1459D05 Cys mutant] ATI001063* [1784F03 Cys mutant] MGVSDVPRDLEVVA ATPTSLLISWVPPSDDYGY YRITYGETGGNSPVQEFTV PIGKGTATISGLKPGVDYT ITVYAVEFPWPHAGYYHRP ISINYRTGSGCHHHHHH (SEQ ID NO:84) MGVSDVPRDLEVVA ATPTSLLISWPPPSHGYGY YRITYGETGGNSPVQEFTV PPGKGTATISGLKPGVDYT ITVYAVEYPYKHSGYYHRP ISINYRTEIDKPCQHHHHH H (SEQ ID NO:86) MGVSDVPRDLEVVA ATPTSLLISWRPPIHAYGY YRITYGETGGNSPVQEFTV PIVEGTATISGLKPGVDYT ITVYAVEYTFKHSGYYHRP ISINYRTEIDKPCQHHHHH H (SEQ ID NO:88) ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGAT CAGCTGGGTCCCGCCTTCAGATGATTACGGTTATTACCGCATCACTTACGGCGAAACAGGAGGCAATAGCC CTGTCCAGGAGTTCACTGTGCCTATTGGTAAAGGAACAGCTACCATCAGCGGCCTTAAACCTGGCGTTGAT TATACCATCACTGTGTATGCTGTCGAG TTTCCGTGGCCACATGCTGGTTACTATCATCGGCCAATTTCCAT TAATTACCGCACAGGTAGCGGTTGCCACCATCACCACCATCAC (SEQ ID NO :85) ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGAT CAGCTGGCCGCCGCCGTCTCATGGTTACGGTTATTACCGCATCACTTACGGCGAAACAGGAGGCAATAGCC CTGTCCAGGAGTTCACTGTGCCGCCTGGTAAAGGTACAGCTACCATCAGCGGCCTTAAACCTGGCGTTGAT TATACCATCACTGTGTATGCTGTCGAATA CCCGTACAAACATTCTGGTTACTACCATCGTCCAATTTCCAT TAATTACCGCACAGAAATTGACAAACCATGCCAGCACCATCACCACCACCAC (SEQ ID NO :87) ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGAT CAGCTGGAGGCCGCCGATTCATGCTTACGGGTATTACCGCATCACTTACGGCGAAACAGGAGGCAATAGCC CTGTCCAGGAGTTCACTGTGCCTATTGTTGAAGGTACAGCTACCATCAGCGGCCTTAAACCTGGCGTTGAT TATACCATCACTGTGTATGCTGTCGA ATATACATTTAAACATTCCGGTTACTACCATCGTCCAATTTCCAT TAATTACCGCACAGAAATTGACAAACCATGCCAGCACCATCACCACCACCAC (SEQ ID NO :89)

- 98 044608

ATU0SH9* [2012A04 Cys mutant] MGVSDVPRDLEVVA ATPTSLLISWRPPSNGHGY YRITYGETGGNSPVQEFTV PVNEGTATISGLKPGVDYT ITVYAVEFPFKWSGYYHRP ISINYRTGSGCHHHHHH (SEQ ID NO:90) ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGAT CAGCTGGCGGCCCCCCTCTAATGGTCACGGTTATTACCGCATCACTTACGGCGAAACAGGAGGCAATAGCC CTGTCCAGGAGTTCACTGTGCCTGTTAATGAAGGTACAGCTACCATCAGCGGCCTTAAACCTGGCGTTGAT TATACCATCACTGTGTATGCTGTCGAAT TCCCCTTCAAGTGGTCGGGCTACTACCATCGACCAATTTTCCAT TAATTACCGCACAGGTAGCGGTTGCCACCATCACCACCATCAC (SEQ ID NO :91) ATI001117* [2011H05 Cys mutant] MGVSDVPRDLEVVA ATPTSLLISWVPSSHAYGY YRITYGETGGNSPVQEFTV PVGVGTATISGLKPGVDYT ITVYAVEYAFEGAGYYHRP ISINYRTGSGCHHHHHH (SEQ ID NO:92) ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGAT CAGCTGGGTTCCGTCGTCTCATGCCTACGGTTATTACCGCATCACTTACGGCGAAACAGGAGGCAATAGCC CTGTCCAGGAGTTCACTGTGCCTGTGGGGGTAGGTACAGCTACCATCAGCGGCCTTAAACCTGGCGTTGAT TATACCATCACTGTGTATGCTGTCGA ATACGCTTTCGAAGGGGCTGGTTACTACCATCGTCCAATTTCCAT TAATTACCGCACAGGTAGCGGTTGCCACCATCACCACCATCAC (SEQ ID NO :93) ATI001194* [2011HO5(V23D) Cys mutant] MGVSDVPRDLEVVA ATPTSLLISWDPSSHAYGY YRITYGETGGNSPVQEFTV PVGVGTATISGLKPGVDYT ITVYAVEYAFEGAGYYHRP ISINYRTEGSGCHHHHHH (SEQ ID NO:94) ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGAT CAGCTGGGACCCGTCGTCTCATGCCTACGGTTATTACCGCATCACTTACGGCGAAACAGGAGGCAATAGCC CTGTCCAGGAGTTCACTGTGCCTGTGGGGGTAGGTACAGCTACCATCAGCGGCCTTAAACCTGGCGTTGAT TATACCATCACTGTGTATGCTGTCGA ATACGCTTTCGAAGGGGCTGGTTACTACCATCGTCCAATTTCCAT TAATTACCGCACAGAAGGTAGCGGTTGCCACCATCACCACCATCAC (SEQ ID NO :95) 2011Н05 (V23E) Cys mutant MGVSDVPRDLEVVA ATPTSLLISWEPSSHAYGY YRITYGETGGNSPVQEFTV PVGVGTATISGLKPGVDYT ITVYAVEYAFEGAGYYHRP ISINYRTEGSGCHHHHHH (SEQ ID NO:96) ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGAT CAGCTGGGAACCGTCGTCTCATGCCTACGGTTATTACCGCATCACTTACGGCGAAACAGGAGGCAATAGCC CTGTCCAGGAGTTCACTGTGCCTGTGGGGGTAGGTACAGCTACCATCAGCGGCCTTAAACCTGGCGTTGAT TATACCATCACTGTGTATGCTGTCGA ATACGCTTTCGAAGGGGCTGGTTACTACCATCGTCCAATTTCCAT TAATTACCGCACAGAAGGTAGCGGTTGCCACCATCACCACCATCAC (SEQ ID NO :97) ATI001112 [1923B02 Cys mutant] MGVSDVPRDLEVVA ATPTSLLISWTPPPKGYGY YRITYGETGGNSPVQEFTV PVGEGTATISGLKPGVDYT ITVYAVEYTYNGAGYYHRP ISINYRTGSGCHHHHHH (SEQ ID NO:98) ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGAT CAGCTGGACGCCTCCCCCTAAAGGGTATGGTTATTACCGCATCACTTACGGCGAAACAGGAGGCAATAGCC CTGTCCAGGAGTTCACTGTGCCTGTTGGTGAAGGTACAGCTACCATCAGCGGCCTTAAACCTGGCGTTGAT TATACCATCACTGTGTATGCTGTCGA ATACACGTACAACGGTGCCGGTTACTACCACCGGCCAATTTCCAT TAATTACCGCACAGGTAGCGGTTGCCACCATCACCACCATCAC (SEQ ID NO :99) ATI001110 [1922G04 Cys mutant] MGVSDVPRDLEVVA ATPTSLLISWRPPSHAYGY YRITYGETGGNSPVQEFTV PIGKGTATISGLKPGVDYT ITVYAVEYPWKGSGYYHRP ISINYRTGSGCHHHHHH (SEQ ID NO:100) ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGAT CAGCTGGCGGCCGCCATCTCATGCTTATGGTTATTACCGCATCACTTACGGCGAAACAGGAGGCAATAGCC CTGTCCAGGAGTTCACTGTGCCTATTGGGAAAGGTACAGCTACCATCAGCGGCCTTAAACCTGGCGTTGAT TATACCATCACTGTGTATGCTGTCGAATACC CGTGGAAAGGTTCTGGTTACTACCATCGGCCAATTTCCAT TAATTACCGCACAGGTAGCGGTTGCCACCATCACCACCATCAC (SEQ ID NO:101)

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ATI001128 [1922G04 Cys mutant] MGVSDVPRDLEVVA ATPTSLLISWRPPSHAYGY YRITYGETGGNSPVQEFTV PIGKGTATISGLKPGVDYT ITVYAVEYPWKGSGYYHRP ISINYRTEIDKPCQHHHHH H (SEQ ID NO:102) ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGAT CAGCTGGCGGCCGCCATCTCATGCTTATGGTTATTACCGCATCACTTACGGCGAAACAGGAGGCAATAGCC CTGTCCAGGAGTTCACTGTGCCTATTGGGGAAAGGTACAGCTACCATCAGCGGCCTTAAACCTGGCGTTGAT TATACCATCACTGTGTATGCTGTCGAATACCCGTGGAAAGGTTCTGGTTACTACCATCGGCCAATTTCCAT TAATTACCGCACAGAAATTGACAAACCATGCCAGCACCACCACCACCACCAC (SEQ ID NO:103) ATI001184 * [1922G04(R23E) Cys mutant] MGVSDVPRDLEVVA ATPTSLLISWEPPSHAYGY YRITYGETGGNSPVQEFTV PIGKGTATISGLKPGVDYT ITVYAVEYPWKGSGYYHRP ISINYRTEGSGCHHHHHH (SEQ ID NO:104) ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGAT CAGCTGGGAACCGCCATCTCATGCTTATGGTTATTACCGCATCACTTACGGCGAAACAGGAGGCAATAGCC CTGTCCAGGAGTTCACTGTGCCTATTGGGAAAGGTACAGCTACCATCAGCGGCCTTAAACCTGGCGTTGAT TATACCATCACTGTGTATGCTGTCGAATACC CGTGGAAAGGTTCTGGTTACTACCATCGGCCAATTTCCAT TAATTACCGCACAGAAGGTAGCGGTTGCCACCATCACCACCATCAC (SEQ ID NO:105) 2381D04- Cys MGVSDVPRDLEVVA ATPTSLLISWEPFSRLPGG GEYYRITYGETGGNSPLQQ FTVVPGSKGTATISGLKPGV DYTITVYAVEFPYDHSGYY HRPISINYRTGSGCHHHHH H (SEQ ID NO:291) ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGAT CAGCTGGGAGCCGTTCAGCCGGTTGCCCGGGGGCGGCGAGTATTACCGGATCACTTACGGCGAAACAGGAG GCAATAGCCCTCTGCAGCAGTTCACTGTGCCTGGTTCTAAAGGTACAGCTACCATCAGCGGCCTTAAACCT GGCGTTGATTATACCATCACTGTGTATGCTGTCGAATTCCCGTACGACCATTCTGGTTACTACCATCGGCC CATTTCCATTAATTACCGCACAGGTAGCGGTTGCCACCATCACCACCATCAC (SEQ ID NO:292) 2382D09- Cys MGVSDVPRDLEVVA ATPTSLLISWDAPAEGYGY YRITYGETGGNSPVQEFTV PVSKGTATISGLKPGVDYT ITVYAVEFDFPGAGYYHRP ISINYRTGSGCHHHHHH (SEQ ID NO:293) ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGAT CAGCTGGGACGCTCCGGCTGAAGGGTACGGTTATTACCGCATCACTTACGGCGAAACAGGAGGCAATAGCC CTGTCCAGGAGTTCACTGTGCCTGTTTCTAAAGGTACAGCTACCATCAGCGGCCTTAAACCTGGCGTTGAT TATACCATCACTGTGTATGCTGTCGA ATTCGACTTCCCCGGCGCCGGTTACTACCATCGTCCAATTTCCAT TAATTACCGCACAGGTAGCGGTTGCCACCATCACCACCATCAC (SEQ ID NO:294) 2451В06- Cys MGVSDVPRDLEVVA ATPTSLLISWDAPADGGYG YYRITYGETGGNSPVQEFT VPVSKGTATISGLKPGVDY TITVYAVE FDFPGAGYYHR PISINYRTGSGCHHHHHH (SEQ ID NO:295) ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGAT CAGCTGGGACGCTCCGGCTGATGGTGGTTACGGTTATTACCGCATCACTTACGGCGAAACAGGAGGCAATA GCCCTGTCCAGGAGTTCACTGTGCCTGTTTCTAAAGGTACAGCTACCATCAGCGGCCTTAAACCTGGCGTT GATTATACCATCACTGTGTATGCTGTC GAATTCGACTTCCCGGGCGCCGGTTACTACCATCGTCCAATTTC CATTAATTACCGCACAGGTAGCGGTTGCCACCATCACCACCATCAC (SEQ ID NO:296) 2382E05- Cys MGVSDVPRDLEVVA AT PT S LLISWDAPAE GGY G YYRITYGETGGNSPVQEFT VPVSKGTATISGLKPGVDY TITVYAVE FDFPGAGYYHR PISINYRTGSGCHHHHHH (SEQ ID NO:297) ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGAT CAGCTGGGACGCTCCGGCTGAGGGCGGCTACGGTTATTACCGCATCACTTACGGCGAAACAGGAGGCAATA GCCCTGTCCAGGAGTTCACTGTGCCTGTTTCTAAAGGTACAGCTACCATCAGCGGCCTTAAACCTGGCGTT GATTATACCATCACTGTGTATGCTGTCGAATTCGACTTCCCCGGCGCCGGTTACTACCATCGTCCAATTTC CATTAATTACCGCACAGGTAGCGG TTGCCACCATCACCACCATCAC (SEQ ID NO:298) 2382B09- Cys MGVSDVPRDLEVVA ATPTSLLISWDAPADAYGY YRITYGETGGNSPVQEFTV PVSKGTATISGLKPGVDYT ITVYAVEFDYPGSGYYHRP ISINYRTGSGCHHHHHH (SEQ ID NO:299) ATGGGAGTTTCTGATGTGCCGCGCGACCTGGAAGTGGTTGCTGCCACCCCCACCAGCCTGCTGAT CAGCTGGGACGCTCCGGCTGACGCGTACGGTTATTACCGCATCACTTACGGCGAAACAGGAGGCAATAGCC CTGTCCAGGAGTTCACTGTGCCTGTTTCTAAAGGTACAGCTACCATCAGCGGCCTTAAACCTGGCGTTGAT TATACCATCACTGTGTATGCTGTCGA ATTCGACTACCCCGGCTCCGGTTACTACCATCGTCCAATTTCCAT TAATTACCGCACAGGTAGCGGTTGCCACCATCACCACCATCAC (SEQ ID NO:300)

*Note. Some of the proteins listed have not been pegylated but are suitable for pegylation due to the presence of a mutated cysteine residue. Proteins that have been pegylated are marked with an asterisk.

Technology of fusion of nucleotide and protein sequences.

One aspect of the present invention is adnectin, which contains a fibronectin type III domain that binds PCSK9. One way to quickly obtain and test Fn3 domains with specific binding properties is the nucleotide-protein sequence fusion technology of the Adnexus division of the Bristol-Myers Squibb R&D Company. This application describes an in vivo expression and tag insertion technology called PROfusion, which uses protein-nucleic acid fusion (RNA- or DNA-protein fusion sequences) to identify novel polypeptides and amino acid motifs that are essential for protein binding. . The technology of fusion of nucleotide and protein sequences is a technology of covalent pairing of a protein with a nucleic acid encoding the given protein. Detailed information on nucleotide-protein sequence technology and methods for screening protein libraries based on the structural part of fibronectin is given in Szostak et al., U.S. patent No. 6258558, 6261804, 6214553, 6281344, 6207446, 6518018 and 6818418; And

Roberts et al., Proc. Natl. Acad. Sci., 94:12297-12302 (1997) and is incorporated herein by reference.

Vectors and polynucleotide variants.

Nucleic acids encoding any of the proteins or polypeptides described herein can be chemically synthesized. Codon usage can be optimized to increase product expression in the cell. The frequency of use of certain codons depends on the cell type selected. Codon usage patterns are known for E. coli and other bacteria, as well as mammalian cells, plant cells, yeast cells and insect cells. See Mayfield et al., Proc. Natl. Acad. Sci., USA, 100(2):438-442 (Jan. 21, 2003); Sinclair et al., Protein Expr. Purif, 26(I):96-105 (Oct. 2002); Connell, N.D., Curr. Opin. Biotechnol., 12(5):446-449 (Oct. 2001); Makrides et al., Microbiol. Rev., 60(3):512-538 (Sep. 1996); and Sharp et al., Yeast, 7(7):657-678 (Oct. 1991).

General principles for working with nucleic acids are described, for example, here: Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition, vol. 1-3, Cold Spring Harbor Laboratory Press (1989) or Ausubel, F. et al., Current Protocols in Molecular Biology, Green Publishing and Wiley-Interscience, New York (1987), and in periodic updates included herein via link. In general, a DNA sequence encoding a polypeptide is linked through certain manipulations to a sequence of suitable transcriptional or translational regulatory elements from mammalian, viral or insect genes. Such regulatory elements include a transcriptional promoter, an optional operator sequence to control transcription, a suitable sequence for the ribosome binding site on the mRNA, sequences encoding transcription and translation termination. Additionally, an origin of replication sequence is introduced, which ensures the ability of the construct to reproduce in the host cell, and a gene encoding a selective trait that facilitates the selection of transformed cells.

The proteins described in this application can be produced using recombinant DNA technology not only in intact form, but also as fusion polypeptides with heterologous polypeptides, which are preferably signal sequences or other polypeptides having specific cleavage sites at the N-terminus of the mature protein or polypeptide. The heterologous signal sequences selected should preferably be recognized and processed (ie, cleaved by a signal peptidase) by the host cell. An example of an N-terminal signal sequence for producing polypeptides in mammalian cells is the sequence METDTLLLWVLLLWVPGSTG (SEQ ID NO: 326), which is removed by the host cell after expression of the polypeptide.

For prokaryotic cells that do not recognize natural signal sequences, such sequences are replaced by prokaryotic signal sequences selected, for example, from the group of alkaline phosphatase, penicillinase, LPP or heat-stable enterotoxin II leader sequences.

For secretion into the external environment during production of the polypeptide in yeast cells, the native signal sequence can be replaced, for example, by the signal sequence of yeast invertase, factors (including leader sequences of Saccharomyces and Kluyveromyces alpha factors), acid phosphatase, C. albicans glucosamylase or signal sequences, described in the U.S. patent No. 5631144. When expressed in mammalian cells, mammalian signal sequences are used, as well as viral signal sequences for secretion, for example the signal sequence of the herpes simplex virus gD. Such DNA sequences can be ligated in frame with the DNA sequence encoding the protein.

Vector systems for cloning and expression contain sequences that ensure replication of the vector in one or more types of host cells. In general, vector cloning systems contain a sequence that allows the vector to replicate independently of the chromosomal DNA of the host cell, and include an origin of replication sequence or autonomously replicating sequences. Such sequences are well known for various bacteria, yeasts and viruses. Plasmid replication origin pBR322 is suitable for most gram-negative bacteria, 2-micron plasmid origin is suitable for yeast systems, various viral origins (SV40, polyomavirus, adenovirus, vesicular stomatitis virus, bovine papillomavirus) are suitable for cloning vectors amplified in mammalian cells .

Expression and cloning vectors may contain selection genes, also called selectable marker genes. Typical selectable marker genes encode proteins that a) provide resistance to antibiotics and other toxins, for example ampicillin, neomycin, methotrexate, tracycline; b) restore the defect of auxotrophic genes; c) provide the synthesis of necessary nutrients that are not available from the cultivation environment, for example, the gene encoding

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D-alanine racemase for Bacilli bacteria.

Expression and cloning vectors typically contain a promoter sequence recognized by host cell systems and fused directly to a sequence encoding a protein of the invention, for example a fibronectin structural moiety protein. Promoter sequences useful for prokaryotic systems include the phoA gene promoter, the beta-lactamase and lactose system promoter, the alkaline phosphatase promoter, the tryptophan system (tip) promoter, hybrid promoters such as the tan promoter. However, other known bacterial promoters are also applicable. Promoters for use in bacterial systems also contain a Shine-Dalgarno (S.D.) sequence attached directly to the DNA encoding the protein described in the present invention. Promoter sequences for eukaryotic systems are known. Almost all eukaryotic genes have an AT-rich region located 25-30 bp. to the transcription initiation point. Another sequence located 70-80 bp. to the transcription initiation point for many genes, CNCAAT, in which N represents any nucleotide. At the 3'-end of most eukaryotic genes there is the sequence AATAAAA, which is probably a signal for polyadenylation (adding poly-A) to the 3'-end of the coding sequence (mRNA). All of these sequences are suitable for addition to the structure of expression vectors in mammalian cells.

Examples of useful promoter sequences for use in yeast systems include the promoters of 3-phosphoglycerate kinase or other glycolytic enzymes such as enolases, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase , triosephosphate isomerase, phosphoglucose isomerase and glucokinase.

The process of transcription from expression vectors in mammalian cells can be controlled, for example, by promoters from viral genomes such as polyomavirus, fowlpox virus, adenovirus (such as adenovirus 2), bovine papillomavirus, avian sarcoma virus, cytomegalovirus, retrovirus, virus hepatitis B and, most preferably, the SV40 virus; due to heterologous mammalian promoters compatible with the host cell, for example, actin or immunoglobulin promoters; due to the promoters of heat shock proteins.

The level of transcription of DNA encoding the proteins described in this invention by cells of higher eukaryotes, in most cases, increases due to the introduction of special enhancer sequences into the vector sequence. Currently, many sequences of mammalian gene enhancers are known (globin, elastase, albumin, alpha-fetoprotein, insulin). However, viral enhancers are usually used. Examples include the SV40 virus enhancer located at the end of the origin of replication sequence (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma virus enhancer located at the end of the origin of replication sequence, adenovirus enhancers. See Yaniv, Nature, 297:17-18 (1982) on enhancer elements for activating eukaryotic promoters. Enhancers can be introduced into the vector sequence at a 5' or 3' position relative to the peptide coding sequence, however, it is preferred that the enhancer be located at a 5' position relative to its promoter.

Expression vectors used in eukaryotic cells (eg, yeast, fungi, insects, plants, humans, or the nuclear cells of other multicellular organisms) also contain sequences necessary to terminate transcription and stabilize the mRNA in the cell. Such sequences are usually found in the 5' and less commonly 3' untranslated regions of eukaryotic or viral DNA or cDNA. Such regions contain nucleotide segments transcribed as polyadenylation signals in the untranslated portion of the mRNA encoding the protein described in this invention. One useful transcription termination sequence is the bovine growth hormone polyadenylation region sequence. See WO 94/11026 and the expression vectors described here.

Recombinant DNA can also include any type of tag sequence that is used to purify the protein being produced. Examples of tag sequences include, but are not limited to, histidine tag, FLAG tag, myc tag, HA tag, GST tag. Suitable vector systems for use in bacterial, fungal, yeast and mammalian cells are described in Cloning Vectors: A Laboratory Manual, (Elsevier, New York (1985)), and the corresponding description of which is incorporated herein by reference.

The expression construct is introduced into cells in a manner appropriate to the host cell type as will be apparent to those skilled in the art. The relevant field of knowledge has developed a wide range of methods for introducing nucleic acids into cells, including, but not limited to, electroporation; transfections with calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran or other substances; ballistic transfection; lipofection; infections (when the vector is an infectious agent).

Suitable host cells are prokaryotic, yeast, mammalian or bacterial cells. Suitable bacteria are gram-positive or gram-negative organisms,

- 102 044608 e.g. E.coli or Bacillus spp. Yeast cells, mainly species of the genus Saccharomyces, for example S. cerevisiae, can also be used to produce polypeptides. Various mammalian or insect cell cultures can also be used to produce recombinant proteins. Baculovirus systems for producing heterologous proteins in insect cells are discussed in Luckow et al. (Bio/Technology, 6:47 (1988)). Examples of useful mammalian cell lines include endothelial cells, monkey kidney cells line COS-7, CV-1, L cells, C127, 3T3, Chinese hamster ovary (CHO) cells, human embryonic kidney cells, HeLa, 293, 293T and BHK. Purified polypeptides are obtained by culturing cells of a suitable vector/host system to produce the recombinant protein. For most applications, the small size of many of the polypeptides described in this application makes it preferable to produce the product in E. coli cells. The resulting protein is then isolated from the culture medium or cell extracts.

Protein production.

Host cells are transformed with the protein expression vectors described herein and cultured in conventional media suitably modified to activate promoters, select transformants, or amplify genes encoding the target sequence. In the examples presented here, bacterial cells of strain HMS174 were used for high-throughput protein production (HTPP) and medium-scale production. Cells of a given strain can be cultured in various media to produce the proteins described in this invention. For cells of this strain, commercially available media are applicable, such as Ham's F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma)), RPMI-1640 (Sigma) and Dulbecco's Modified Eagle's Medium ((DMEM), (Sigma)) . In addition, for culturing cells of this strain, the medium described in Ham et al., Meth. Enzymol., 58:44 (1979), Barites et al., Anal. Biochem., 102:255 (1980), U.S. patent No. 4767704, 4657866, 4927762, 4560655, 5122469, 6048728, 5672502, or U.S. Patent No. RE 30985. Any of these culture media may be supplemented, if necessary, with hormones and/or other growth factors (such as insulin, transferrin or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium and phosphate ), buffers (such as HEPES), nucleotides (such as adenosine or thymidine), antibiotics (such as Gentamicin), trace components (defined as inorganic substances whose final concentration is usually in the micromolar range), glucose or an equivalent energy source. Any other necessary components may also be included in the medium in suitable concentrations known to those skilled in the art. Cultivation conditions such as temperature, pH, etc. are the same as those used for this type of host cell and are known to those skilled in the art.

The proteins described in this application can also be produced using cell-free translation systems. To do this, the nucleic acid sequences encoding the polypeptide must be modified for in vitro transcription and translation in a particular cell-free system (eukaryotic, such as a mammalian or yeast cell-free system, or prokaryotic, such as a bacterial cell-free translation system).

The proteins described in this invention can also be prepared by chemical synthesis (eg, using the methods described in Solid Phase Peptide Synthesis, 2nd edition, The Pierce Chemical Co., Rockford, IL (1984)). Modifications to these proteins can also be made by chemical synthesis methods.

The proteins described in the present invention can be purified by generally known protein chemistry isolation and purification methods. Non-limiting examples include extraction, recrystallization, salting out (eg with ammonium or sodium sulfates), centrifugation, dialysis, ultrafiltration, adsorption chromatography, ion exchange chromatography, hydrophobic interaction chromatography, normal phase chromatography, reverse phase chromatography, gel filtration, gel permeation chromatography, affinity chromatography, electrophoresis, countercurrent distribution or any combination of these methods. After purification, the polypeptides can be transferred into various buffer solutions and/or concentrated using any of various methods known in the relevant field, including, but not limited to, filtration and dialysis.

The purity of the polypeptide is preferably at least 85%, or preferably at least 95%, and most preferably at least 98%. Regardless of the exact purity number, the polypeptide is sufficiently purified for use as a pharmaceutical product.

A platform production method was used to produce anti-PCSK9 adnectin. AntiPCSK9 adnectin is produced in Escherichia coli (E.coli) cells. E. coli strain BLR (DE3) cells were transformed with an expression vector (pET9d/ATI001173) encoding a protein that accumulates inside the cell in a soluble form. Cells of this recombinant strain are cultured with stirring in a fermenter tank. At the end of the fermentation process, the cells are collected, lysed, and the lysate is then clarified for further purification. ATI001173 is

- 103 044608 variant ATI001114 without His tag. Purified anti-PCSK9 adnectin is conjugated to a 40 kDa branched methoxyPEG using a maleimide linker. The conjugated protein is re-purified to remove unbound PEG and anti-PCSK9 adnectin as well as impurities. Quality control is carried out with bulk medicinal substances.

Therapeutic applications in vivo.

One of the objects of the invention is the use of anti-PCSK9 adnectin in the treatment of atherosclerosis, hypercholesterolemia and other diseases associated with high cholesterol levels. This application also describes methods of administering anti-PCSK9 adnectin to a subject. In some embodiments, the subject is a human. In some embodiments, anti-PCSK9 adnectin is pharmaceutically acceptable for mammals, particularly humans. The term pharmaceutically acceptable polypeptide refers to polypeptides that do not cause significant side effects when administered to animals. Such polypeptides may be polypeptides that are almost free of endotoxin or contain very low levels of endotoxin.

Dosage form and administration.

This application also describes pharmaceutically acceptable compositions containing anti-PCSK9 adnectin or anti-PCSK9 adnectin fusion proteins described herein, in which the composition is substantially free of endotoxin. Therapeutic dosage forms containing anti-PCSK9 adnectin or fusion proteins with anti-PCSK9 adnectin are prepared for storage by mixing the described polypeptide at the desired degree of purity with optional physiologically acceptable carriers, excipients or stabilizers (Osol, A., Remington's Pharmaceutical Sciences, 16th edition (1980)) in the form of aqueous solutions, lyophilized or dried dosage forms. Acceptable carriers, excipients or stabilizers are non-toxic to the subject at the dosages and concentrations used and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecydimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkylparabens such as methyl or propylparaben, catechol, resorcinol, cyclohexanol, 3-pentanol and m-cresol); low molecular weight (less than 10 amino acid residues) polypeptides; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates, including glucose, mannose or dextrans; chelators such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (eg Zn-protein complexes); and/or non-ionic surfactants such as Tween, PLURONIC® or polyethylene glycol (PEG).

The dosage forms described in this application may also contain more than one active substance, depending on the specific indication for use, preferably having complementary activities and not undesirably influencing one another. Such molecules are suitable for combination in amounts that are effective to achieve the intended use.

Dosage forms for in vivo use must be sterile. This is achieved by filtration through sterile filtration membranes.

Qualified personnel should understand that the dosage of each therapeutic agent will depend on the properties of that agent.

For therapeutic applications, anti-PCSK9 adnectin or fusion proteins containing anti-PCSK9 adnectin are administered to a subject by a pharmaceutically acceptable route. It can be given intravenously as a bolus or by continuous infusion over a period of time, or subcutaneously. Suitable pharmaceutically acceptable carriers, excipients and diluents are well known and can be determined by those skilled in the art based on the clinical situation. Examples of suitable carriers, diluents and/or excipients include (1) Dulbecco's Phosphate Saline Solution; (2) 0.9% saline solution (0.9% w/v NaCl); and (3) 5% (w/v) dextrose.

The method described in this invention can be practiced in vitro, in vivo or ex vivo.

Administration of anti-PCSK9 adnectin or anti-PCSK9 adnectin fusion proteins and one or more additional therapeutic agents, administered either together or sequentially, can be carried out as described above for therapeutic applications. Suitable pharmaceutically acceptable carriers, diluents and excipients for co-administration will be determined by those skilled in the art depending on the properties of the particular therapeutic substances administered.

In the case of an aqueous dosage form, rather than in the case of a lyophilized form, the protein will generally be present at a concentration of from 0.1 to 100 mg/ml. However, variations within a wide range from the specified range are acceptable. When treating a disease, the appropriate dosage of anti-PCSK9 adnectin or anti-PCSK9 adnectin fusion proteins will depend on the type, severity and progress of the disease being treated, the course of previous therapy, medical history, the body's response to protein and the decisions of the supervising physician, regardless of the purpose of administration (prevention or other therapeutic purposes). This protein is suitable for administration to a patient once or for a series of administrations.

Serum albumin binding adnectins (SABA) fusions.

Other objects of the present invention are fusion proteins comprising anti-PCSK9 adnectin fused to ¹⁰ Fn3 human serum albumin binding domains (Serum Albumin Binding Adnectin ( ¹⁰ Fn3 domain) or SABA). Such fusion proteins have a longer plasma half-life in the presence of albumin compared to non-fusion anti-PCSK9 adnectins (eg, those not conjugated to a PK group).

Due to their small size (~10 kDa), Fn3 domains ¹⁰ are rapidly cleared from the circulation by renal filtration degradation (t1/2=15-45 min in mice; 1-3 h in monkeys). Fusion of Fn3 domain ¹⁰ , such as anti-PCSK9 adnectin, with a second polypeptide containing Fn3 domain ¹⁰ that specifically binds serum albumin, such as human serum albumin (HSA), can be used to increase the t1/2 of anti-PCSK9 adnectin.

In certain embodiments, the plasma half-life of anti-PCSK9 adnectin fused to SABA is increased compared to the plasma half-life of anti-PCSK9 adnectin not conjugated to SABA. In certain embodiments, the plasma half-life of the SABA fusion protein is at least 20, 40, 60, 80, 100, 120, 150, 180, 200, 400, 600, 800, 1000, 1200, 1500, 1800, 1900, 2000, 2500 or 3000% higher than the plasma half-life of non-SABA-conjugated anti-PCSK9 adnectin. In other embodiments, the plasma half-life of the SABA fusion protein is at least 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 10, 12, 13, 15, 17, 20, 22, 25, 27, 30, 35, 40 or 50 times higher than the plasma half-life of anti-PCSK9 adnectin not conjugated to SABA. In some embodiments, the plasma half-life of the SABA fusion protein is at least 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 135, 140, 150, 160 or 200 hours.

In certain embodiments, the serum albumin binding portion of the fusion protein binds HSA with a K _D of less than 3, 2.5, 2, 1.5, 1 μM, 500, 100, 50, 10, 1 nM, 500, 100, 50, or 10 pM. In certain embodiments, the serum albumin binding portion of the fusion protein binds HSA with a K _D of less than 3, 2.5, 2, 1.5, 1 μM, 500, 100, 50, 10, 1 nM, 500, 100, 50, or 10 pM per pH range 5.5-7.4 at a temperature of 25 or 37°C. In certain embodiments, the serum albumin binding portion of the fusion protein binds to HSA more strongly at pH less than 7.4 compared to binding at pH 7.4.

Accordingly, the SABA fusion molecules described herein are useful for increasing the plasma half-life of anti-PCSK9 adnectin by creating anti-PCSK9 adnectin fusion proteins with SABA. The present invention contemplates the use of SABA fusion molecules in diseases caused by dysregulation of PCSK9.

The fusion can be performed by attaching anti-PCSK9 adnectin to either end of the SABA sequence, i.e. Any configuration can be obtained: SABA-anti-PCSK9 adnectin or anti-PCSK9 adnectin-SABA.

In humans, the concentration of HSA in plasma is 600 μM, at _1/2 19 days. The increase in the t _1/2 parameter is possibly due to the recycling of HSA due to the neonatal Fc receptor (FcRn). Following endosomal uptake by endothelial cells, HSA binds to FcRn, resulting in HSA being released back into the bloodstream without lysosomal degradation. This binding is pH-dependent. The FcRn receptor is expressed by a large number of cells and this HSA recycling pathway is considered constitutive. In most cell types, almost all of the FcRn receptor is located within intracellular sorting endosomes. HSA is readily internalized through a nonspecific mechanism during pinocytosis and is protected from degradation in lysosomes by interacting with FcRn. Under acidic pH conditions inside the endosome, the binding affinity of HSA to FcRn increases (5 μM at pH 6.0). Binding of HSA to FcRn abolishes the lysosomal degradation of HSA and leads to the release of the HSA molecule to the cell surface through transcytosis.

In certain embodiments, a portion of the SABA fusion protein described herein is capable of binding monkey and/or rat and/or mouse serum albumin. In certain embodiments, the human serum albumin (HSA) binding portion of the SABA fusion protein described herein is capable of binding rhesus monkey serum albumin (RhSA) or cynomolgus serum albumin (CySA) with a K _D constant of less than 3.2.5 , 2, 1.5, 1 µM, 500, 100, 50, 10, 1 nM, 500 or 100 pM.

In certain embodiments, the serum albumin binding portion of the SABA fusion protein described herein is capable of binding domains I and/or II of HSA. In one embodiment, the HSA binding portion of the SABA fusion protein does not bind to domain III of HSA.

In certain embodiments, the serum albumin binding portion of the SABA fusion protein comprises a sequence having at least 40, 50, 60, 70, 75, 80, or 85%

- 105 044608 identical to domain ¹⁰ of wild type Fn3 (SEQ ID NO: 1). In one embodiment, at least one of the BC, DE, or FG loops is altered compared to domain ¹⁰ of wild-type Fn3. In another embodiment, at least two of the BC, DE or FG loops are altered compared to wild type Fn3 domain ¹⁰ . In another embodiment, all three loops BC, DE or FG are changed compared to domain ¹⁰ of wild type Fn3. In other embodiments, SABA contains a sequence having at least 40, 50, 60, 70, 75, 80, 85, 90 or 95% identity to any of the 26 SABA sequences listed in table. 6 (ie SEQ ID NO: 334, 338, 342, 346 and 348-370) or to any of the extended SABA sequences shown in table. 6 (i.e. SEQ ID NO: 420-447, excluding 6xШS tag).

In certain embodiments, the serum albumin binding adnectin sequences based on the Fn3 domain ¹⁰ structure can be generally expressed as

EVVAAT(X) _a SLLI(X) _x YYRITYGE(X)6QEFTV(X) _y ATI(X) _c DYTITVYAV(X) _z IS

INYRT (SEQ ID NO: 328)

In certain embodiments, the serum albumin binding adnectin sequences based on the Fn3 domain ¹⁰ structure may be generally expressed as EVVAATPTSLLI(X) _x YYRITYGETGGNSPVQEFTV(X) _y ATISGLKPGVDYTIT

VYAV(X)zISINYRT (SEQ Ш NO: 329)

As described herein for anti-PCSK9 adnectins, the sequences are SEQ ID NO: 328 and

329 can be applied to SABA molecules in the same way. In embodiments of the invention, the BC, DE or FG loops represented by (X) _x , (X) _y and (X) _z , respectively, are replaced by polypeptides containing the BC, DE or FG loop sequences from any other HSA binding protein, the sequences of which are given in table 6 below (i.e., SEQ ID NOs: 330, 334, 338, 342, 346 and 348-370 in Table 6). In certain embodiments, the sequences of loops BC, DE or FG, given in table. 6 may contain one or more amino acid residues flanking N- or

C-end. In particular, the BC loop may contain SW residues at the N-terminus of the BC loop sequence given in Table. 6 when replacing (X)x in the sequence SEQ ID NO: 328. Similarly, the DE loop may contain a P residue before the DE loop sequence and a T residue after the DE loop when replacing (X) _y in the sequence SEQ ID NO: 328. An FG loop may contain a P residue after an FG loop sequence when it replaces (X) _z in SEQ ID NO: 328. For example, SEQ ID NO: 330 shows that BC, DE or FG loops contain the sequence

HSYYEQNS (SEQ ID NO: 638), YSQT (SEQ ID NO: 639) and YGSKYYY (SEQ ID NO: 640), respectively.

However, when replacing (X) _x , (X) _y and (X) _z in the sequence SEQ ID NO: 328, i.e. loops BC, DE and FG, sequence (X) _x may be SWHSYYEQNS (SEQ ID NO: 641), sequence (X) _y may be PYSQTT (SEQ ID NO: 642), sequence (X) _z may be YGSKYYYP (SEQ ID NO: 643).

In certain embodiments, the SABA sequence used to create the fusion proteins described in this application may contain the sequences of SEQ ID NO: 328 or 329, in which the loop sequences BC, DE or FG, respectively represented by the designations (X)x, (X) y and (X)z are replaced by the corresponding set of BC, DE or FG loop sequences from any of the 26 SABA core sequences (i.e. SEQ ID NOs: 330, 334, 338, 342, 346 and 348-370 in Table 6 ) or sequences that are at least 75, 80, 85, 90, 95, 97, 98 or 99% identical to the BC, DE or FG loop sequences of the 26 SABA core sequences. In exemplary embodiments, the SABA sequence as described herein is defined by SEQ ID NO: 329 and includes a set of BC, DE or FG loop sequences from any of the 26 SABA core sequences (i.e., SEQ ID NO: 330, 334, 338, 342, 346 and 348-370 in Table 6), optionally with N- or C-terminal additions to the loop sequences as described above. For example, SABA1 has a core sequence defined by SEQ ID NO: 330 and contains the BC, DE or FG loop sequences defined by SEQ ID NO: 331-333, respectively. Thus, a SABA sequence based on the SABA1 core sequence may comprise the sequence SEQ ID NO: 328 or 329, in which (X) _x contains the sequence SEQ ID NO: 331, (X) _y contains the sequence SEQ ID NO: 332, (X ) _z contains the sequence SEQ ID NO: 333. In some embodiments, the sequences that replace (X)x, (X)y and (X)z contain additional amino acid residue(s) at one or both ends of the loops, as described above . The present invention contemplates the use of BC, DE or FG loop sequences from other SABA core sequences for similar constructions of this set. Structural regions of such a SABA sequence may contain anywhere from 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 substitution, conservative substitution, deletion or insertion with respect to structural amino acid residues of SEQ ID NO: 1. Such structural modifications may be made as long as the SABA polypeptide remains capable of binding serum albumin, such as HSA, to the desired value K _D .

In certain embodiments, the SABA sequence (for example, the afterbirth

- 106 044608 SABA activity or a sequence based thereon as described above) may be modified to contain an N-terminal extension sequence and/or a C-terminal extension sequence. Examples of extension sequences are given in table. 6. For example, the sequence SEQ ID NO: 420 designated SABA1.1 contains the core sequence SABA1 (SEQ ID NO: 330) with the N-terminal sequence MGVSDVPRDLE (SEQ ID NO: 371 designated AdNT1) and the C-terminal sequence EIDKPSQ ( SEQ ID NO: 380, designated AdCT1). The SABA1.1 sequence also contains a 6His tag at the C-terminus, however, it should be understood that the 6His tag sequence is completely optional and can be located anywhere in the N- and C-terminal extension sequences, or may be completely absent. Any of the examples of N- and C-terminal extension sequences given in table. 6 (SEQ ID NO: 371-395), as well as any of their variants, can be used to modify the SABA core sequence shown in table. 6.

In certain embodiments, the C-terminal extension sequences (also called tails) contain residues E and D and can be 8 and 50, 10 and 30, 10 and 20, 5 and 10, and 2 and 4 amino acid residues in length. In some embodiments, the tail sequences include ED linkers, in which the sequence consists of tandem ED repeats. In exemplary embodiments of the invention, the tail sequences contain 2-10, 2-7, 2-5, 3-10, 3-7, 3-5, 3, 4 or 5 ED repeats. In certain embodiments, these ED-based tail sequences may also include additional amino acid residues, such as, for example, EI, EID, ES, EC, EGS, and EGC. Such sequences are based, in part, on known adnectin tail sequences, such as the sequence EIDKPSQ (SEQ ID NO: 380), in which the D and K residues are deleted. In exemplary embodiments, the tail sequences based on the ED sequence contain E, I, or EI residues before the ED repeats.

In other embodiments, the tail sequences may be combined with other known linker sequences (eg, SEQ ID NOs: 396-419 in Table 6) as necessary when constructing a SABA fusion molecule.

Conjugation/linkers.

SABA-containing fusion proteins can be linked covalently or non-covalently. In some embodiments, serum albumin binding domain ¹⁰ of Fn3 may be covalently or non-covalently linked to anti-PCSK9 adnectin via a polypeptide linker. Useful linkers for joining Fn3 domains are those that allow the domains to be separated, allowing independent folding and forming a spatial structure capable of binding the target molecule with high affinity.

This application describes a number of suitable linkers that meet these criteria, including glycine-serine linkers, glycine-proline linkers, and a linker with the amino acid sequence PSTSTST (SEQ ID NO: 416). The examples described herein demonstrate that Fn3 domains connected via polypeptide linkers retain target molecule binding properties. In some embodiments, the linker is a glycine-serine linker. These linkers contain glycine and serine residues and can be between 8 and 50, 10 and 30, and 10 and 20 amino acid residues in length. Examples include linkers with amino acid sequence (GS) ₇ (SEQ ID NO: 403), G(GS) ₆ (SEQ ID NO: 398) and G(GS) ₇ G (SEQ ID NO: 400).

Other linkers contain glutamic acid and include, for example, the sequences (GSE) ₅ (SEQ ID NO: 405) and GGSE GGSE (SEQ ID NO: 409).

Other examples of glycine-serine linkers include (GS) ₄ (SEQ ID NO: 402), (GGGGS) ₇ (SEQ ID NO: 411), (GGGGS) ₅ (SEQ ID NO: 412) and (GGGGS) ₃ G (SEQ ID NO: 413).

In some embodiments, the linker is glycine-proline.

Such linkers contain glycine and proline residues and can be between 3 and 30, 10 and 30, 3 and 20 amino acid residues in length. Examples of such linkers include those with the sequences (GP) _3G (SEQ ID NO: 414), (GP) _5G (SEQ ID NO: 415) and gpg.

In other embodiments, the linker can be proline-alanine in length between 3 and 30, 10 and 30, 3 and 20 amino acid residues. Examples of proline-alanine linkers include the sequences (P\E (SEQ ID NO: 417), (PA) ₆ (SEQ ID NO: 418) and (PA) ₉ (SEQ ID NO: 419).

The present invention assumes that the optimal length and amino acid composition of the linker can be determined using standard experiments taking into account the information provided in this application. In some embodiments, anti-PCSK9 adnectin is linked to SABA via a polypeptide linker containing a protease cleavage site that can be cleaved by a protease in blood or tissue. Such embodiments may be used to release anti-PCSK9 adnectin for ease of delivery, improved therapeutic properties, or more efficient production.

Additional linkers or spacers may be introduced at the C-terminus of the Fn3 domain between the Fn3 domain sequence and the polypeptide linker sequence. Additional linkers or spacers may be introduced at the N-terminus of the Fn3 domain between the Fn3 domain sequence and the polypeptide linker sequence.

In some embodiments, the anti-PCSK9 adnectin may be directly or indirectly linked to SABA via a polymer linker. Polymeric linkers can be used to optimally vary the distance between components of a fusion protein to create a fusion protein with one or more of the following characteristics: 1) reduced or increased steric mismatch of binding of one or more protein domains upon binding of a target protein; 2) increased protein stability or solubility; 3) reduced level of protein aggregation; and 4) increased overall avidity or affinity of the protein.

In some embodiments, the anti-PCSK9 adnectin is linked to SABA via a biocompatible polymer, such as a polymeric sugar. Such polymeric sugar may include a cleavage site that is cleaved by an enzyme in the blood or target tissue. Such embodiments can be used to release anti-PCSK9 adnectin for easier delivery, improved therapeutic properties, or more efficient production.

Sequence summary data.

Many of the sequences referenced in the Serum Albumin Binding Adnectin (SABA) Fusion Proteins and Conjugation/Linkers sections are collected in Table 1. 6 below. Unless otherwise noted, all N-terminal extension sequences are indicated with a single underline, all C-terminal tail/extension sequences are indicated with a double underline, and linker sequences are enclosed in parentheses. The BC, DE, and FG loop regions are shaded for each SABA core sequence. In addition to the above, these sequence modifications (eg, N- or C-terminal extensions and linkers) can also be used to modify anti-PCSK9 adnectin molecules.

Table 6

Summary of Example Sequences

EQ ID Named out of sequence Description Subsequence Examples of Serum Albumin Binding Adnectins (SABA) 27 ¹⁰ Fn3WT core sequence Human Fn3 domain ¹⁰ wild-type core sequence EVVAATPTSLLISWDAPAVTVRYYRITYG ETGGNSPVQE FTVPGSKSTATISGLKPGVDYTITV YAVTGRGDSPASSKPISINYRT 28 ¹⁰ Fn3v6 Fn3 domain analogue ¹⁰ containing 6 variable loops EVVAAT(X)aSLLI(X) _x YYRITYGE(X) _ь QEFTV (X) _y ATI (X) _C DYTITVYAV(X) _Z ISINYRT 29 ¹⁰ Fn3v3 Fn3 domain analogue ¹⁰ containing 3 variable loops EVVAATPTSLLI (X) _X YYRITYGETGGNSP VQEFTV(X) _y ATISGLKPGVDYTITVYAV(X) _Z IS INYRT thirty SABA1 Core sequence 1 Adnectina EVVAATPTSLLISWHSYYEQNSYYRITYG ETGGNSPVQE FTVPY SQTTATISGLKPGVDYTITV YAVYGSKYYYPISINYRT 31 SABA1 Sun Core sequence 1 loop BC HSYYEQNS 32 SABA1 DE Core sequence 1 loop DE YSQT 33 SABA IF G Core sequence 1 loop FG YGSKYYY

- 108 044608

34 SABA2 Core sequence 2 Adnectin EVVAATPTSLLISWPKYDKTGHYYRITYG ETGGNSPVQE FTVPTRQTTATISGLKPGVDYTITV YAVSKDDYYPHEHRPISINYRT 35 SABA2 Sun Core sequence 2 loops BC PKYDKTGH 36 SABA2 DE Core sequence 2 loops DE TRQT 37 SABA2F G Core sequence 2 loops FG SKDDYYPHEHR 38 SABA3 Core sequence 3 Adnectin EVVAATPTSLISWSNDGPGLSYYRITYG ETGGNSPVQE FTVPS SQTTATISGLKPGVDYTITV YAVSYYTKKAYSAGPISINYRT 39 SABA3 Sun Core sequence 3 loops BC SNDGPGLS 40 SABA3 DE Core sequence 3 loops DE SSQT 41 SABA3F G Core sequence 3 loops FG SYYTKKAYSAG 42 SABA4 Core sequence 4 Adnectin; containing mutations in the structural part (highlighted in bold); structurally the best option SABA5 EMVAATPTSLLISWEDDSYYSRYYRITYG ETGGNSPVQEFTVPSDLYTATISGLKPGVDYTITV YAVTYDVTDLIMHEPISINYRT

- 109 044608

43 SABA4 Sun Core sequence 4 loops BC EDDSYYSR 44 SABA4 DE Core sequence 4 loops DE SDLY 45 SABA4F G Core sequence 4 loops FG YDVTDLIMHE 46 SABA5 Core sequence 5 Adnectin; see description for SABA4; adjusted residuals are in bold EWAATPTSLLISWEDDSYSRYYRITYG ETGGNSPVQEFTVPSDLYTATISGLKPGVDYTITV YAVTYDVTDLIMHEPISINYRT 47 SABA5 Sun Core sequence 5 loops BC EDDSYYSR 48 SABA5 DE Core sequence 5 loop DE SDLY 49 SABA5F G Core sequence 5 loops FG YDVTDLIMHE 50 SABA6 Adnectin core sequence 6 EVVAATPTSLLISWYMDEYDVRYYRITYG ETGGNSPVQE FTVPNYYNTATISGLKPGVDYTITV YAVTRIKANNYMYGPISINYRT 51 SABA7 Adnectin core sequence 7 EVVAATPTSLLISWNHLEHVARYYRITYG ETGGNSPVQE FTVPEYPTTATISGLKPGVDYTITV YAVTITMLKYPTQSPISINYRT 52 SABA8 Adnectin core sequence 8 EVVAATPTSLLISWGHYRRSGHYYRITYG ETGGNSPVQEFTVDPSSYTATISGLKPGVDYTITV YAVSKDDYYPHEHRPISINYRT

- 110 044608

53 SABA9 Core sequence 9 Adnectin EVVAATPTSLLISWDASHYERRYYRITYG ETGGNSPVQE FTVPRYHHTATISGLKPGVDYTITV YAVTQAQEHYQPPISINYRT 54 SABA 10 Core sequence 10 Adnectin EVVAATPTSLLISWNSYYHSADYYRITYG ETGGNSPVQE FTVPY PPTTATISGLKPGVDYTITV YAVYSAKSYYPISINYRT 55 SABAH Core sequence 11 Adnectin EVVAATPTSLLISWSKYSKHGHYYRITYG ETGGNSPVQE FTVPSGNATATISGLKPGVDYTITV YAVEDTNDYPHTHRPISINYRT 56 SABA 12 Core sequence 12 Adnectin EVVAATPTSLLISWHGEPDQTRYYRITYG ETGGNSPVQE FTVPPYRRTATISGLKPGVDYTITV YAVTSGYTGHYQPISINYRT 57 SABA 13 Core sequence 13 Adnectin EVVAATPTSLLISWSKYSKHGHYYRITYG ETGGNSPVQEFTVDPSSYTATISGLKPGVDYTITV YAVSKDDYYPHEHRPISINYRT 58 SABA 14 Core sequence 14 Adnectin EVVAATPTSLLISWYEPYTPIHYYRITYG ETGGNSPVQE FTVPGYYGTATISGLKPGVDYTITV YAVYGYYQYTPISINYRT 59 SABA 15 Core sequence 15 Adnectin EVVAATPTSLLISWSKYSKHGHYYRITYG ETGGNSPVQE FTVPSGNATATISGLKPGVDYTITV YAVSDDNKYYHQHRPISINYRT 60 SABA 16 Core sequence 16 Adnectin EVVAATPTSLLISWGHYRRSGHYYRITYG ETGGNSPVQEFTVDPSSYTATISGLKPGVDYTITV YAVSKDDYYPHEHRPISINYRT 61 SABA 17 Core sequence 17 Adnectin EVVAATPTSLLISWSKYSKHGHYYRITYG ETGGNSPVQE FTVPSGNATATISGLKPGVDYTITV YAVEDTNDYPHTHRPISINYRT 62 SABA 18 Core sequence 18 Adnectin EVVAATPTSLLISWYEPGASVYYYRITYG ETGGNSPVQE FTVPSYYHTATISGLKPGVDYTITV YAVYGYYEYEPISINYRT 63 SABA 19 Core sequence 19 Adnectin EVVAATPTSLLISWQSYYAHSDYYRITYG ETGGNSPVQE FTVPY PPQTATISGLKPGVDYTITV YAVYAGSSYYPISINYRT

- 111 044608

64 SABA20 Core sequence 20 Adnectin EVVAATPTSLLISWGHYRRSGHYYRITYG ETGGNSPVQEFTVDPSSYTATISGLKPGVDYTITV YAVSKDDYYPHEHRPISINYRT 65 SABA21 Core sequence 21 Adnectina EVVAATPTSLLISWPEPGTPVYYYRITYG ETGGNSPVQE FTVPAYYGTATISGLKPGVDYTITV YAVYGYYDYSPISINYRT 66 SABA22 Core sequence 22 Adnectin EVVAATPTSLLISWYRYEKTQHYYRITYG ETGGNSPVQEFTVPPESGTATISGLKPGVDYTITV YAVYAGYEYPHTHRPISINYRT 67 SABA23 Core sequence 23 Adnectin EVVAATPTSLLISWVKSEEYYRYYRITYG ETGGNSPVQE FTVPYYVHTATISGLKPGVDYTITV YAVTEYYYAGAVVSVPISINYRT 68 SABA24 Core sequence 24 Adnectin EVVAATPTSLLISWYDPYTYGSYYRITYG ETGGNSPVQE FTVGPYTTTATISGLKPGVDYTITV YAVSYYYSTQPISINYRT 69 SABA25 Core sequence 25 Adnectin EVVAATPTSLISWSNDGPGLSYYRITYG ETGGNSPVQE FTVPS SQTTATISGLKPGVDYTITV YAVSYYTKKAYSAGPISINYRT 70 SABA26 Core sequence 26 Adnectin EVVAATPTSLLISWPDPYYKPDYYRITYG ETGGNSPVQE FTVPRDYTTATISGLKPGVDYTITV YAVYSYYGYYPISINYRT Examples of N-terminal extension sequences of Adnectins 71 AdNTl Example of a leader sequence MGVSDVPRDL 72 AdNT2 Example of a leader sequence GVSDVPRDL 73 AdNT3 Example of a leader sequence VSDVPRDL 74 AdNT4 Example of a leader sequence SDVPRDL

- 112 044608

75 AdNT5 Example of a leader sequence DVPRDL 76 AdNT6 Example of a leader sequence VPRDL 77 AdNT7 Example of a leader sequence PRDL 78 AdNT8 Example of a leader sequence RDL 79 AdNT9 Example of a leader sequence D.L. Examples of Adnectin C-terminal extension sequences 80 AdCTl Example of a tail sequence EIDKPSQ 81 AdCT2 Example of a tail sequence EIDKPS 82 AdCT3 Example of a tail sequence EIDKPC 83 AdCT4 Example of a tail sequence EIDKP 84 AdCT5 Example of a tail sequence EIDK 85 AdCT6 Example of a tail sequence EI

- 113 044608

86 AdCT7 Example of a tail sequence EIEKPSQ 87 AdCT8 Example of a tail sequence EIDKPSQLE 88 AdCT9 Example of a tail sequence EIEEDEDEDEDED 89 AdCTIO Example of a tail sequence EIEKPSQEDEDEDEDED 90 AdCTll Example of a tail sequence EGSGS 91 AdCT12 Example of a tail sequence EIDKPCQ 92 AdCT13 Example of a tail sequence EIEKPCQ 93 AdCT14 Example of a tail sequence GSGC 94 AdCT15 Example of a tail sequence EGSGC 95 AdCT16 Example of a tail sequence EIDKPCQLE 96 LI G(GS) ₂ GGSGS 97 L2 G(GS) ₄ GGSGSGSGS

- 114 044608

98 L3 G(GS) ₆ GGSGSGSGSGSGS 99 L4 G(GS) ₇ GGSGSGSGSGSGSGS 00 L5 G(GS) _7G GGSGSGSGSGSGSGSG 01 L6 GSGS GSGS 02 L7 (GS) ₄ GSGSGSGS 03 L7 (GS) ₇ GSGSGSGSGSGSGS 04 L9 GS(A)9GS GSAAAAAAAAAGS 05 LIO (GSE) ₅ GSEGSEGSEGSEGSE 06 Lil (PAS) ₅ PAS PAS PAS PAS PAS 07 L12 (GSP) ₅ GSPGSPGSPGSPGSP 08 L13 GS(TVAAPS) ₂ GSTVAAPSTVAAPS 09 L14 (GGSE) ₂ GGSEGGSE 10 L15 (ST) _3G STSTSTG eleven L16 (GGGGS) ₇ GGGGSGGGGSGGGGSGGGGSGGGGSGGGG SGGGGS 12 L17 (GGGGS) ₅ GGGGSGGGGSGGGGSGGGGSGGGGSGGGG S 13 L18 (GGGGS) _3G GGGGSGGGGSGGGGSG

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14 L19 (GP) _3G GPGPGPG 15 L20 (GP) _5G GPGPGPGPGPG 16 L21 P(ST) ₃ PSTSTST 17 L22 (RA) ₃ RARARA 18 L23 (ΡΑ)β RARARARARAR 19 L24 (RA) ₉ RARARARARARARARAR Examples of Adnectin core sequence extensions 20 SABA1. 1 Adnectin core sequence 1 containing terminal AdNTl and AdCTl sequences with His6-ToroM MGVSDVPRDLEVVAATRTSLLISWНSYYE QNSYYRITYGETGGNSPVQEFTVPYSQTTATISGL KPGVDYTITVYAVYGSKYYYPISINYRTEIDKPSQ HHHHHH 21 SABA1. 2 Adnectin core sequence 1 containing the terminal sequences AdNTl and AdCT8 MGVSDVPRDLEVVAATRTSLLISWНSYYE QNSYYRITYGETGGNSPVQEFTVPYSQTTATISGL KPGVDYTITVYAVYGSKYYYPISINYRTEIEDEDE DEDED 22 SABA1. 3 Adnectin core sequence 1 containing the terminal sequences AdNTl and AdCT9 with His6-ToroM MGVSDVPRDLEVVAATRTSLLISWНSYYE QNSYYRITYGETGGNSPVQEFTVPYSQTTATISGL KPGVDYTITVYAVYGSKYYYPISINYRTEIEDEDEDE DEDEDHHHHHH

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23 SABA2. 1 Adnectin core sequence 2 containing terminal AdNTl and AdCTl sequences with His6-ToroM MGVSDVPRDLEVVAATPTSLLISWPKYDK TGHYYRITYGETGGNSPVQEFTVPTRQTTATISGL KPGVDYTITVYAVSKDDYYPHEHRPISINYRTEID KPSQHHHHHH 24 SABA3. 1 Adnectin core sequence 3 containing terminal AdNTl and AdCTl sequences with His6-ToroM MGVSDVPRDLEVVAATPTSTSLLISWSNDGP GLSYYRITYGETGGNSPVQEFTVPSSQTTATISGL KPGVDYTITVYAVSYYTKKAYSAGPISINYRTgXg KPSQHHHHHH 25 SABA4. 1 Adnectin core sequence 4 containing terminal AdNTl and AdCTl sequences with His6-ToroM MGVSDVPRDLEΜVAATPTSLLISWEDDSY YSRYYRITYGETGGNSPVQEFTVPSDLYTATISGL KPGVDYTITVYAVT Y DVT DLIMHE PISINYRTEID KPSQHHHHHH 26 SABA5. 1 Core sequence 5 of Adnectin containing the terminal sequences AdNTl and AdCTl with the H18b tag MGVSDVPRDLEVVAATPTSLLISWEDDSY YSRYYRITYGETGGNSPVQEFTVPSDLYTATISGL KPGVDYTITVYAVT Y DVT DLIMHE PISINY RTEID KPSQHHHHHH

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27 SABA6. 1 Adnectin core sequence 6 containing terminal sequences AdNTl and AdCTl with His6-ToroM MGVSDVPRDLEVVAATPTSLLISWYMDEY DVRYYRITYGETGGNSPVQEFTVPNYYNTATISGL KPGVDYTIТVYAVTRIKANNYMYGΡISINYRTEID KPSQHHHHHH 28 SABA7. 1 Core sequence 7 of Adnectin containing the terminal sequences AdNTl and AdCTl with the H18b tag MGVSDVPRDLEVVAATPTSLLISWNHLEN VARYYRITYGETGGNSPVQEFTVPEYPTTATISGL KPGVDYTITVYAVTITMLKYPTQSPISINYRTgID KPSQHHHHHH 29 SABA8. 1 Core sequence 8 of Adnectin containing the terminal sequences AdNTl and AdCTl with the H18b tag MGVSDVPRDLEVVAATPTSLLISWGHY RR SGHYYRITYGETGGNSPVQEFTVDPSSYTATISGL KPGVDYTITVYAVSKDDYYPHEHRPISINYRTEID KPSQHHHHHH thirty SABA9. 1 Core sequence 9 Adnectin, MGVSDVPRDLEVVAATPTSLLISWDAS HY ERRYYRITYGETGGNSPVQEFTVPRYHHTATISGL KPGVDYTITVYAVTOAOEHYQPPISINYRTEIDKP containing the terminal sequences AdNTl and AdCTl with the H18b tag SQHHHHHH

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31 SABA 10 .1 Adnectin core sequence 10 containing terminal AdNTl and AdCTl sequences with His6-ToroM MGVSDVPRDLEVVAATRTSLLISWNSYYН SADYYRITYGETGGNSPVQEFTVPYPPTTATISGL KPGVDYTIТVYAVY SAKS YY РISINY RTEIDKPSQ NNNNNNN 32 SABAH .1 Adnectin core sequence 11 containing terminal AdNTl and AdCTl sequences with His6-ToroM MGVSDVPRDLEVVAATRTSLLISWSKYSК HGHYYRITYGETGGNSPVQEFTVPSGNATATISGL KPGVDYTITVYAVEDTNDYPHTHRPISINYRTglg KPSQHHHHHH 33 SABA 12 .1 Core sequence 12 Adnectin, MGVSDVPRDLEVVAATPTSLISWHGEPD QTRYYRITYGETGGNSPVQEFTVPPYRRTATISGL KPGVDYTITVYAVTSGYTGHYQPISINYRTEIDKP containing the terminal sequences AdNTl and AdCTl with His6-ToroM SQHHHHHH 34 SABA 13 .1 Adnectin core sequence 13 containing terminal AdNTl and AdCTl sequences with His6-ToroM MGVSDVPRDLEVVAATPTSLLISWSKYSК HGHYYRITYGETGGNSPVQEFTVDPSSYTATISGL KPGVDYTITVYAVSKDDYYPHEHRPISINYRTglg KPSQHHHHHH

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SABA 14 Cow MGVSDVPRDLEVVAATPTSLLISWYEPYT 35 .1 sequence 14 Adnectina, PIHYYRITYGETGGNSPVQEFTVPGYYGTATISGL KPGVDYTITVYAVYGYYQYTPISINYRTEIDKPSQ containing the terminal sequences AdNTl and AdCTl with His6-T3roM NNNNNNN SABA 15 Cow MGVSDVPRDLEVVAATPTSLLISWSKYSK 36 .1 Adnectin sequence 15 containing the terminal sequences AdNTl and AdCTl with His6-T3roM HGHYYRITYGETGGNSPVQEFTVPSGNATATISGL KPGVDYTITVYAVSDDNKYYHQHRPISINYRTglg KPSQHHHHHH SABA 16 Cow MGVSDVPRDLEVVAATPTSLLISWGHYRR 37 .1 Adnectin sequence 16 containing the terminal sequences AdNTl and AdCTl with His6-T3roM SGHYYRITYGETGGNSPVQEFTVDPSSYTATISGL KPGVDYTITVYAVSKDDYYPHEHRPISINYRTEID KPSQHHHHHH SABA 17 Cow MGVSDVPRDLEVVAATPTSLLISWSKYSK 38 .1 Adnectin sequence 17 containing the terminal sequences AdNTl and AdCTl with His6-T3roM HGHYYRITYGETGGNSPVQEFTVPSGNATATISGL KPGVDYTITVYAVEDTNDYPHTHRPISINYRTglg KPSQHHHHHH

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39 SABA 18 .1 Adnectin core sequence 18 containing terminal AdNTl and AdCTl sequences with His6-ToroM MGVSDVPRDLEVVAATРSLLISWYEPGA SVYYYRITYGETGGNSPVQEFTVPSYYHTATISGL KPGVDYTITVYAVYGYYEYEPISINYRTEIDKPSQ NNNNNNN 40 SABA 19 .1 Adnectin core sequence 19 containing terminal AdNTl and AdCTl sequences with His6-ToroM MGVSDVPRDLEVVAATPTSLLISWQSYYA HSDYYRITYGETGGNSPVQEFTVPYPPQTATISGL KPGVDYTIТVYAVYAGSSYYРISINYRTEIDKPSQ NNNNNNN 41 SABA20 .1 Adnectin core sequence 20 containing terminal AdNTl and AdCTl sequences with His6-ToroM MGVSDVPRDLEVVAATPTSLLISWGHY RR SGHYYRITYGETGGNSPVQEFTVDPSSYTATISGL KPGVDYTITVYAVSKDDYYPHEHRPISINYRTEID KPSQHHHHHH 42 SABA21 .1 Adnectin core sequence 21 containing terminal AdNTl and AdCTl sequences with His6-ToroM MGVSDVPRDLEVVAATRTSLLISWPERGT PVYYYRITYGETGGNSPVQEFTVPAYYGTATISGL KPGVDYTIТVYAVYGYYDYSPISINYRTEIDKPSQ NNNNNNN

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43 SABA22 .1 Adnectin core sequence 22 containing terminal AdNTl and AdCTl sequences with His6-ToroM MGVSDVPRDLEVVAATPTSLLISWYRYEK TQHYYRITYGETGGNSPVQEFTVPPESGTATISGL KPGVDYTITVYAVYAGYEYPHTHRPISINYRTEID KPSQHHHHHH 44 SABA23 .1 Adnectin core sequence 23 containing terminal AdNTl and AdCTl sequences with His6-ToroM MGVSDVPRDLEVVAATPTSLLISWVKSEE YYRYYRITYGETGGNSPVQEFTVPYYVHTATISGL KPGVDYTIТVYAVT ЕYYYAGAVVSVPISINYRTEI DKPSQHHHHHH 45 SABA24 .1 Adnectin core sequence 24 containing terminal AdNTl and AdCTl sequences with His6-ToroM MGVSDVPRDLEVVAATPTSLLISWYDPYT YGSYYRITYGETGGNSPVQEFTVGPYTTTATISGL KPGVDYTITVYAVSYYYSTQPISINYRTEIDKPSQ HHHHHH 46 SABA25 .1 Adnectin core sequence 25 containing terminal AdNTl and AdCTl sequences with His6-ToroM MGVSDVPRDLEVVAATPTSTSLLISWSNDGP GLSYYRITYGETGGNSPVQEFTVPSSQTTATISGL KPGVDYTITVYAVSYYTKKAYSAGPISINYRTglg KPSQHHHHHH 47 SABA26 .1 Adnectin core sequence 26 containing terminal AdNTl and AdCTl sequences with His6-ToroM MGVSDVPRDLEVVAATPTSLLISWPDPYY KPDYYRITYGETGGNSPVQEFTVPRDYTTATISGL KPGVDYTITVYAVYSYYGYYPISINYRTEIDKPSQ HHHHHH

Examples

Example 1. Materials and methods used for this invention.

High-performance protein production technology (HTPP).

Selected sequences encoding binding proteins were cloned into the pET9d vector and transformed into E. coli strain HMS174 cells. Next, the cells were inoculated in 5 ml of LB medium containing kanamycin at a concentration of 50 μg/ml into 24-well plates and incubated at 37°C overnight. To prepare for induction of expression, 200 μl of the overnight culture was transferred to 5 ml of fresh LB medium (50 μg/ml kanamycin) in a new well. Cultures were grown at 37°C to an optical density A ₆₀₀ of 0.6-0.9. Expression was then induced by adding isopropyl-β-thiogalactoside to a concentration of 1 mM and incubated at 30°C for 6 hours, after which the cells were collected by centrifugation for 10 minutes at 2750 g and 4°C.

Cell pellets (in a 24-well _plate ) were lysed by adding 450 µl Lysis Buffer (50 mM _NaH2PO4 , 0.5 M NaCl, 1x Complete Protease Inhibitor Cocktail-EDTA free (Roche), 1 mM PMSF, 10 mM CHAPS , 40 mM Imidazole, 1 mg/ml lysozyme, 30 μg/ml DNase, 2 μg/ml aprotonin, pH 8.0) and stirring at room temperature for 1-3 hours. Lysates were clarified and transferred to 9-well format by filtration at elevated pressure through a 96-well Whatman GF/D UNIFILTER® filter with a 96-well collection plate volume

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1.2 ml. The clarified lysates were transferred to a 96-well plate with Ni-chelating resin equilibrated with the buffer _: 50 mM _NaH2PO4 , 0.5 M NaCl, 40 mM imidazole, pH 8.0, and incubated for 5 min. Unbound substances were removed under vacuum. The resin was washed twice with 0.3 ml/well with wash buffer No. 1 (50 mM NaH ₂ PO ₄ , 0.5 M NaCl, 5 mM CHAPS, 40 mM imidazole, pH 8.0) under vacuum. Before elution, 50 μl of elution buffer (PBS+20 mM EDTA) was added to each well, incubated for 5 min and removed using vacuum. Protein was eluted by adding an additional 100 μl of elution buffer to each well. After 30 min of incubation at room temperature, the plate(s) were centrifuged for 5 min at 200 g, and the eluted protein was collected using a 96-well collection plate containing 5 μl of 0.5 M MgCl ₂ at the bottom of the wells. The amount of eluted protein was measured by a standard BCA assay using SGE as a standard.

Medium-scale expression and purification of insoluble binding proteins based on fibronectin structural moieties.

To express insoluble protein-producing clone sequences, the 6His-terminated clone(s) sequences were cloned into the pET9d vector (EMD Bioscience, San Diego, Calif.) and expressed in E. coli strain HMS174 cells. To do this, 20 ml of cell culture (obtained from one colony grown by sieving on one dish) is inoculated into 1 liter of LB medium containing 50 µg/ml carbenecycline and 34 µg/ml chloramphenicol. The culture is incubated at 37°C until the optical density A ₆₀₀ reaches 0.6-1.0. After induction of expression by the addition of 1 mM isopropyl-β-thiogalactoside (IPTG), the culture is incubated for 4 h at 30°C and cells are collected by centrifugation for 30 min at >10,000 g at 4°C. Cell pellets are frozen at -80°C. Cell pellets are then resuspended in 25 ml of lysis buffer (20 mM NaH ₂ PO ₄ , 0.5 M NaCl, 1x Complete Protease Inhibitor Cocktail-EDTA free (Roche), 1 mM PMSF, pH 7.4) using an ULTRA-TURRAX® homogenizer (IKA ) on ice. Cell lysis is achieved using a high pressure homogenizer (>18,000 psi) using a Model M-110S MICROFLUIDIZER® (Microfluidics). The insoluble fraction is separated by centrifugation for 30 minutes at an acceleration of 23,300 g at 4°C. The resulting precipitate is washed with a buffer containing 20 mM sodium phosphate/500 mM NaCl, pH7.4. Then the precipitate is dissolved in a 6.0 M solution of guanidine hydrochloride in a buffer containing 20 mM sodium phosphate/500 mM NaCl, pH7.4 with ultrasound treatment and subsequent incubation at 37°C for 1-2 hours. The dissolved precipitate is filtered through filters with 0.45 μm pore size and applied to a Histrap column pre-equilibrated with 20 mM sodium phosphate/500 mM NaCl/6.0 M guanidine buffer, pH7.4. After application, the column is washed with 25 column volumes of equilibration buffer. The bound protein is eluted with a buffer containing 50 mM imidazole and 20 mM sodium phosphate/500 mM NaCl/6.0 M guanidine chloride, pH7.4. Next, the purified protein is refolded by dialysis against a buffer with 50 mM sodium acetate/150 mM NaCl, pH 4.5.

Medium-scale expression and purification of soluble binding proteins based on fibronectin structural moieties.

To express the sequences of the clones producing insoluble proteins, the sequences of the clone(s) with a 6His tag at the end were cloned into the pET9d vector (EMD Bioscience, San Diego, CA) and expressed in E. coli strain HMS174 cells. To do this, 20 ml of a cell culture (obtained from one colony grown by sieving on one dish) was inoculated into 1 liter of LB medium containing 50 µg/ml carbencycline and 34 µg/ml chloramphenicol. The culture was incubated at 37°C until the optical density A ₆₀₀ reached 0.6-1.0. After induction of expression by the addition of 1 mM isopropyl β-thiogalactoside (IPTG), the culture was incubated for 4 h at 30°C and cells were harvested by centrifugation for 30 min at >10,000 g at 4°C. Cell pellets were frozen at -80°C. Cell pellets were then resuspended in 25 ml of lysis buffer (20 mM NaH ₂ PO ₄ , 0.5 M NaCl, 1x Complete Protease Inhibitor Cocktail-EDTA free (Roche), 1 mM PMSF, pH 7.4) using an ULTRA-TURRAX® homogenizer (IKA) on ice. Cell lysis was achieved using a high pressure homogenizer (>18,000 psi) using a Model M-110S MICROFLUIDIZER® (Microfluidics). The soluble fraction was separated by centrifugation for 30 min at an acceleration of 23,300 g at 4°C. The resulting supernatant was clarified by filtration through a filter with a pore size of 0.45 μm. The clarified lysate was applied to a Histrap (GE) column pre-equilibrated with 20 mM sodium phosphate/500 mM NaCl buffer, pH 7.4. After application, the column was washed with 25 column volumes of equilibration buffer containing 20 mM sodium phosphate/500 mM NaCl, pH 7.4, followed by a wash with 20 column volumes of buffer containing 20 mM sodium phosphate/500 mM NaCl/25 mM imidazole, pH 7.4. followed by washing with 35 volumes of buffer containing 20 mM sodium phosphate/500 mM NaCl/40 mM imidazole, pH7.4. The protein was eluted with 15 column volumes of buffer containing 20 mM sodium phosphate/500 mM NaCl/500 mM imidazole, pH 7.4. Fractions were pooled based on absorbance at 280 nm and dialyzed against 1x PBS, 50 mM Tris, 150 mM NaCl buffers; pH 8.5 or 50 mM NaOAc; 150 mM NaCl; pH 4.5. Any precipitate was removed by filtration through a 0.22 μm filter.

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Example 2. Preclinical pharmacology in vitro.

KD measurement using the SPR method.

The binding characteristics were determined by surface plasmon resonance (SPR). Human and cynomolgus PCSK9 were immobilized in separate channels in one dimension on the surfaces of a ProteOn XPR chip (Bio-Rad) and treated with 6 different concentrations of 2013E01 in another dimension of the surface of the same SPR chip. This made it possible to determine the kinetics in the absence of regeneration. To determine the kinetics of human and cynomolgus PCSK9 at 25°C, the experiment was performed in duplicate. The kinetic parameters were estimated using the Langmuir interaction model and constant point-fitting parameters of the ProteOn Manager software.

Under these conditions, anti-PCSK9 adnectins bind to human PCSK9 with dissociation constants (KD) ranging from 80 to 1.6 nM and to cynomolgus PCSK9 with dissociation constants (KD) ranging from 8 to 24 nM (Table 7). Association rates were approximately estimated at 10 ⁵ M' ¹ s' ¹ , along with dissociation rates, which were typically 10' ³ -10' ⁵ s ¹ . For some adnectins, the dissociation rates from human PCSK9 were low (on the order of 10 ^-5 s ¹ ), which is close to the detection limit using SPR technology. Thus, it is possible that these dissociation constants from human PCSK9 are lower than the estimates reported.

Table 7

Kinetic parameters of anti-PCSK9 adnectin relative to directly immobilized human and cynomolgus PCSK9 determined using SPR technology

w clone organism- PCSK9 source kon (Μ ⁴ S' ¹ ) koff (s' ¹ ) K _D (nM) 14 59D05 Human 1.13E+05 1.80E-04 1.58 + 0.176 20 13E01 Human 7.03 ± 0.1 E+05 2.42 + 0.3 E-05 0.292 ± 0.008 20 13E01 Javanese macaques 2.19 E+05 1.77 E- 03 8.1 19 22G04 Human 5.41E+05 5.08E-05 0.094 ± 0.009 19 22G04 Javanese macaques 4.65E+05 7.00E-03 15.03 20 11Н05 Human 1.18E+05 9.76E-06 0.079 ± 0.038 20 11Н05 Javanese macaques 1.90E+05 4.40E-03 23.1 20 12A04 Human 2.59E+05 6.47E-05 0.251 ± 0.011 20 12A04 Javanese macaques 1.95E+05 4.75E-03 24.32

KD measurements using the BLI method.

The binding characteristics of adnectins and human PCSK9 were also determined by Bio-Layer Interferometry (BLI). Biotinylated human PCSK9 was immobilized onto superstreptavidin-coated sensor tips, which were then dipped into wells containing adnectin dilutions for the time required for binding. The tips were then placed into wells filled with pure buffer to observe adnectin dissociation. Experiments were performed at controlled temperature, either 25°C or 37°C. The set oscillation frequency was 1500 rpm.

Binding analysis was carried out using appropriate software from Fortebio (Fortebio Data Analysis Software version 6.3.0.36). An overall curve fit was generated for all samples using a 1:1 binding model. From these overall correspondences, a pair of association and dissociation rates was obtained that were mutually consistent. Affinity (K _D ) association and dissociation rate values were averaged across the different loading levels used in the analysis. Under these conditions, adnectins bind human PCSK9 with affinities ranging from 200 to 7.5 nM, as shown in Table 1. 8 below. Association rates varied in the range of 104-10 ⁵ M' ¹ s' ¹ and corresponded to dissociation, which was usually 10' ³ -10' ⁵ s ^-1 .

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Table 8

Kinetic parameters of binding of PCSK9 adnectins to human PCSK9 determined using the BLI method

Clone number or ATI number kon (M ¹ s' ¹ ) koff (c' ¹ ) K _D (nM) 2381D04 2.66E+05 ± 6.4 E+04 6.90E-05 ± 6.9E-05 0.237 ±0.08 2382D09 5.33E+05 1.64E-04 0.304 2382D05 5.05E+05 ± 2.0E+05 1.57E-04± 4.2E-05 0.314 ±0.06 2381В04 3.09E+05 ± 2.1 E+04 1.60E-04± 1.6E-04 0.527 ±0.11 2382E05 4.36E+05 ± 1.6E+05 2.50E-04 ± 1.1E-05 0.604 ±0.21 2382В09 4.51E±05± 4.7E+04 2.91E-04 + 4.0E-05 0.656 ±0.02 2382N03 4.71E+05 ± 1.4E+05 5.57E-04± 2.3E-04 0.677 ±0.02 2382С09 3.49E+05 2.33E-04 0.757 2971A03 5.74E+05 4.20E-04 0.806 2382G04 4.19E+05 4.64E-04 1.11 2381G09 2.71E+05 2.91E-04 1.12 2451С06 3.47E+05 ± 5.4E+04 4.35E-04± 5.4E+04 1.27 ± 0.14 2382N10 2.88E+05 3.82E-04 1.40 2013E01 5.46E+05 8.37E-04 1.51 2382D03 3.54E+05 6.63E-04 1.53

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2381F11 3.23E+05 5.22E-04 1.59 2971E02 3.55E+05 5.55E-04 1.78 2382Н09 2.51E+05 4.83E-04 1.86 2451Н07 4.48E+05 9.40E-04 2.08 23 82В10 3.94E+05 9.77E-04 2.35 2382С05 3.68E+05 8.79E-04 2.49 2971A09 3.99E+05 1.05E-03 2.79 2382E03 2.20E+05 5.96E-04 2.94 2381Н09 1.36E+05 4.60E-04 3.23 2381В02 2.65E+05 8.31E-04 3.29 2381В08 2.13E+05 9.40E-04 4.11 2382F05 2.44E+05 1.09E-03 4.54 1459D05 1.49E+05± 2.5E+04 2.02E-03 ± 2.0E-03 14.4 ± 0.2 ATI 1091 2.863E+05 8.201E-05 0.293 ATI001117 1.451E+05 5.074E-05 0.554 ATI001057 5.325E+05 1.403E-04 0.255 ATI001119 8.190E+04 4.450E-04 5.296 ATI001168 1 5.829E+05 3.335E-04 0.586 ATI001175 2 6.558E+05 3.522E-04 0.543 ATI 1081 8.29E+05 ± 8.1E+05 3.49E-04± ZE-04 0.479 ±0.11 ATI891 1.08E+05 4.03E-04 3.56 ATI1114 8.072E+04 3.952E-04 4.876 ATI1174 1.265E+05 9.012E-04 7.397

¹ ATI001168 is a deimmunized variant of 1922G04 with an R23E substitution.

² ATI001175 is a deimmunized variant of 1922G04 with an R23D substitution.

Affinity in solution.

KinExA.

The affinity of ATI001081 and ATI001174 for human PCSK9 in solution was measured using the Kinetic Exclusion Assay (KinExA). Relative concentrations of unbound adnectin were measured by binding to a solid support with immobilized hPCSK9 and subsequent detection using fluorescently labeled antibodies recognizing structural regions of adnectin. Due to technical limitations, the lowest concentration of adnectins measured was 1 nM. The KD value for ATI001081 was 70 nM (28-127 nM 95% CI) and for ATI001174 was 223 nM (54-585 nM 95% CI) based on the overall KD analysis.

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Table 9

Measurements of the affinity of adnectins for PCSK9 in solution

ATU 01081 ATU 01174 K _D 69.5 PM 223 PM 95% confidence interval: Kd high 127 PM 585 PM Kd low 28 PM 54 PM

ITC.

The stoichiometry and thermodynamics of binding of human adnectins ATI001174 to ATI001081 PCSK9 were characterized using the isothermal titration calormetry (ITC) method. Solution binding measurements were carried out in a buffer containing 25 mM HEPES, pH 7.41, 150 mM NaCl at 37°C. The average single-molecule binding constant was observed to be in the range of 1.3±0.2 nM for ATI001174 and 1.4±0.4 nM for ATI001081. Detailed thermodynamic analysis is given in table. 10 and fig. 13. The difference in observed enthalpy (-3.3 kcal/mol) for the two adnectins suggests that the order of magnitude decrease in magnitude is due to PEGylation of ATI001174, which is at least partially offset by the corresponding difference in entropy (-10.4 kcal/mol) mol K).

Table 10

Adnectin Stoichiometry (N) K _A (Μ' ¹ ) Kd (nM) AN (kcal/mol) AS (cal/mol °C) ATU01081 0.926 7.1E8±2.1E8 1.4±0.4 (±30%) -26.6 ±0.2 -45.4 ATI001174* 0.857 7.5E8± 1.5E8 1.3 ±0.2 -29.9 ±0.1 -55.8

Average after three experiments.

Fluorescence resonance energy transfer (FRET) analysis.

Three FRET-based systems were developed to determine the binding affinity and potential of PCSK9-binding adnectins, adapted from the general method described by Maio et al. (See, Miao, V. et al., Meth. Enzymol., 357:180-188 (2002)). The PCSK9:EGFA FRET assay (Figures 2 and 3) measured inhibition of PCSK9 binding to the low-density lipoprotein receptor (LDLR) homologous epidermal growth factor precursor domain (EGFA domain), using recombinant human PCSK9 expressed in the baculovirus system and synthetic 40 -member EGFA peptide (biotinylated). EGFA has been shown to be a key domain for the interaction of LDLR with PCSK9 (Kwon, H.J. et al., Proc. Natl. Acad. Sci., USA, 105(6):1820-1825 (2008)). This assay used a monoclonal antibody (mAb 4H5) that binds the C-terminal domain of PCSK9 tagged with Eu-chelator to mediate FRET interaction with biotinylated EGFA via a streptavidin/allophycocyanin fluorophore complex.

Two other similar PCSK9-dependent FRET systems were also constructed for analysis. One of these systems quantified the competitive displacement of biotinylated adnectins ATI000972 or ATI001125 by adnectins (results for ATI000972 are shown in Fig. 4). ATI000972 is a biotinylated variant of 1459D05, and ATI001125 is a biotinylated variant of ATI001081. In another system, direct binding of adnectin (His-tagged) to PCSK9 was analyzed using an αanti-6His-tagged antibody (Fig. 5). In each of these analytical FRET systems, the concentration of human PCSK9 was 1 or 5 nM. In some cases, human PCSK9 was replaced by cynomolgus PCSK9. In table Figure 11 presents summary data obtained using all three FRET systems.

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Table 11

Summary of adnectin testing using 3 FRET systems using human PCSK9 and in one case cynomolgus PCSK9

Sh clone hP CSK9: EGFA IC50 (nM) hP CSK9: ATI000972 IC50 (nM) hP CSK9: ATI001125 IC50 (nM) Direct hPCSK9 binding EC50 (nM) cPCSK9:EGFA IC50 (nM) 1459D05 4.0 14 nd 3.9 2300 1784F03 2 1.9 nd 1.6 106 1813E02 1.3 2.4 nd 2.3 118 1923B02 2.3 3.2 nd 3.2 53.5 1922G04 1.2 2.0 nd 2.5 26.6 2012A04 2.1 1.2 nd 1.4 70.3 2011Н05 2.7 1.4 nd 2.0 17.2 2013E01 1.6 1.8 nd 0.90 12.5 1922G04 (R25D) 2.5 nd nd nd 48.1 1922G04 (R25E) 3.5 nd nd nd 58.5 2382E03 2.4 nd 0.5 3.7 10.8 2382E05 2.8 nd 0.4 3.8 12.6 2381В08 2.6 nd 1 4.1 27.1 2381В02 2.5 nd 0.5 8.4 9.8 2381В04 2.4 nd 0.5 4.2 17.3 2451Н07 0.2 nd 0.2 13.2 27.1 2381D04 2.9 nd 0.5 3.7 12 2381F11 4 nd 0.8 < 1 26.5 2381G09 3.1 nd 1.1 4.5 25.2 2381Н09 3.4 nd 0.5 4.1 20.2 2382В09 3.8 nd 0.6 4.0 37.2 23 82В10 3 nd 0.5 3.1 18.1 2382С05 4 nd 0.9 3.4 27.2 2382С09 3.5 nd 0.6 2.9 23.9 2382D03 3.3 nd 0.6 3.3 11.8 2382D09 2.6 nd 0.4 3.7 13.9 2382F05 2.4 nd 0.9 3.8 14.6 2382G04 2.9 nd 0.5 3.6 20.1 2382N03 3.4 nd 0.3 3.8 19.8 2382Н09 0.9 nd 0.3 3.6 17.9 2382N10 2.6 nd 0.4 4.6 18.2 2382D02 4.5 nd 0.5 5.3 57.8 2451С06 1.2 nd 0.4 5.5 24.7

Analysis of PCSK9 inhibition by adnectins of PCSK9 activity using cell culture.

DiI-LDL uptake assay.

To analyze the ability of adnectins to inhibit the activity of PCSK9 in relation to LDLR

- 128 044608 Cell culture-based approaches have been developed. Effective methods for measuring cellular LDL activity are methods based on the uptake of labeled LDL, as shown by Lagace, TA et al. (J. Clin. Invest., 116(11):2995-3005 (2006)). This work adapts a method for assessing the functional activity of LDLR using the fluorescently labeled LDL (DiI-LDL) uptake method, transferred from the method originally developed by Teupser et al. (Biochim. Biophys. Acta, 1303(3):193-198 (1996)). Initially, as shown, cells are incubated with recombinant human PCSK9 (10 mg/ml, 135 nM) in the presence and absence of adnectins. After 2 h, residual LDL activity is assessed by incubation with DiI-LDL (5 μg) for 2 h and subsequent assessment of intracellular DiI-LDL accumulation using high-resolution fluorescence microscopy and subsequent image analysis (Cellomics). In fig. Figure 6 shows the effect of several adnectins that inhibit PCSK9 activity and restore the functional activity of LDLR in HepG2 cells. In this assay, adnectins that inhibited PCSK9 and restored DiI-LDL uptake had the following EC ₅₀ characteristics: 1459D05, EC ₅₀ =190 nM; 1784F03, 210 nM; 2012A04, 130 nM; 2013E01, 160 nM.

Analysis of the reduction in the amount of LDL cholesterol.

HepG2 cells were grown in Eagle's Minimum Essential Medium (EMEM, ATCC®) with 10% FBS (Hyclone) treated with 0.25% trypsin (Invitrogen) twice a week. To induce an increase in LDL receptor expression, cells were incubated overnight in LPDS medium [RPMI (ATCC) with 5% lipoprotein-free serum (Intracel), 100 nM superstation (BMS) and 50 μM sodium mevalonate (Sigma)]. The next day, cells were briefly treated with 0.05% trypsin (Invitrogen) and resuspended at 2 x 106 cells per ml, and then aliquoted at 100 μl per well into 96-well V-bottom plates. In parallel, adnectins were preincubated with PCSK9 in LDPS medium for 1 hour at 37°C. After an hour, the cells were centrifuged and resuspended in 100 μl of adnectin/PCSK9 mixture and incubated overnight at 37°C. The next day, cells were labeled with anti-LDL receptor antibody (BAF 2148 jn R&D) followed by treatment with phycoerythrin (PE)-streptavidin-conjugated secondary antibodies (BD554061 from BD Pharmingen) and analyzed on a FACS Canton flow cytometer (BD).

Before addition to HepG2 cells, a 10 nM solution of PCSK9 was incubated with increasing concentrations of candidate adnectins. After overnight incubation, LDL levels were measured using a flow cytometer. The percentage of PCSK9 inhibition of LDLR reduction was plotted and EC ₅₀ was determined using PRISM. Clone 1459D05 had the least inhibitory potency (inhibition did not reach a maximum), while the other clones reached a maximum of 150-200% PCSK9 inhibition (Fig. 7). Summary data on the pharmacology of PCSK9 adnectin in vitro are given below in table. 12.

Table 12

th clone % monomeric form (HPLC X-gel filtration) Tsh (°C, DSC) KD hPCSK9 (37°C, Octet Red) (nM) K _D hPCSK9 (25 °C, ProteOn) (nM) Cross-reactivity with cynomolgus protein PCS K9: GFA FRET (EC ₅ o, nM) Decline amount of LDLP K _D CPCSK9 (25 °C, ProteOn) (nM) PCSK9 macaque: EGFA FRET (EC ₅ o, nM) % inhibition at 75 nM EC ₅₀ (nM) 14 59D05 >95 63 14.4 1.58 >1000 >1000 5.8 66.8 >200 18 13E02 >95 70 nd nd nd 118.5 2.7 nd 16 17 84F03 >95 65 nd nd nd 106.5 2.01 150.2 26±13 19 23В02 >95 73 nd 0.17 nd 53.5 2.3 178 23±7 19 22G04 100 83 nd 0.09 15.0 26.6 1.2 105.1 10±2 20 13E01 100 81 nd 0.29 8.1 12.5 1.6 165.5 10±4

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20 12A04 100 84 nd 0.25 24.3 70.3 2.1 144.5 12± 6 20 11Н05 100 76 nd 0.08 23.1 17.2 2.7 197.6 12± 5 23 82D05 97 86 0.314 nd nd 57.8 2.6 140.5 nd 23 82E03 96 83 2.94 nd ND 10.8 2.4 89.5 ND 23 82E05 95 84 0.604 nd ND 12.6 2.8 103.5 ND 23 81В02 96 68 3.29 nd ND 9.8 2.5 125.4 ND 23 81В04 98 77 0.527 nd ND 17.3 2.4 121.6 ND 23 81В08 97 78 4.11 nd ND 27.1 2.6 124.8 ND 23 81D04 98 70 0.237 nd ND 12 2.9 119.2 ND 23 81F11 95 79 1.59 nd ND 26.5 4 110.2 ND 23 81G09 96 78 1.12 nd ND 25.2 3.1 133.0 ND 23 81Н09 77 63 3.23 nd ND 20.2 3.4 94.4 ND 23 82B 09 99 88 0.656 ND ND 37.2 3.8 105.0 nd 23 82В10 99 82 2.35 ND ND 18.1 3 100.2 nd 23 82С05 97 85 2.49 ND ND 27.2 4 105.3 nd 23 82С09 96 85 0.757 ND ND 23.9 3.5 121.7 nd 23 82D03 97 84 1.53 ND ND 11.8 3.3 80.4 nd 23 82D09 93 84 0.304 ND ND 13.9 2.6 109.1 nd 23 82F05 99 83 4.54 ND ND 14.6 2.4 72.2 ND 23 82G04 95 81 1.11 ND ND 20.1 2.9 146.1 ND 23 82N03 96 85 0.677 ND ND 19.8 3.4 102.1 ND 23 82N09 95 81 1.86 ND ND 17.9 0.9 101.2 ND 23 82N10 97 87 1.40 ND ND 18.2 2.6 118.6 ND 24 51B06 nd nd nd ND ND 57.8 4.5 92.2 ND 24 51С06 96 87 1.27 ND ND 24.7 1.2 89.4 ND 24 51N07 97 87 2.08 ND ND 27.1 0.2 88.8 ND

Example 3. Pharmacodynamic properties of PCSK9 adnectins in vivo.

Studies using human PCSK9 transgenic mouse models.

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The in vivo pharmacodynamic properties of PCSK9 adnectins were studied in two different transgenic mouse models. One mouse model expressed human PCSK9 at high levels, resulting in hypercholesterolemia (Lagace, T.A. et al., J. Clin. Invest., 116(11):2995-3005 (2006)). In another mouse model, hPCSK9 was integrated into the genome (using BAC), its expression level was regulated in the liver similar to mouse PCSK9, and hPCSK9 was expressed in plasma at a level close to normal in humans. An ELISA test was developed to measure the level of unbound human PCSK9 in plasma (i.e., hPCSK9 not complexed with administered adnectin), as an indicator of the amount of the target protein. based on species-specific site-specifically labeled antibodies and adnectins.

Single doses of PCSK9 adnectins in pegylated form were administered intraperitoneally to transgenic mice with an increased level of hPCSK9 expression, at the dosages shown in Fig. 8 and 9. PBS or dialysate samples were also administered as controls. Administration of adnectin 1459D05-PEG (100 mg/kg i.p.) resulted in a rapid decrease in plasma total cholesterol and LDL-C (Figure 8A) to more than 35% below basal levels within 4 hours. Cholesterol levels in administered mice adnectin remained below the level in the control group throughout 48 hours of measurements. This reduction was accompanied by a dramatic decrease in circulating levels of unbound hPCSK9 in adnectin-treated transgenic mice (Fig. 8B). Western blot analysis of liver tissue samples taken after 6 hours in a parallel study showed that LDL levels were increased ~2-fold in Adnectin-treated mice (results not shown). In further studies on mice of this transgenic line, ATI001114 was administered in dosages of 10 or 60 mg/kg. There was a significant dose-dependent rapid decrease in plasma cholesterol levels, accompanied by a dose-dependent decrease in the level of unbound hPCSK9 (Fig. 9). These studies provide an in vivo experimental test of the ability of PCSK9 adnectins to effectively reduce cholesterol levels in hPCSK9 transgenic hypercholesterolemic mice.

In vivo studies were performed on linear transgenic mice that normally express hPCSK9. Administration of single doses of 1459D05-PEG or ATI001114 (5 mg/kg) resulted in a rapid and pronounced decrease in plasma unbound hPCSK9 levels (Fig. 10). This pharmacodynamic effect on unbound hPCSK9 was longer lasting with ATI001114 compared to to1459D05-PEG, as greater magnitude and duration of effect was observed for this higher affinity/potential adnectin. Further dose-response studies revealed that the 50% inhibitory dose was less than 0.1 mg/kg for ATI001114 from 3 to 48 hours after administration, as shown in FIG. 11. Data obtained in a transgenic mouse model expressing normal levels of hPCSK9 show that PCSK9 inhibitory adnectins have significant, affinity- and dose-dependent effects on pharmacodynamic endpoints that correlate with the regulation of LDL-C and the reduction of LDL-cholesterol.

Studies on cynomolgus macaques.

Pharmacodynamic studies were conducted in normal cynomolgus monkeys. Adnectin ATI001114 was administered intravenously to monkeys at a dosage of 5 mg/kg, after which plasma samples were collected at regular intervals to determine LDL cholesterol levels and evaluate pharmacokinetics. Administration of a single dose of ATI001114 rapidly reduced plasma LDL-C levels by more than 50% compared to basal levels (or compared to the PBS control group) over 48 hours (Fig. 12). The duration of the effect on LDL-C levels was more than a week, followed by mandatory recovery to basal levels within 3 weeks. This effect was observed for anti-PCSK9 adnectin variants PEGylated with two- and four-strand PEG weighing 40 kDa (ATI001114 and ATI001211, respectively). ATI001211 is a variant of ATI001081 with a 4-strand PEG (NOF) group of 40 kDa. Total cholesterol levels changed in a similar manner, but no effect was observed on HDL or other metabolic parameters (data not shown). Pharmacokinetic analysis showed a plasma half-life of approximately 80-120 hours, which is consistent with the pharmacodynamics of LDL-lowering in cynomolgus monkeys. The findings demonstrate that PCSK9 adnectin is an effective and rapidly acting agent that produces a rapid, significant and specific LDL-C lowering effect in the cynomolgus monkey model.

Example 4 In vitro and in vivo pharmacological evaluation of PCSK9 adnectin-Fc fusion protein (PRD460).

PRD460 operating time.

HEK-293 6E cells were transfected with a vector encoding PRD460 using polyethylenimine (PEI). Cells were incubated for 5 days at 37°C, 80% humidity and 5% CO ₂ . After this, the cells were pelleted, the supernatant was filtered through a filter with a pore size of 0.22 μm and applied to a column with protein A. The column was washed with PBS buffer and the protein was eluted with a buffer containing 20 mM glycine and 150 mM NaCl pH 2.8. The eluted protein preparation was concentrated and passed through a superdex200 column in a buffer containing 50 mM MES, 100 mM NaCl pH 5.8.

Determination of K _D PRD460 by SPR method.

Binding characteristics were determined by surface plasmon resonance (SPR).

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Antibody against human proteins was immobilized on the surface of the BIACORE® chip substrate, after which the PRD460 protein (sequence shown in SEQ ID NO: 322) was placed on the substrate for binding. Various concentrations of hPCSK9 were added to the wash solution using 3M MgCl ₂ solution to regenerate the substrate between cycles. For comparison, the ATI-1081 protein was bound to a BIACORE® chip substrate with immobilized antibodies to the 6His tag. Experiments for PRD460 were performed twice on different days. The kinetics were determined at 25°C. Kinetic parameters were assessed using BIACORE® Evaluation software and a 1:1 coupling algorithm.

Under these conditions, ATI-1081 bound to human PCSK9 with a dissociation constant ( _KD ) of 6.7 nM at 25°C. The dissociation constant (K _D ) of PRD460 with human PCSK9 under these conditions was 3.29 ± 0.55 nM (Table 13). Determination of the dissociation rate using this assay format may be limited due to the rate of dissociation of the bound ligand from the immobilized binding antibody. Therefore, an assay format that uses direct immobilization of PCSK9 is more accurate for estimating the dissociation constant (K _D ) for ATI-1081.

Table 13 Kinetic binding parameters for

PRD460 and ATI-1081 with immobilized human PCSK9

ka (1/Ms) kd (1/s) KD (nM) PRD460 3.75 +/-0.7 E+04 1.21 +/-0.05 E-04 3.29+/-0.55 ATI-1081 3.65E+04 2.45E-04 6.7

FRET-based PCSK9 binding assays.

Two fluorescence resonance energy transfer (FRET) assay systems were used to determine the binding potential of PRD460 and other adnectins to hPCSK9 under competitive conditions. The PCSK9:EGFA system measures the binding of PCSK9 to LDLR using soluble epidermal growth factor precursor homologous domain A peptide (EGFA) and recombinant human PCSK9. The PCSK9:ATI972 system measures competitive adnectin displacement of biotinylated ATI-972 adnectin on the PCSK9 molecule.

In the PCSK9:EGFA system (at a PCSK9 concentration of 5 nM), PRD460 completely replaced EGFA bound to PCSK9 with an EC ₅₀ of 0.7 nM (Fig. 1, left). In this assay format, PRD460 had greater activity than ATI-1174 (EC ₅₀ =1.9 nM) or ATI-1081 (EC ₅₀ =3.7 nM) (Fig. 14). The greater activity of PRD460 in this assay format may be due to the (theoretically) bivalent binding of PRD460 adnectin to PCSK9 compared to the monovalent (1:1) binding of ATI-1081 and ATI-1174.

In the PCSK9:ATI972 system (at a PCSK9 concentration of 5 nM), PRD460 inhibited binding with an EC of 0.3 nM, compared to 0.8 nM for ATI-1114 and 2.8 nM for ATI-1081 (Figure 15). These data demonstrate that PRD460 actively displaces adnectin ATI-972 at its PCSK9 binding sites. The greater activity of PRD460 compared to ATI-1081 and ATI-1174 is consistent with the bivalent binding mode of PRD460.

Inhibition of PCSK9-induced decrease in LDLR levels in HepG2 cells.

Human PCSK9 induces a decrease in the presence of LDLR on the surface of HepG2 cells. Preincubation of PCSK9 with PCSK9 adnectins inhibits the binding of PCSK9 to LDLR and prevents a decrease in the presence of LDLR on the cell surface. This assay system was used to measure the inhibition activity of ATI-1081, ATI-1174, and PRD460 on PCSK9-induced reduction in cell surface LDLR.

Serial dilutions of PCSK9 adnectins were incubated with human PCSK9 at a concentration of 10 nM for 1 h at 37°C. Then HepG2 cells were added to the mixtures and the mixtures were further incubated for 24 hours. After this, the level of LDL on the cell surface of HepG2 cells was measured using flow cytometry. The graph indicated the calculated level of inhibition of the decrease in LDL cholesterol induced by PCSK9 (Fig. 15). In this assay, ATI-1081, ATI-1174, and PRD460 inhibited PCSK9 activity with comparable EC50 values (9, 8, and 6 nM, respectively). However, for PRD460 a shift of this curve to the left was repeatedly observed. The EC ₅₀ values stated represent the measurement limit for this analysis.

Analysis of PCSK9 in HepG2 cells.

The binding of PCSK9 to LDLR on the surface of hepatocytes leads to the internalization of the LDLR-PCSK9 complex during endocytosis of LDLR, which leads to accelerated degradation of LDLR. A specific assay was developed to measure LDLR-mediated intracellular entry of fluorescent PCSK9. Human PCSK9 molecules were covalently labeled with the fluorophore ALEXA FLUOR®-647(AF647). The resulting PCSK9-AF647 was incubated with HepG2 cells in the presence and absence of PCSK9-adnectins, and intracellular fluorescence was measured using high-resolution fluorescence microscopy and image analysis (Cellomics). Depend

- 132 044608 the possibility of PCSK9-AF647 uptake by cells from endocytosis of LDLR was shown in preliminary experiments. HepG2 cells were incubated with PCSK9-AF647, at a concentration of 10 nM, and various amounts of adnectins for 4 hours at 37°C. Based on the results of this analysis, active inhibition of intracellular fluorescence of PCSK9-AF647 was observed for PRD460 (EC ₅₀ =6 nM) and for ATI-1174 (EC ₅₀ =10 nM) (Fig. 16). These data in a cultured human hepatocyte line demonstrate that the adnectins PRD460 and ATI-1174 effectively block the binding of PCSK9 to LDLR on the cell surface, thereby reducing the level of internalization of PCSK9-AF647 during LDLR endocytosis.

In vivo studies on transgenic mouse lines.

In vivo studies were performed in a transgenic mouse line expressing hPCSK9 at normal levels. It is hypothesized that the binding of adnectins to PCSK9 in plasma results in a decrease in the measured amount of unbound (free) circulating PCSK9. This reduction in unbound PCSK9 is an initial pharmacodynamic effect that results in inhibition of the interaction of PCSK9 with LDL and a reduction in LDL cholesterol. Administration of single doses of PRD460 (i.p., at dosages ranging from 0.6 to 1.8 mg/kg) to transgenic mice resulted in a rapid, pronounced decrease in plasma levels of unbound hPCSK9 (FIG. 17). This effect was observed to be dose dependent with an _{ED50 of} <0.6 mg/kg at 3 hours. These results, obtained in a transgenic mouse line with normal hPCSK9 expression, demonstrate active and pronounced binding of PRD460 to circulating hPCSK9 in vivo.

In vivo pharmacodynamic studies in cynomolgus monkeys The pharmacodynamic parameters of PCSK9 adnectin PRD460 were determined in studies in normal cynomolgus monkeys. To analyze LDL-C and free PCSK9 levels, PRD460 was administered intravenously to monkeys at a dosage of 15 mg/kg, and plasma samples were collected at specified time intervals for 4 weeks. A single dose of PRD460 resulted in a rapid decrease in LDL-C levels in monkeys, peaking at 42% of baseline LDL-C levels (58% reduction, n=3 monkeys) on the third day after administration (FIG. 18). LDL-C levels were reduced by 50% or more within a week after administration, remaining significantly lower than baseline for 3 weeks and reaching baseline levels 4 weeks after administration. Total cholesterol levels changed in a similar manner, but HDL cholesterol levels did not (data not shown). Administration of PRD460 resulted in an immediate decrease to almost 0 (below the limit of quantitation) in the plasma level of unbound free PCSK9 (FIG. 18). Free PCSK9 levels remained below the limit of detection for several days and then gradually increased to baseline levels by the end of week 4, consistent with changes in LDL-C levels with PRD460. The findings demonstrate that the decrease in plasma LDL levels reflects a decrease in free PCSK9 levels, consistent with inhibition of PCSK9 regulating LDL function following PRD460 administration in vivo. Pharmacokinetic analysis in these monkey studies indicated that the plasma half-life of adnectin PRD460 was approximately 70 hours. These data demonstrate the high efficacy and rapidity of the PCSK9 adnectin-Fc fusion protein in significantly, specifically, and long-lastingly reducing LDL-C levels in a study in cynomolgus monkeys. .

Pharmacological evaluation of unmodified PCSK9 adnectin ATI-1081 in vivo.

In addition to modified adnectins containing a PK group (eg, pegylated or Fc-fused adnectins), unmodified (free) PCSK9 adnectin can also be administered. Strategies for treatment with unmodified PCSK9 adnectin include more frequent dosing, given the shorter plasma half-life, or the use of subcutaneous extended-release dosage forms to prolong the absorption phase and prolong pharmacodynamics. Many such dosage forms can be developed, including, as a simple example, propylene glycol/PBS solutions to reduce the rate of absorption and increase the exposure time of adnectin in the bloodstream.

Unmodified adnectin ATI-1081 was administered intravenously to cynomolgus monkeys at a dosage of 10 mg/kg in PBS. For comparison, ATI-1114 (a pegylated version of the same adnectin) was administered at a dosage of 1 mg/kg in PBS. Administration of ATI-1081 caused a rapid, transient decrease in the level of unbound PCSK9 in the bloodstream. Within 30 minutes the reduction rate reached 100% (below the limit of quantitative detection) followed by a return to the baseline level within several hours (Fig. 19). At the same time, there was a trend towards a decrease in LDL-C levels during the first 24 hours after administration of ATI-1081 to monkeys (Fig. 20).

Unmodified adnectin ATI-1081 was also administered to transgenic mice with normal hPCSK9c expression as a simple sustained-release subcutaneous dosage form: a 50:50 mixture of polypropylene glycol and PBS, for comparison with PBS vehicle. It was expected that this dosage form would moderately reduce the rate of adnectin absorption and increase the exposure time of ATI-1081 in the bloodstream, thereby improving pharmacodynamics. Administration (i.p.) of ATI-1081 at 1 mg/kg in PBS resulted in a 50% reduction in plasma unbound PCSK9 at 3 hours, compared with an 85% reduction with ATI-1114 at 0.3 mg/kg. (Fig. 21). During

- 133 044608 of the second experiment ATI-1081 was administered to transgenic mice by subcutaneous injection with a polypropylene glycol (PG) vehicle, which resulted in approximately equivalent reduction in unbound LDL compared to ATI-1174 with an increased duration of this effect during the first 6 hours of the study (Fig. 22 ). These data demonstrate binding of unmodified PCSK9 adnectin ATI-1081 to human PCSK9 in vivo and improvement in initial pharmacodynamics when unmodified PCSK9 adnectin ATI-1081 is administered subcutaneously with a simple vehicle providing extended release time. The time course of the effect was consistent with the increased release time and duration of effect of unmodified PCSK9 adnectin.

Example 5: Serum albumin binding adnectins (SABA).

Example 5A. Search and selection of candidate adnectins that bind serum albumin.

Review.

A selection method called PROfusion (see, for example, Roberts et al., Proc. Natl. Acad. Sci., USA, 94(23):12297-12302 (1997); and WO 2008/066752) was applied to the libraries DNA containing engineered variable regions in the BC, DE and FG loops of domain ¹⁰ of Fn3. From these constructs, libraries containing over 10 ¹³ random sequences were generated and then selected against the biotinylated form of HSA to select candidate serum albumin binding adnectins (SABA) with the desired binding properties.

The process of producing proteins using High Throughput Protein Production (HTTP) technology.

Various HSA-binding adnectins were obtained using HTTP high-throughput protein production technology. Selected sequences encoding HSA binding proteins were cloned into the pET9d vector containing the 6His tag, after which E. coli strain BL21(DE3) pLysS cells were transformed with this vector. The resulting transformants were inoculated into 5 ml of LB medium containing 50 μg/ml kanamycin in 24-well plates. Cultures were incubated at 37°C overnight. Subsequent cultures were then prepared for expression induction by adding 200 μl of overnight cultures to fresh 5 ml of LB medium (50 μg/ml kanamycin) in the appropriate wells. The cultures were incubated at 37°C until an optical density A6 ₀₀ of 0.60.9 was reached. Expression was induced by adding isopropyl β-thiogalactoside (IPTG) to the culture to a concentration of 1 mM, after which the cultures were incubated for 4 hours at 30°C. After this, the cells were pelleted by centrifugation for 10 min at an acceleration of 3200 g and a temperature of 4°C. The resulting cell pellets were frozen at -80°C.

Cell pellets (in ₂₄ -well format) were lysed by resuspension in 450 μl of Lysis Buffer (50 mM _NaH2PO4 , 0.5 M NaCl, 1x Complete Protease Inhibitor Cocktail-EDTA free form (Roche), 1 mM PMSF, 10 mM CHAPS, 40 mM imidazole, 1 mg/ml lysozyme, 30 μg/ml DNase, 2 μg/ml aprotonin, pH 8.0) followed by stirring at room temperature for 1 hour. Lysates were clarified and the assay was converted to 96-well format by transferring the lysates to Whatman GF/D UNIFILTER® 96-well filtration plate combined with a 650 µl filtrate collection plate followed by centrifugation for 5 min at 200 g. The clarified lysates were transferred to a 96-well plate with Ni-chelating sorbent, pre-equilibrated with equilibration buffer (50 mM _NaH2PO4 , 0.5 M NaCl, 10 mM CHAPS, 40 mM imidazole, pH _8.0 ), and incubated for 5 min. Unbound material was removed. The resin was washed twice with 0.3 _ml /well of washing buffer No. 1 (50 mM _NaH2PO4 , 0.5 M NaCl, 5 mM CHAPS, 40 mM imidazole, pH 8.0). The resin was then washed three times with 0.3 ml/well of PBS. Before elution, 50 μl of Elution Buffer (PBS+20 mM EDTA) was added to each well. After incubation for 5 min, the liquid was removed using vacuum. The protein was eluted by adding 100 μl/well of Elution Buffer. After incubation for 30 min at room temperature, the plate(s) were centrifuged for 5 min at 200 g. The eluted protein was collected into a 96-well collection plate containing 5 µl of 0.5 M MgCl2 solution at the bottom of the wells. The eluted protein concentration was measured using the BCA method using SGE (adnectin control) as a protein standard. SGE adnectin is a wild-type ¹⁰ Fn3 domain (SEQ ID NO: 1) in which the integrin binding domain (RGD amino acid residues at positions 78-80) has been replaced by SGE residues.

HAS, RhSA and MuSA proteins in direct ELISA.

To analyze direct binding to HSA, MaxiSorp plates (Nunc International, Rochester, NY) were adsorbed with HSA (Sigma, St. Louis, MO) at a concentration of 10 μg/ml overnight at 4°C. After adsorption, nonspecific binding sites were blocked by incubating the plates with casein blocking buffer (Thermo Scientific, Rockford, IL) for 1–3 h at room temperature. For single-point screening, HTTP-purified adnectin was diluted 1:20 in casein blocking buffer and added to the wells for HSA binding. The plates were incubated for 1 hour at room temperature. Concentrations ranging from 0.1 nM to 1 μM were used in the concentration-dependent binding analysis. After incubation, plates

- 134 044608 were washed from unbound adnectin with PBST buffer, after which, to bind to adnectin with His tag, a conjugate of anti-His monoclonal antibodies with HRP (R&D Systems, MN) was added to the wells at a dilution of 1:2500 in casein blocking buffer and the plates were incubated in for 1 hour at room temperature. Excess conjugate was removed by washing with PBST, and adnectin binding was detected using TMB-containing reagents (BD Biosciences) according to the manufacturer's recommendations.

Measuring the degree of aggregation using gel filtration chromatography.

Gel filtration chromatography (Size exclusion chromatography (SEC)) was performed on SABA obtained via HTTP. For this purpose, SUPERDEX® 200 5/150 or SUPERDEX® 75 5/150 columns (GE Healthcare) were used on Agilent 1100 or 1200 HPLC systems with detection at A ₂₁₄ nm and A ₂₈₀ nm, as well as with a fluorescence detection system (excitation at 280 nm, emission at 350 nm). The following buffer was used for chromatography: 100 mM sodium sulfate, 100 mM sodium phosphate, 150 mM sodium chloride, pH 6.8; at a flow rate appropriate to the columns used. Gel filtration molecular weight standards (Bio-Rad Laboratories, Hercules, CA) were used for molecular weight calibration.

By gel filtration chromatography, isolated and HTTP purified SABAs were predominantly monomeric and eluted in the 10 kDa region compared to globular molecular weight gel filtration standards (BioRad).

Identification of candidate serum albumin binding adnectins (SABAs).

Through screening for HSA/RhSA/MuSA binding and biophysical criteria, four unique serum albumin binding adnectins (SABA) were identified and selected for further half-life evaluation in a mouse model. For in vitro and in vivo characterization, SABA data were accumulated in medium-scale quantities. In table Figure 6 shows the sequences of 26 unique SABA core sequences (designated by numbers from 1 to 26), identified using PROfusion technology. In the SABA4 sequence, before production in medium-scale quantities, a mutation was recorded in the structural part. The best variant of the structural part of the SABA4 sequence is SABA5. Both variants, SABA4 and SABA5, have identical sequences of the BC, DE and FG loops.

Example 5B. SABA candidate products and dosage forms.

Medium-scale production of SABA proteins.

Selected SABA sequences described in example 5A, with a 6His tag at the end, were cloned into the pET9d vector and expressed in E. coli strain BL21(DE3) pLysS cells (Table 6 shows the sequences of all SABAs with a His tag, designated SABA1 .1, SABA2.1, SABA3.1 and SABA5.1). 20 ml of cell culture (obtained from one colony scattered on a plate) was inoculated into 1 liter of LB medium with 50 μg/ml kanamycin. The culture was incubated to an optical density A6 ₀₀ of 0.6-1.0 at 37°C. After induction of expression by adding isopropyl-β-thiogalactoside (IPTG) to a concentration of 1 mM, the culture was incubated for 4 h at 30°C, after which the cells were collected by centrifugation for 30 min at an acceleration of >10,000 g and a temperature of 4°C. The resulting cell pellets were frozen at -80°C. Cell pellets were resuspended in 25 ml of lysis buffer (20 mM NaH ₂ PO ₄ , 0.5 M NaCl, 1x Complete Protease Inhibitor Cocktail-EDTA free form (Roche), pH 7.4) and cells were lysed on ice using an ULTRA-TURRAX® homogenizer (IKA works). Cell lysis was achieved by high homogenization pressure (>18,000 psi) using a Model M-110S MICROFLUIDIZER® (Microfluidics). The soluble fraction was separated by centrifugation for 30 min at an acceleration of 23300 g and a temperature of 4°C. The resulting supernatant was clarified by filtration through a filter with a pore size of 0.45 μm. The clarified lysate was applied to a HISTRAP® (GE) column, _pre -equilibrated with a buffer containing 20 mM _NaH2PO4 , 0.5 M NaCl, pH 7.4. After application, the column was washed with 25 column volumes of buffer 20 _{mM NaH2PO4} , 0.5 M NaCl, pH 7.4, then with 20 volumes of buffer ₂₀ mM _NaH2PO4 , 0.5 M NaCl, 25 mM imidazole pH 7.4, then with 35 volumes of 20 mM _NaH2 PO ₄ , 0.5 M NaCl, 40 mM imidazole pH 7.4. _the protein was eluted in 15 column volumes with a buffer containing 20 mM _NaH2PO4 , 0.5 M NaCl, 500 mM imidazole pH 7.4. Fractions were pooled based on absorbance values at _A280 , after which the resulting protein preparations were dialyzed against buffer: 1x PBS, 50 mM Tris, 150 mM NaCl pH 8.5 or 50 mM NaOAc; 150 mM NaCl; pH 4.5. The precipitate that formed was removed by filtration through a filter with a pore size of 0.22 μm.

Medium-scale expression and purification resulted in highly purified and active adnectins expressed in soluble form and purified from soluble fractions of the bacterial lysate. Analysis using gel filtration on SUPERDEX® 200 or SUPERDEX® 75 10/30GL columns with a mobile phase of 100 mM NaPO ₄ , 100 mM NaSO ₄ , 150 mM NaCl, pH 6.8 (GE Healthcare) demonstrated the predominant presence of the monomeric form of adnectins.

Dosage form SABA 1.2.

A specific SABA, SABA1.2 (SEQ ID NO: 411), was selected for preliminary formulation selection. The SABA1.2 sequence contains extension (ED) ₅ in core sequence 1 of Fn3 sequence ¹⁰ (see SEQ ID NO: 421 in Table 6). For SABA1.2 it was determined

- 135 044608 stable dosage form in the form of a solution containing 10 mM succinic acid, 8% sorbitol, 5% glycine and 5 mg/ml SABA1.2 protein, pH 6.0. The melting point of this protein in this dosage form was 75°C as determined by differential scanning calorimetry (DSC) at a protein concentration of 1.25 mg/ml. This dosage formula provided satisfactory physical and chemical stability at 4 and 25°C with an initial aggregation level of 1.2%. After a month of storage, the level of aggregation was very low (1.6% at 4°C and 3.8% at 25°C). The protein in this dosage form also remained stable after 4 freeze-thaw cycles when warmed from -80 and -20°C to room temperature. Also in this dosage form, SABA1.2 was soluble to a concentration of at least 20 mg/ml at 4°C and room temperature, without the formation of precipitate or increase in the number of aggregates.

Example 5C. Determination of biophysical characteristics of candidate SABAs.

Gel filtration chromatography.

Samples of medium-scale candidate SABAs were subjected to gel filtration chromatography (SEC). Gel filtration chromatography was performed on SUPERDEX® 200 10/30 or SUPERDEX® 75 10/30 columns (GE Healthcare) using Agilent 1100 or 1200 HPLC systems and a detection system for absorbance at wavelengths of 214 and 280 nm, as well as fluorescence (excitation =280 nm, emission =350 nm). For chromatography, a buffer solution of the following composition was used: 100 mM sodium sulfate, 100 mM sodium phosphate, 150 mM sodium chloride, pH 6.8. Chromatography was carried out at flow rates depending on the type of column. A set of molecular weight standards (Bio-Rad Laboratories, Hercules, CA) was used to calibrate molecular weights.

Gel filtration chromatography results demonstrated that in the samples tested, adnectin was predominantly in monomeric form and eluted in the approximate 10 kDa range compared to a set of globular gel filtration standards (BioRad).

Thermal stability.

To determine the Tm of SABA proteins produced in medium-scale quantities, the differential scanning calorimetry (DSC) method was used. Solutions with a concentration of 1 mg/ml were analyzed using an N-DSC II calorimeter (Calorimetry Sciences Corp) in the temperature range from 5 to 95°C, with a rate of change of 1 degree per minute and at a pressure of 3 atm. The obtained data were compared with the control measurement for the corresponding buffer and the best-fit algorithm of the Orgin software (OrginLab Corp). The results of DSC gel filtration chromatography are summarized in table. 14.

Table 14

Summary of Gel Filtration Chromatography and DSC Data for SABA Candidates

Clone Gel filtration chromatography DSC (Tm) Monomer (%) Dimer (%) SABA1.1 92.3 7.7 63.9 °C SABA5.1 88 12 70.1 °C SABA2.1 91 9 58.5/78.2°С SABA3.1 99 <pd* 65.2 °C

* pd - detection limit.

Example 5D. Characteristics of serum albumin binding of the SABA1 candidate variant.

The binding kinetics of selected SABA clones obtained using HTTP technology or produced in medium-scale quantities (described in Examples 5A and 5B) were determined by binding the corresponding serum albumin (HSA/RhSA/MuSA) to the surface of the Biasensor CM5 chip and passing a stream of solutions with serial dilutions of SABA through the reference cell and the cell with bound albumins. In addition, binding to albumins was carried out at different pH levels, ranging from 5.5 to 7.4. The HSA-binding adnectins SABA2.1, SABA3.1, SABA4.1, and SABA1.1 were cross-reactive to RhSA but did not bind to MuSA. Binding of SABA2 and SABA4 to HSA is pH-sensitive, whereas binding to HSA of the SABA3 variant is resistant to pH reductions down to 6.0. The SABA1.1 variant satisfies biochemical criteria for pH stability and affinity/kinetics when pH is reduced to 5.5.

Domain mapping was performed using BIACORE®. Selected SABA clones, purified using HTTP technology or produced in medium-scale quantities, were analyzed by binding HSA or constructs containing HSA domains I and II or HSA domain III on the surface of a Biasensor CM5 chip and then passing serial dilutions of SABA variants through a reference cell. and a cell with bound albumins. Clones SABA2 and SABA1 bound to HSA and HSA domain 1-domain II constructs, but not to domain III alone. Clones SABA3 and SABA4 bound to HSA but to none of the above constructs. The results obtained are summarized in table. 15.

Table 15

Binding affinity and kinetics of candidate SABA variants (SABA1.1, 2.1, 3.1 and 4.1)

Adnectin Target protein K _D (hM) Koff (c' ¹ ) pH stability 7.4—>5.5? Epitope on HSA SABA2 HSA 33.8 +/-20.5 (n=6) 1.71E-04 Domain I-II RhSA 63.6 4.42E-04 SABA3 HSA 863 6.82E-02 (at reduction to 6.0) None of the constructs: domain I-Π or domain III (interdomain region?) RhSA 431 3.37E-02 SABA4 HSA 412+/-8 (n=4) 7.82E-04 None of the constructs: domain I-Π or domain III (interdomain region?) RhSA >1000 3.83E-03 SABA1 HSA 47.2+/-18.2 (n=9) 4.57E-04 +++ Domain I-II RhSA 778+/-313 (n=4) 5.45E-03

Example 5E. In vivo study of the t _1/2 parameter of candidate SABA variants.

Determination of the parameters of HSA-binding adnectins was carried out in mice, since these adnectins do not cross-react with MuSA. To do this, the half-life of HSA in mouse plasma was determined. 6-week-old female Ncr nude mice were injected into the tail vein with HSA at dosages of 20 mg/kg (Fig. 23A) and 50 mg/kg (Fig. 23B), after which the concentration of HSA in blood samples taken through certain time intervals after injection. The t _1/2 parameter of the administered HSA was determined for a dosage of 20 mg/kg after 24 hours, for a dosage of 50 mg/kg 20 hours after administration.

Determination of the half-life of SABA1-4 in mice.

In a volume of one liter of E. coli culture, proteins of clones SABA1.1, SABA2.1, SABA3.1 and SABA4.1 were produced, isolated, purified from impurities and endotoxin. Each of the SABA variants was injected into the tail vein of mice, after which the concentration of the injected proteins was assessed using ELISA in blood samples taken at certain time intervals after injection.

Studies of the pharmacokinetic profiles of each SABA variant in the presence and absence of HSA were carried out on 6-week-old female Ncr nude mice. When administering additional SABA to mice, HSA was first prepared with a mixture of HSA and SABA (HSA in 3-4 molar excess). The SABA clones tested were specific for HSA and RhSA but did not bind MuSA. The plasma half-life of SABA1.1 protein (clone 1318H04) in mice was 0.56 hours, whereas co-injection of SABA1.1 with HSA resulted in a ~10-fold increase in half-life, which was 5.6 hours (Fig. 24A). The plasma half-life of SABA2.1 protein in mice was 0.24 hours, whereas co-injection of SABA2.1 with HSA resulted in a ~12-fold increase in half-life, which was 2.8 hours (Fig. 24B). The half-life of SABA3.1 protein (clone 1245H07) in mouse plasma was

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0.28 h, whereas co-injection of SABA3.1 with HSA resulted in a ~2-fold increase in half-life, which was 0.53 h (Fig. 24C). The mouse plasma half-life of SABA4.1 was 0.66 hours, whereas co-injection of SABA4.1 with HSA resulted in a ~7-fold increase in half-life of 4.6 hours (FIG. 24D). A summary of the examples given is shown in FIG. 25. In table. 16 summarizes similar data for variants SABA1.1, SABA2.1, SABA3.1 and SABA5.1; Where possible, comparisons are made with the half-life of the cynomolgus monkey.

Table 16

CLONE RK (T1/2) Notes Mice Javanese macaques SABA1.1 5.6 h 96-137 4 T1/2 = 96-137 4 SABA5.1 4.6 h 12 h Low binding affinity for RhSA. At pH<6.0 there is 2x increase in KD SABA2.1 2.8h ND No binding at pH<6.5 SABA3.1 32 min ND Little knowledge of T1/2 is observed in mice

Determination of the half-life of SABA1.1 and SABA5.1 variants in cynomolgus macaques.

To preliminary evaluate pharmacokinetics, a study was conducted in 2 cynomolgus monkeys for the SABA1.1 (Fig. 26A) and SABA5.1 (Fig. 26B) variants. Proteins were administered intravenously once at a dose of 1 mg per kg (mg/kg, mpk). Pharmacokinetics were assessed using a quantitative ELISA assay developed for the detection of adnectin in plasma samples. The SABA1.1 variant has a half-life in the range of 96-137 hours (Fig. 26A and Table 17A). The half-life of the SABA5.1 variant is approximately 12 hours. The plasma concentration of SABA5.1 was reliably measured by ELISA only up to 120 hours after injection (Fig. 26B). In table 17A summarizes the data obtained for option SABA1.1 in table. 17V - for SABA5.1.

Table 17A

SABA1.1

Monkey tl/2 (n) Stach (mcg/ml) AUCall (h* µg/ml) Clobs (ml/h/kg) Vzobs (ml/kg) #1 95.8 9.03 673.7 1.45 200.8 #2 136.6 7.24 625.1 1.60 315.2

Table 17B

SABA5.1

H.L.Lambdaz (h) Stach (mcg/ml) AUCall (h*µg/ml) Cl_obs (ml/h/kg) Vzobs (ml/kg) N 2 2 2 2 2 Average 12.186 17.358 246.882 4.089 72.507 SD 1.451 3.08 36.245 0.596 19.045 Min 11.16 15.18 221.25 3.67 59.04 Max 13.21 19.54 272.51 4.51 85.97 CV% 11.9 17.7 14.7 14.6 26.3

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Example 5F. Binding characteristics of SABA1 to serum albumin.

SABA1.1 and 1.2 bind to HAS and RhSA.

SABA1.2 is the Fn3 domain ¹⁰ core sequence 1 containing extension (ED)5 (SEQ ID NO: 421 in Table 6). SABA1.2 bound to human serum albumin (HSA) at neutral pH and 25°C with an average association rate constant (ka) of 8.21E+03 M ^-1 s ^-1 and an average dissociation rate constant (kd) of 4.34E -04c ^-1 , with a calculated average KD of 55.3 nM (Table 18). For rhesus monkey serum albumin, the measured association rate constant was 6.6E+03 M ^-1 s ^-1 and the dissociation rate constant was 3.78E -03 s ^-1 , giving an estimated average K _D value of 580 nM. No measurable interaction was observed between SABA1.2 and mouse or rat serum albumins up to a concentration of 1 μM (Table 18 and FIG. 27). At 37°C, the values of ka and kd increased from 2 to 5 times, which led to an approximately 2-fold increase in affinity for HSA and a 1/ ₂ -fold increase in affinity for RhSA (Table 18).

Table 18

Kinetic parameters of albumin binding for SABA1.2 in HBS-P b ufere Albumin Tem ka (1/Ms) kd (1/s) KD temperature (nM) (°C) Person 25 8.21 ±1.19 E+03 4.43 ±0.65 E-04 55.3 ±13.7 Rhesus monkey 6.60 ±1.18 E+03 3.78 ±0.45 E-03 580±62.6 Mouse no observed binding Person 37 3/38E+04 8/15E-04 24/1 Rhesus monkey 1/89E+04 1/85E-02 977/4 Mouse no observed binding

Additionally, calorimetric titration was performed to determine the stoichiometric parameters of SABA1 and HSA binding. The variant used for this study was SABA1.1, which is the Fn3 domain ¹⁰ core sequence 1 containing a 6His tag extension (SEQ ID NO: 420 in Table 6). HSA (10 μL per injection of 115 μM solution) was injected into a calorimetric cell containing SABA1.1 at a concentration of 8.1 μM. This experiment was carried out at 37°C in PBS at pH 7.4. In fig. Figure 28 shows data demonstrating a 1:1 stoichiometry of SABA1.1 binding to HSA.

SABA1.2 actively binds to HSA at low pH.

The long half-life of albumins (e.g. t _1/2 HSA is 19 days) is largely due to the fact that albumins bind to the neonatal Fc receptor (FcRn) at low pH values within the endosome, which avoids degradation and leads to the return of the molecule albumin into the bloodstream. Data in table. 19 show that the SABA1.2 variant was actively associated with HSA at pH5.5, which coincides with the pH in the endosome, suggesting that the t1 _/2 of SABA bound to HSA will increase due to recycling of the molecule upon interaction with FcRn.

Table 19

Comparison of albumin binding kinetics at pH 7.4 and 5.5 in MES buffer

Albumen pH ka (1/Ms) kd (1/s) KD (nM) Human 7.4 9.26 E+03 3.88 E-04 41.9 5.5 9.44 E+03 2.70 E-04 28.6 Macakaresus 7.4 6.16 E+03 2.95 E-03 479 5.5 7.57 E+03 2.72 E-03 359

SABA1.2 binds to domains I and II, but not III, of the HAS molecule.

The SABA1.2 binding site on the albumin molecule was mapped using recombinant-139-

Claims (3)

ny fragments of HSA. The binding site is located at the end of domains I or II, while no binding is detected with domain III (Fig. 29). Since domain III of the HSA molecule is primarily involved in interaction with FcRn, the likelihood of SAA1.2 competing for HSA binding to FcRn is low, which also raises the possibility of using the FcRn recycling mechanism to increase half-life. Example 5G. Pharmacology of the SABA1.2 variant in an in vivo study. To evaluate the pharmacokinetics and immunogenicity of SABA1.2, four-week preliminary toxicology studies were conducted in cynomolgus monkeys in which SABA1.2 was administered as a single dose at 2 concentrations. Pharmacokinetics and immunogenicity were also assessed in three-week preliminary toxicology studies, which included two study variants in which the protein was administered as a single intravenous or subcutaneous dose. The pharmacokinetics of SABA1.2 were also evaluated in two independent single-dose toxicology studies in cynomolgus monkeys. Detection of SABA1.2 in plasma samples was carried out using a developed quantitative ELISA-based assay. SABA1.2 was administered to monkeys at dosages of 1 and 10 mg/kg intravenously. Non-compartmental analysis using WINNONLIN® software was used to evaluate pharmacokinetic parameters. As shown in FIG. 30 and according to the parameters below, the pharmacokinetics of SABA1.2 in the described study are dose-dependent, as determined by the area under the concentration-time curve (AUC). Clearance (CL) of SABA1.2 at 10 mg/kg was 0.15 ml/h/kg, beta phase half-life (t _1/2 ) was 143 h, volume of distribution (Vz) was 30 ml/kg, total drug exposure substance (AUCall) was 5609457 h-nmol/l (Table 20). Clearance (CL) of SABA1.2 at 1 mg/kg was 0.4 ml/h/kg, beta phase half-life (t _1/2 ) was 124 h, volume of distribution (Vz) was 72 ml/kg, total drug exposure substance (AUCall) was 214,636 h-nmol/l (Table 20). Following subcutaneous or intravenous administration of SABA1.2, the beta phase pharmacokinetic profiles were similar (Figure 31). Clearance (CL) of SABA1.2 when administered intravenously at 1 mg/kg was 0.22 ml/h/kg, beta phase half-life (t _1/2 ) was 125 h, volume of distribution (Vz) was 40 ml/kg, total drug exposure (AUCall) was 357,993 h-nmol/l (Table 20). The clearance (CL) of SABA1.2 with subcutaneous administration of 1 mg/kg was 0.32 ml/h/kg, beta phase half-life (t _1/2 ) was 134 h, volume of distribution (Vz) was 62 ml/kg, total drug exposure (AUCall) was 251,339 h-nmol/l (Table 20). The relative bioavailability (F) for subcutaneous versus intravenous administration was 0.7. Table 20 Pharmacokinetic parameters for SABA1.2 in monkey studies Study # 1 2 Dose (mg/kg) 1 10 1 1 Way introduction i.v. i.v. i.v. S.C. N 3 3 1 2 CL (ml/h/kg) 0.4 0.15 0.22 0.32 Vz (ml/kg) 72 thirty 40 62 AUCall (h*nmol/l) 214,636 5,609,457 357,993 251.339 beta T1/2 (h) 124 143 125 134 Bioavailability(F) n/a n/a n/a 0.7 CLAIM 1. An isolated polypeptide containing the tenth type III domain of fibronectin ( ¹⁰ Fn3) fused to serum albumin or serum albumin binding protein, wherein the sequence ¹⁰ Fn3 has loops BC, DE and FG, wherein the loop BC contains SEQ ID NO: 112, loop DE contains SEQ ID NO: 138 and loop FG contains SEQ ID NO: 156 and wherein the polypeptide binds proprotein convertase subtilisin/kexin type 9 (PCSK9). 2. The isolated polypeptide according to claim 1, wherein the polypeptide binds PCSK9 with a KD of less than 10 nM. 3. The isolated polypeptide according to claim 1, wherein the polypeptide additionally contains polyethylene glycol. - 140 -